Micro Test Final PDF

PROTECTION MEASURES TO PREVENT AND CONTROL
MICROBIAL CONTAMINATION
The first step in protecting a public water supply is the development of a watershed or wellhead protection
program. Controlling or eliminating microbial sources before they contaminate a water supply goes a long way
toward simplifying treatment and reducing costs associated with a contaminated supply.
An effective protection program must address a variety of sources of microbial contamination. Following is a list of
those sources and suggested protection measures aimed at reducing the risk they pose to drinking water.
SEWAGE DISPOSAL SYSTEMS STORMWATER RUNOFF area (around an intake or in areas where the nuisance
Failing sewage disposal systems represent the major Rainwater and snowmelt flow over the wildlife population is concentrated). Federal and/or
source of microbial contamination from human waste. land picking up pollutants and then state permits are often required for wildlife control
Contamination sources include large municipal waste depositing them into water supplies. harassment programs.
treatment systems, pump stations, and on-site Runoff can also collect microbial ! Monitor wildlife populations in and around water
sewage treatment systems such as cesspools and contaminants (such as pet waste) from suburban supplies.
septic tanks and leachfields. environments. ! Maintain a daily human presence.
! Implement proper planning for ! Minimize impervious surfaces within your watershed. ! Employ scare techniques such as pyrotechnics.
sewage systems within your ! Install catch basins and settling basins to slow down ! Modif y habitat (shoreline fencing, mowing,
watershed. flows and filter out contaminants. landscaping changes, tree branch pruning).
! Ensure septic systems are ! Use landscaping techniques such as native plants, ! Prohibit the public from feeding wildlife, especially
inspected and serviced on a regular basis. low maintenance grasses, shrubs, and rock gardens waterfowl.
! Promote public education on how to care for a that conserve water and limit runoff. ! Reduce food sources such as palatable plant
septic system. ! Require the proper removal and disposal of pet species.
waste. ! Keep beavers and muskrats from building dams/
AGRICULTURE dens by installing fencing or drainage devices.
Runoff carrying animal waste from barnyards, manure WILDLIFE ! Consider permitted trapping or hunting.
storage areas, dairy farms, pig farms, pastures, and Wildlife is an integral part of a
the land application of manure is a significant source balanced watershed. However,
of microbial contamination. birds and small mammals can
! Ensure animal waste runoff introduce microorganisms into a
is properly collected, settled, water supply either through direct
and stored. contact or from watershed runoff. Giardia, CONCLUSION
! Manure storage sheds cr yptosporidium, salmonella, campylobacter, and When it comes to the protection of our drinking water
should be sited away from surface waters and Escherichia coli (E.coli) are the most commonly identified resources, an ounce of prevention is worth a pound of
should have impermeable floors and roofs. microorganisms found in mammals and birds. Wildlife cure. For more information about some of the most
! Install vegetative buffer strips to slow runoff flow commonly associated with microbial contamination of prevalent threats to drinking water, request the
and to act as a filter for microbial contaminants. drinking water supplies include deer, beavers, muskrats, following brochures from NEIWPCC:
! Livestock should be kept out of streams and water gulls, and geese. What Do You Know About...Nonpoint Source
bodies. Pollution?
The following protection measures should not be What Do You Know About...Septic Systems?
considered as general practice but should be carefully What Do You Know About...CSOs?
deployed in specific areas of a water supply protection
WHAT ARE MICROORGANISMS?
Microorganisms are microscopic creatures such as
bacteria, viruses, and protozoa. Because some
National
CONTACT INFORMATION
What Do You
Know About...
American Water Works Association
microorganisms are known pathogens (disease
www.awwa.org
causing agents), microbial contamination of public
drinking water supplies can threaten human health. Association of State Drinking Water Administrators
www.asdwa.org
Contaminants of concern in drinking water include:
US EPA, New England Regional Office
! Bacteria: Heliobacter pylori, the Salmonella family,
www.epa.gov/region01/eco/drinkwater/
and Escherichia coli (E.coli).
index.html
! Viruses: Hepatitis A, Norwalk type viruses,
rotaviruses, adenoviruses, enteroviruses, and Interstate
reoviruses.
! Protozoa: Giardia lamblia and Cryptosporidium
parvum.
NEIWPCC
978/323-7929
Microbial
WHAT IS THE THREAT TO HUMAN HEALTH?
When ingested in drinking water, bacteria, viruses, and
www.neiwpcc.org
NEWWA Contamination?
protozoa can cause a number of infectious waterborne 508/893-7979
diseases such as cholera, typhoid, hepatitis, and www.newwa.org
infectious gastrointestinal diseases like cryptosporidiosis
State
and giardiasis. Symptoms of waterborne disease may
include fever, fatigue and weight loss (common in viral Connecticut Department of Environmental
cases), vomiting, abdominal cramping, diarrhea, and Conservation Source Water Assessment Program
stomachaches. In the most severe cases waterborne dep.state.ct.us/wtr/SWAP/swapsumm.htm
disease can prove lethal. Maine Department of Human Services/Division of
Health Engineering, Drinking Water Program
WHO IS AT RISK? janus.state.me.us/dhs/eng/water/index.htm
Waterborne diseases pose a risk to everyone. Some
individuals are more sensitive to infection and suffer more Massachusetts Department of Environmental
pronounced symptoms. These sensitive groups include Protection, Drinking Water Program
infants, young children, older persons, and immuno- www.state.ma.us/dep/brp/dws
compromised individuals such as persons with HIV/AIDS New Hampshire Department of Environmental
and those undergoing chemotherapy. Services Water Division Drinking Water Source
Protection Program
HOW CAN WE PROTECT AGAINST MICROBIAL www.des.state.nh.us/dwspp/
CONTAMINATION? Rhode Island Department of Environmental
Treatment Management Office of Water Resources
Some microbial contaminants can be removed by water www.state.ri.us/dem/programs/benviron/water/
treatment coagulation and filtration processes. quality/index.htm New England Interstate Water
Disinfection has proven effective against bacteria and
Vermont Agency of Natural Resources Water Pollution Control Commission
viruses, but protozoa such as Giardia and especially Boott Mills South
Supply Division
Cryptosporidium are very resistant to chlorination alone. 100 Foot of John Street
www.anr.state.vt.us/dec/watersup/wsd.htm
Prevention Lowell, MA 01852
The most important and cost effective protection for water Phone: 978/323-7929 ! Fax: 978/323-7919
suppliers is to prevent pathogen entry into source water. www.neiwpcc.org ! mail@neiwpcc.org
Menu Background
Background
• United States Pharmacopeia (USP)
Personnel Cleansing and Gowning
– Non-governmental non-profit organization
Responsibilities of Compounding Personnel – Primary activities are creation of standards, patient safety,
healthcare information, and verification of products
Risk Level Classifications
• Quality and consistency of medicines
Verification of Accuracy and Sterilization – Prescription
– Non-prescription
Personnel Training and Assessment – Dietary supplements
– Veterinary drugs
Environmental Quality and Control – Healthcare products
Equipment • Safe and proper use of medications

– USP standards are developed by a unique process of public
Storage and Beyond-Use Dating involvement
University of Kentucky / NetLearning CBL ‹#› of 46 University of Kentucky / NetLearning CBL ‹#› of 46
Background (cont.) Background (cont.)

USP Legal and Regulatory Basis USP Legal and Regulatory Basis (cont.)
• Food and Drugs Act – 1906 • Each general chapter of the USP/NF is assigned a
– US Pharmacopeia (USP/NF) became the official standard for
number which appears in brackets
drugs in the United States
• Chapter <1> to <999> are required
• Federal Food, Drug and Cosmetic Act – 1938 – Pharmacies are subject to inspection for compliance with
– USP/NF official compendia of drug standards required standards by:
– Food and Drug Administration (FDA) responsible for • Boards of Pharmacy
enforcement of the act • FDA
• Joint Commission on Accreditation of Healthcare Organizations
– FDA may enforce required standards in USP/NF (JCAHO)
• Includes Chapter <797>
• Chapter <1000> to <1999> are informational
Background (cont.) Background (cont.)

Goal of Chapter <797> Definition of Compounded Sterile Products (CSP) – USP27 <797>
• Goal of USP Chapter <797> is to prevent potential • Preparations prepared according to the manufacturer’s
patient harm or death that could result from: labeled instructions and other manipulations that expose
– Microbial contamination contents to potential contamination
– Excessive bacterial endotoxins • Preparations containing nonsterile ingredients or employ
– Large content errors in the strength of correct ingredients nonsterile components or devices that must be sterilized
– Incorrect ingredients before administration
• Biologics, diagnostics, drugs, nutrients, and
radiopharmaceuticals that possess either of the above
two characteristics
1
Background (cont.) Selected Sections of Chapter <797>
Scope of USP <797> Multidisciplinary • Personnel cleansing and gowning

Chapter USP <797> applies to pharmacists, physicians, • Responsibilities of compounding personnel
nurses, and allied health team members. • Risk level classification of Compounded Sterile Products
(CSP) and quality assurance
• Verification of accuracy and sterilization of CSP
• Personnel training and assessment
• Environmental quality and control
• Equipment
• Storage and beyond-use dating
Personnel Cleansing and Gowning Personnel Cleansing and Gowning (cont.)
• Hand Washing • After washing hands, put on

– Wash hands, nails and arms up to the elbow with soap and non-shedding uniform
water components in this order:
– Knee-length coats or coveralls
– Wash for at least 15 seconds (Sing alphabet song)
– Hair cover
– Use a disposable scrub brush to clean nails and between fingers – Shoe covers
– Dry hands with non-shedding towel – Protective gloves
– Face mask when in hood
– Turn off faucet with towel or use foot pedals
Personnel Cleansing and Gowning (cont.) Personnel Cleansing and Gowning (cont.)
• Every time you leave the buffer area you must remove
• Hair Covers
– Must cover all hair and discard your:
– Beards and long sideburns require use of beard cover – Hair cover
– Gloves
– Face mask
• Gloves
– Powder free
– Clean new gloves with 70% isopropyl alcohol before use • Must remove lab coat when leaving buffer area
– Avoid touching non-sterile surfaces – May hang coat inside out
– Intermittently sanitize gloves with 70% isopropyl alcohol – Must discard coat at the end of each shift
2
Personnel Cleansing and Gowning (cont.) Selected Sections of Chapter <797>
• Face mask must be worn • Personnel cleansing and gowning

while in hood • Responsibilities of compounding personnel
• Risk level classification of Compounded Sterile Products
– Minimizes airborne
contaminants while talking,
sneezing and coughing • Verification of accuracy and sterilization of CSP
– Must cover mouth and • Personnel training and assessment
nose completely • Environmental quality and control
• Equipment
Responsibilities of Compounding Personnel Responsibilities of Compounding Personnel (cont.)
• Manipulate sterile products • Ensure products are correctly • Open or partially used • Before dispensing and
aseptically – Purified packages for subsequent use administration, products are
• Ensure products are accurately – Sterilized are: visually inspected for clarity
– Identified – Packaged – Properly stored
– Measured – Sealed – Clearly labeled with date and • Beyond-use dates are
– Labeled time opened or date and time assigned based on direct
– Diluted
of expiration
– Mixed – Stored testing or extrapolation from
• Labels on products must list reliable literature and other
• Maintain appropriate – Dispensed
names, amounts added and documentation
cleanliness conditions – Distributed
concentrations of all
ingredients
Selected Sections of Chapter <797> Risk Level Classifications of Compounded Sterile Products
• Personnel cleansing and gowning • Appropriate risk level (low, medium, high) assigned
• Responsibilities of compounding personnel according to corresponding probability of contamination
• Risk level classification of Compounded Sterile Products with:
(CSP) and quality assurance – Microbial (organisms, spores, endotoxins)
– Chemical or Physical (foreign chemicals or physical
• Personnel training and assessment matter)
• Environmental quality and control • Characteristics serve as guide and are not prescriptive
• Equipment • Risk level ultimately determined by professional
• Storage and beyond-use dating judgment
3
Risk Level Classifications of Compounded Sterile Products (cont.) Risk Level Classifications of Compounded Sterile Products (cont.)
Low-Risk Level Characteristics Low-Risk Level Characteristics (cont.)
• Low-Risk Conditions • Examples of Low-Risk Compounding

– Products compounded with aseptic manipulations entirely within – Single transfers of sterile dosage forms from ampules, bottles,
ISO class 5 quality air using only sterile ingredients, products, bags, and vials using sterile syringes with sterile needles, and
components or devices other sterile containers. The contents of ampules requires sterile
– Involves only transfer, measuring, and mixing manipulations with filtration to remove glass particles
closed or sealed packaging systems performed promptly and – Manually measuring and mixing no more than three
attentively manufactured products to compound drug admixtures
– Manipulations limited to aseptically opening ampules, – Tobramycin piggyback
penetrating sterile stoppers on vials with sterile needles and
– Morphine drip
syringes, and transferring sterile liquids in sterile syringes to
other sterile products
Low-Risk Level Characteristics (cont.) Medium-Risk Level Characteristics
• Quality Assurance • Medium-Risk Conditions include all low-risk conditions in
– Routine disinfection of the direct compounding environment to
addition to one or more of the following:
minimize microbial surface contamination – Multiple individual or small doses of sterile products are
combined or pooled to prepare a product that will be
– Visual conformation that personnel are properly garbed with hair administered to multiple patients or the same patient on multiple
covers, gloves, masks, etc. occasions
– Review of all products to ensure correct identity and amounts of – Compounding includes complex aseptic manipulations other
ingredients were compounded than single volume transfer
– Visual inspection of products to ensure the absence of – Compounding requires unusually long duration to complete
particulates in solutions, the absence leakage from vials or bags, dilution or homogenous mixing
accuracy of labeling
– The product does not contain bacteriostatic substances and is
administered over several days (e.g. external or implanted
pump)
Medium-Risk Level Characteristics (cont.) Medium-Risk Level Characteristics (cont.)

• Examples Medium-Risk Compounding • Quality Assurance
– Compounding of total parenteral nutrition using manual or – Routine disinfection of the direct compounding environment to
automated devices minimize microbial surface contamination
– Filling of reservoirs of injection and infusion devices – Visual conformation that personnel are properly garbed with hair
• with multiple sterile products covers, gloves, masks, etc.
• administered over several days at ambient temperatures (implanted – Review of all products to ensure correct identity and amounts of
pumps) ingredients were compounded
– Transfer of volumes from multiple ampules or vials into a single, – Visual inspection of products to ensure the absence of
final sterile container or product (terbutaline drip) particulates in solutions, the absence leakage from vials or bags,
– Hydromorphone IVPCA batches using commercial sterile accuracy of labeling
ingredients
4
High-Risk Level Characteristics High-Risk Level Characteristics (cont.)
• High-Risk Conditions include all low-risk and medium- • Examples of High-Risk Compounding
risk conditions in addition to one or more of the following – Dissolving non-sterile bulk drug and nutrient powders to make
– Non-sterile ingredients are incorporated or a non-sterile device is solutions, which will be terminally sterilized
employed before terminal sterilization
– Exposure of sterile ingredients to air less than ISO class 5 (e.g.
– Sterile ingredients, components, devices, and mixtures are nursing preparation on the ward)
exposed to air quality inferior to ISO class 5
– Measuring or mixing of sterile ingredients in non-sterile devices
– Non-sterile preparations are stored greater than 6 hours before before sterilization is performed
being sterilized
Media-Fill Testing
High-Risk Level Characteristics (cont.)
• Low and Medium Risk Levels
• Quality Assurance – Personnel authorized to compound low or medium risk products
– Routine disinfection of the direct compounding environment to are required to perform media-fill testing annually
minimize microbial surface contamination – Test must simulate most challenging and stressful conditions
– Visual conformation that personnel are properly garbed with hair during compounding of low or medium risk products respectively
covers, gloves, masks, etc.
• High-Risk Level
– Review of all products to ensure correct identity and amounts of
– Personnel authorized to compound high-risk products are
ingredients were compounded
required to perform media-fill testing semi-annually
– Visual inspection of products to ensure the absence of
– Test must simulate most challenging and stressful conditions
particulates in solutions, the absence leakage from vials or bags,
during compounding of high-risk products
accuracy of labeling
Selected Sections of Chapter <797> Verification of Accuracy and Sterilization for CSP
• Personnel cleansing and gowning • Physical Inspection

• Responsibilities of compounding personnel – Finished products to be individually inspected for
• Particulates
(CSP) and quality assurance • Foreign matter
• Container-closure integrity
• Other apparent visual defects
• Personnel training and assessment
– Products not immediately distributed must be inspected before
• Environmental quality and control leaving the storage area
• Equipment
5
Verification of Accuracy and Sterilization for CSP (cont.) Verification of Accuracy and Sterilization for CSP (cont.)
• Sterilization and Bacterial Endotoxin Testing

• Compounding Accuracy Checks – Required for high-risk level products that involve nonsterile
– Written procedures for double-checking compounding accuracy products or devices
must be followed – Product must be tested according to USP Chapter <85>
Bacterial Endotoxins Test
– Double check should include label accuracy and accuracy of the
addition of all products or ingredients – Sterilization Methods
• Dry Heat
– Used containers and syringes should be quarantined with the – 250°C for two hours
final product until double check is performed • Steam (autoclave)
– Double check should be performed by person other than the – 121°C at 15 pounds per square inch (p.s.i.) for 20 to 60 minutes
compounder • Filtration
– 0.2 micron filter certified to retain 107 Brevundimonas diminuta per cm2
• Must verify sterilization procedures
– Required to prove it
– Accomplish with media fill testing
Selected Sections of Chapter <797> Personnel Training and Assessment
• Personnel cleansing and gowning

• Personnel who prepare compounded sterile products or
• Responsibilities of compounding personnel parenteral preparations must be provided with
• Risk level classification of Compounded Sterile Products appropriate training in the theoretical principals and
(CSP) and quality assurance practical skills of aseptic manipulations.
• Verification of accuracy and sterilization of CSP • Assessment of knowledge and skills
• Personnel training and assessment – Annually for personnel preparing low and medium-risk level
products
– Semi-annually for personnel preparing high-risk level products
• Equipment
Personnel Training and Assessment (cont.) Selected Sections of Chapter <797>
• Assessment
• Personnel cleansing and gowning
– Written tests
– Media-fill challenge testing • Responsibilities of compounding personnel
– Failure of assessment requires re-instruction and re-evaluation • Risk level classification of Compounded Sterile Products
before being allowed to compound sterile products (CSP) and quality assurance
• Media-fill challenge testing • Verification of accuracy and sterilization of CSP
– Sterile bacterial culture medium transferred via a variety of aseptic • Personnel training and assessment
manipulations
– Test should represent the most challenging products made for a
particular risk level • Equipment
– Products are monitored for microbial growth, indicated by visual • Storage and beyond-use dating
turbidity, for 14 days
6
Environmental Quality and Control Environmental Quality and Control (cont.)
• Critical Site • Cleaning and sanitizing workspaces

– Standard operating procedures are required
– Any opening providing a direct pathway between a sterile product – Pharmacy policy PH06-05 fulfills this requirement and discusses specific
and the environment or any surface coming into direct contact with UKCMC procedures
the product and the environment – Please “click” on policy link above for PH06-05 and read the policy.
– Must protect these sites from environmental contamination Required to answer a test question.
• Personnel garb and hand washing (policy PH06-04)

• Air Quality – Please “click” on policy link above for PH06-04 and read the policy.
– ISO Class 5 (Class 100) required in critical area (area where Required to answer a test question.
sterile products are directly exposed, e.g. hood)
• Environmental monitoring
– ISO Class 8 (Class 100,000) required for buffer area or clean room
– Air quality inspections (every 6 months)
– Minimize air currents from open doors, personnel traffic and – Certification of hoods and barrier isolators (every 6 months)
ventilation ducts – Evaluation of airborne microorganisms
– Air conditioning and humidity control required • Monthly for low and medium risk-level compounding areas
• Weekly for high risk-level compounding areas
Selected Sections of Chapter <797> Equipment
• Personnel cleansing and gowning • Personnel are to be trained to operate any piece of
• Responsibilities of compounding personnel equipment or apparatus they may use to prepare
compounded sterile products
(CSP) and quality assurance • Equipment calibration documentation
• Verification of accuracy and sterilization of CSP • Annual and routine maintenance documentation
• Personnel training and assessment • Monitoring for proper function documentation
• Environmental quality and control • Procedures for use
• Equipment
• Calibration and maintenance reports to be kept on file
Selected Sections of Chapter <797> Storage and Beyond-Use Dating
• Personnel cleansing and gowning In the absence of sterility testing , storage periods (before
administration) shall not exceed the following based on sterility:
• Responsibilities of compounding personnel
• Risk level classification of Compounded Sterile Products Low-risk Medium-risk High-risk
• Verification of accuracy and sterilization of CSP Room Temp <48 hours <30hours <24 hours
• Personnel training and assessment >8ºC
Refrigerated <14 days <7 days <3 days
2º to 8ºC
• Equipment
Freezer <45days <45days <45days
• Storage and beyond-use dating <20ºC
7
Storage and Beyond-Use Dating (cont.) Storage and Beyond-Use Dating (cont.)
• Use stABility dating only if shorter than stERility beyond- • Single dose or single use vials shall be discarded within:
use dating recommendations – 24 hours if stored at room temperature
– Low-risk level product “ABC” in refrigerator would receive a – 72 hours if stored in the refridgerator
beyond-use date of <7 days based on stERility • Multi-dose vials shall be discarded within 28 days
– If the stABility of product “ABC” is 24 hours, then product should
• Beyond-use dating can be extended if direct sterility
be labeled with beyond-use date of <24 hours
testing is performed
• Beyond-use dating can be extended if direct sterility
testing is performed
Summary Exit
We hope this Computer Based Learning course has been both informative and helpful.
• USP<797> is an important patient safety initiative Feel free to review the screens of this course until you are confident about your knowledge of
the material presented.
• Adoption of the standards will require personal
motivation and teamwork
Click the Take Test button on the left side of the screen when you are ready to complete the
• For more information on USP<797> visit requirements for this course.
http://www.ashp.org/SterileCpd/
Choose the My Records button to view your transcript.
• USP<797 is under continual revisions. Please obtain
a current copy of the chapter for the most current Select Exit to close the Student Interface.
information.
8
Microbial Control
• Ignatz Semmelweis (1816-1865)
CONTROL OF • In 1847, Dr. Ignaz Semmelweis's close

friend, Jakob Kolletschka, cut his
MICROBIAL GROWTH finger while he's doing an autopsy
• Kolletschka soon died of symptoms

like those of puerperal fever leading
Ignaz Semmelweis to pioneer
antiseptic policy
– Chlorinated lime
Joseph Lister Lister’s Carbolic

acid
• Joseph Lister (1827-1912) sprayer
– Joseph Lister had been convinced of the
importance of scrupulous cleanliness and the
usefulness of deodorants in the operating
room; and when, through Pasteur's
researches, he realized that the formation of
pus was due to bacteria, he proceeded to
develop his antiseptic surgical method.
– Carbolic acid (phenol)

TERMS RELATED TO
DESTRUCTION OF
ORGANISMS
Control of Microbial Control of Microbial

Growth Growth
• Sterilization – killing of all microbial life • Degerming– removal of most microbes by
– (commercial sterilization – only hot enough to swabbing with alcohol
kill Clostridium botulinium canned food)
• Sanitization – removal of most microbes
• Disinfection – destruction of vegetative (non-
spore forming) pathogens mostly chemically –
from surfaces (dishes, glasses, etc.)
disinfectant • Biocide – kills microbes
• Antisepsis – treatment of living tissue to kill • Bacteriostatic – inhibits microbial growth
pathogens - antiseptic
STERILIZATION (cont.)
1. Physical Methods
i. Heat (Moist heat, 121O C for 15 min & Dry TERMS RELATED TO
heat, 170O C for 120 min)
ii. Filtration SUPRESSION OF
2. Chemical Methods ORGANISMS
3. Radiation
4. Gas Sterilization
ANTISEPSIS
DISINFECTION
(anti-putrefaction)
• The process of destroying
microorganisms • Disinfection of skin or
• Usually liquid chemicals tissues
–Some organisms may –Usually wounds
survive disinfection (Legionella
pneumophilia)
–Surgery
BACTERIOSTASIS ASEPSIS (A = without)

• Multiplication of • Absence of
organisms is inhibited microorganisms from an
• May continue when the object or area
bacteriostatic factor is
removed
Sanitization
CONDITIONS
• The reduction of organisms INFLUENCING
on inanimate objects MICROBIAL CONTROL
–Eating utensils
–Dairy equipment, etc.
• Temperature MODE OF ACTION
– low, inhibits growth
• Alteration of lipids and proteins of
– high, promotes disinfectant activity cytoplasmic membrane
• Physiological state – Quaternary ammonium compounds
– Actively growing organisms more

susceptible to disinfection • Alteration of proteins and nucleic acids
– Enzymes
• Environment – Reproduction
– Organic matter protects against chemical – Synthesis of proteins
or physical inactivation
Bacterial Death Rate 1,000,000

1 x 104 E. coli
Number of bacteria
1 x 103
1 x 102
1,000
1 x 101
E. coli
Time
PHYSICAL METHODS Physical Methods

OF DISINFECTION • Radiation
– Ionizing Radiation (<1 nm, high energy) gamma
-Heat -Desiccation rays, X-rays, high energy electron beam
• Creation of hydroxyl radicals from water, DNA
-Filtration -Osmotic pressure damage
• Certain foods, pharmaceuticals, surgical
-Freezing -Radiation supplies, plastic syringes, etc.
– Non-Ionizing Radiation (>1nm, lower energy)
UV light, DNA damage (260 nm Thymine
dimers)
– (microwaves) –inadequate
Physical Methods
• Desiccation
HEAT
– Very variable effect on microbes, many can
survive extended times (months), some endospore • Moist heat
for centuries, others die in minutes
• Dry heat
– Great significance for hospital & nursing home
microbiology, bedding, dust, clothing • Pasteurization
contaminated with mucus, urine, feces or pus
• Osmotic Pressure
– High salt or sugar content (hypertonic conditions)
MOIST HEAT Autoclave

• Boiling (100o
C)
–Kills nonspore-forming
bacteria, viruses, mold and
fungi
• Pressurized (121o C)
–All organisms (but prions)
Physical Methods
DRY HEAT • Heat – destruction of proteins
– Autoclave – 121oC 1 atm pressure for 15 min will
• Flaming kill all organisms ad their endospores
– Pasteurization – milk, yogurt, beer, etc. brief
• Hot air sterilization, 170o C, heating kills most spoiling microbes without
altering taste (milk 72oC 30 s)
2 hours – UHT – ultra high temperature treatment (coffee
creamer, etc. need no refrigeration (140 oC ca. 2 s)
• Dry Heat – direct heat by flame or hot air (170oC)
for 2 h
Sterilizing Physical Methods
Oven • Sterile Filtration
This Class 100 Sterilization – For heat sensitive solutions/liquids
Oven is
used for the depyrogenation – Cellulose esters or plastic polymers (pore
of glass, size from 0.45 – 0.01 µm)
teflon, and other heat
resistant – High Efficiency Particulate Air Filters
materials.
(HEPA)filters (pore size 0.3µm)
• Cooling/Freezing
– Refrigeration –(0-7oC) only bacteriostatic
– Slow freezing kills many pathogens and
parasites
LIQUID FILTRATION GAS

• Culture media FILTRATION
• Laboratory reagents
• Pharmaceutical products • High-Efficiency Particulate
Air (HEPA) filters
–Laminar flow hoods
–Hospital rooms
Used in cell culture

Laminar flow hood
Semiconductor industry
Chemical Methods -Disinfectants
to produce wafers for
cell phones • Heavy Metals
– Ag, Hg, Cu
– Reaction of –SH groups on proteins with minute
amounts of metal ions
– Burn victims, dressings with AgNO3 and a
sulfonamide (antibiotic)
– (Hg in paints prevention of mildew)
– (CuSO4) potent against algae in swimming pools,
fish tanks, etc.
Heavy Metals Chemical Food Preservatives
• Silver (Ag) is an antimicrobial agent • SO2 (wine)
• Ag impregnated dressings for burned victims • Na-benzoate, Ca-propionate, sorbic acid (cheese,
soft drinks, breads)
• Ag is also incorporated into indwelling catheters
• NaNO3 or NaNO2 (ham, bacon, hot dogs)
•CuSO4 used to destroy green algae in swimming – Very effective against Clostridium botulinum
pools and fish tanks
• Nabenzoate in coke/cheese prevents growth of
•Cu + Zn treated shingles are available to create molds
anti-fungal roofs
• Foods w/ low pH tend to be susceptible to molds
•ZnCl2 is a common ingredient in mouthwashes
Nitrates/Nitrites Nitrates in food

•NaNO3/NO2 are added to meat products (ham, bacon, Nitrite in meat greatly delays the development of
hot dogs etc.) botulinal toxin (botulism)
•Salts prevents growth of some types of bacteria that Sugar is added to cured meats as well to reduce the
are responsible for meat spoilage
harshness of salts
•Certain salts produced a pink color
and special flavor (Sodium nitrate) Sodium nitrite (NaNO2), rather than sodium nitrate
(NaNO3), is commonly used for curing
•Nitrate changes to nitrite by
bacteria during processing & storage NO2 is converted to Nitric oxide
Nitric oxide combines with myoglobin (responsible
for the natural red color of uncured meat)
Disk Diffusion Method Disinfectants

Kirby-Bauer Method • Earliest recorded example is use of copper or silver vessels,
instead of pottery ones, to store drinking water to prevent it
becoming foul (450 BC)
• Both copper and silver have significant antimicrobial activity
• Disk of filter paper soaked in
• Other ancient disinfectants, used mainly for topical treatment of
disinfectant or antibiotic wounds were wine, vinegar and honey
• Place on agar previously • Mercuric chloride was introduced as a wound dressing in the
inoculated with test organism Middle Ages by the Arabs
• Incubate at proper temp. • Others include copper sulphate (1767), bleaching powder (1798),
creosote (literally, 'flesh-saviour' from the Greek; 1836), iodine
• Measure inhibition zone (1839), chlorine water (1843) and phenol (1860)
Types of disinfectants Chemical Methods -Disinfectants
• Phenol and phenolics • Alcohols
• Chlorhexidine – 70% ethanol, or isopropanol (“rubbing alcohol)
• Halogens – Mode of action: Protein denaturation &
• Alcohols coagulation therefore membrane disruption
• Heavy metals
– Advantage of evaporation after disinfection
• Surfactants
• Quaternary ammonium compounds (quats) – Poor effect on wounds! Superficial coagulation
• Organic acids of proteins  secludes deeper layers of
• Aldehydes microbes
Effect of Ethanol
• Alcohols kill vegetative forms of bacteria (including How does it work?
TB) and fungi, but have no action on spores or
viruses
Alcohols precipitate proteins and solubilize
• Their effect depend on concentration and type of lipids present in cell membranes
alcohol
It has a rapid action
• The following three solutions have similar effect:
Ethanol 70% , isopropanol 60% and n-propanol 40% Contact time should preferably be 5-30 minutes
70-80% alcohol inactivates HIV and Hepatitis B in 2- (even less exposure kills)
10 minutes.
Phenols Chemical Methods -Disinfectants

• Lister (1867) used phenol (carbolic acid) to kill
microorganisms, many chemicals have been tested • Efficacy evaluated via standard test methods
(standard organisms)
• Derived from coal tar, were first used as wound • Phenol & Phenolics
dressings, but today used as general disinfectants – Mode of action: Damage lipid cell membranes
• e.g.'Lysol' (cresol and soap solution) & 'Stericol' – Quite effective in the presence of organic
(xylenol-rich cresylic acid and soap solution), materials (pus, saliva, feces)
active against viruses and bacteria but less active – E.g. Lysol® (O-phenylphenol)
against bacterial spores. – Very good surface disinfectants
• Phenols are low-intermediate disinfectants
• No longer used as a disinfectant because of its
toxicity to tissues
QUATS and PHENOLICS
PHENOLICS
• cleaning non-critical environmental surfaces
• Products used for this purpose generally contain
• Phenol derivatives
– Less irritant, and more effective
low-level disinfectants such as quaternary
ammonium compounds (quats) or phenolics • Mode of action
– Damages plasma membrane
• effective against easier to kill vegetative bacteria – Enzyme inactivation
• minimum of 10 minutes of contact time – Protein denaturation
• In most situations neither condition is met to make

the disinfection successful • Hexachlorophene
– Against Gram positives
• Such chemicals may also have the potential to – Currently used in hospitals (scrubbing)
make bacteria more resistant to antibiotics – Neurotoxic
SOAPS Chemical Methods -Disinfectants

• mechanical removal of microbes through scrubbing
• Surfactants (surface active agents)
• soap breaks oily film on skin (emulsification); little value – “Soaps” old fashioned Na/K salts of fatty acids (Na/K
as an antiseptic + animal fat= glycerol)
• detergents-more effective against gram positive than gram – Detergents modern surface active agents
negative – Emulsification of skin oils & dead cells  cleansing
of skin and other surfaces
-- nonionic detergents have no germicidal activity – Some destruction of cell membranes (at high
-- anionic (acid) sanitizes food and dairy industry concentrations, cell lysis)
– Active against bacteria and enveloped viruses (such
-- cationic (positive)-antiseptic for skin, instruments, as feline herpesvirus) but not non-enveloped viruses
utensils (such as feline calicivirus)
Types Of Disinfectants (cont.) Chemical Methods -Disinfectants

• Gaseous chemosterilizers • Oxidizing agents
– O3 (Ozone) drinking water
– Ethylene oxide: effective against all – H2O2
forms of life incl. endospores) • effective on inanimate objects, but ineffective on open
• Oxidizing agents (Chlorine) wounds due to destruction by Catalase
– (2 H2O2  2 H2O + O2)
• Contact lens sterilization
– Benzoyl peroxide
• Irrigation of wounds infected by anaerobic bacteria,
acne treatment (anaerobic bacteria in hair follicle)
• Oxidizing agents Organic Acids
– Peroxyacetic acid
• Most effective sporicide, sterilization of food processing Organic Acids - various organic acids and their salts are common
and medical equipment antimicrobials in foods
• Leaves no residue (disintegrates into O2 & AA) - preservatives to control mold growth
• Bisphenols
- sorbic acid (Ca, Na, K) used in cheeses, baked goods, soft drinks,
– E.g. hexachlorophene
fruit juices, jams, jellies
– Mechanism ?
– Used in OR, hospitals, nurseries - benzoates (sodium benzoate, methy-p-hydroxybenzoate
– Very effective against gram+ staphylococci causing skin [methylyparaben]) fruit juices, jam, jellies, soft drinks, salad
infections in newborns dressings, margarine, and many pharmaceutical products
• Biguanides - Boric acid used in eye washes
– E.g. chlorhexidine
- calcium propionate-prevents mold growth in bread
– Damage lipid cell membranes
– Surgical hand scrub, preoperative skin disinfection (combined
with soaps & alcohol)

• Halogens • Halogens
– Iodine I2 – Chlorine Cl2 (gas)
– Halogenation of microbial proteins – Formation of ClO- in water (“bleach”)
– Iodine tincture (solution in diluted alcohols) – Strong oxidizing activity destroys microbial enzymes
– Iodophores – organic molecules slowly releasing I2,
less staining than straight I2 preparations (e.g. – Drinking water, swimming pools, sewage treatment
Betadine®, Isodine®) • Since 1908 main water disinfectant in the USA
– Extremely effective, skin disinfection, wound
treatment (I2 tablets water treatment)
Chemical Methods -Disinfectants GASEOUS

CHEMOSTERILIZERS
• Gas sterilizers
– E.g.: ethylene oxide • Ethylene oxide
– Denatures proteins via alkylation of –SH, -
COOH, -OH groups • Denatures proteins
– Highly penetrating
– Used in closed chambers, industrial sterilization • Sterilizes (4 to 18 hours)
of plastic-wrapped syringes, needles, tubing etc.
– Hospitals (large chambers to treat entire • Disposable plasticware
mattresses)
OXIDIZING AGENTS
• Ozone
–Water disinfection
• Hydrogen peroxide
–Inanimate surfaces
Ignaz Semmelweis,
This slide set “Hand Hygiene in Healthcare Settings-
Supplemental” provides:
1815-1865
1) Slides that may be used in conjunction with the “Hand  1840’s: General Hospital 16
Hygiene in Healthcare Settings-Core” slide set. of Vienna 14
Maternal mortality, 1842

2) Question and answer slides to be used in an interactive 12
 Divided into two clinics,
presentation. 10
alternating admissions 8
3) Slides containing information on topics related to hand
hygiene (e.g., antimicrobial resistance). every 24 hours: 6
4) Slides that you can customize by inserting data specific – First Clinic: Doctors 4
2
to your hospital. and medical students
0
The Hand Hygiene in Healthcare Settings-Supplemental
– Second Clinic: First Clinic Second
Clinic
slide set was developed in conjunction with the Chicago Midwives
Antimicrobial Resistance Project [www.carp-net.org].
The Intervention: Hand Hygiene: Not a New Concept

Hand scrub with chlorinated lime solution Maternal Mortality due to Postpartum Infection
General Hospital, Vienna, Austria, 1841-1850
18 Semmelweis’ Hand
16 Hygiene Intervention
Maternal Mortality (%)
14
12
10
0
1841 1842 1843 1844 1845 1946 1847 1848 1849 1850
MDs Midwives
~ Hand antisepsis reduces the frequency of patient infections ~

Hand hygiene basin at the Lying-In Women’s Hospital in Vienna, 1847.
Adapted from: Hosp Epidemiol Infect Control, 2nd Edition, 1999.
Colonized or Infected: The Iceberg Effect

What is the Difference?
 People who carry bacteria without evidence of Infected
infection (fever, increased white blood cell count)
are colonized
 If an infection develops, it is usually from bacteria
that colonize patients
 Bacteria that colonize patients can be transmitted Colonized
from one patient to another by the hands of
healthcare workers
~ Bacteria can be transmitted even if the
patient is not infected ~
1
Recovery of VRE from Hands
and Environmental Surfaces The Inanimate Environment Can
Facilitate Transmission
 Up to 41% of healthcare worker’s hands X represents VRE culture positive sites
sampled (after patient care and before
hand hygiene) were positive for VRE1
 VRE were recovered from a number of
environmental surfaces in patient rooms
 VRE survived on a countertop for up to 7
days2
~ Contaminated surfaces increase cross-transmission ~
Abstract: The Risk of Hand and Glove Contamination after Contact with a
VRE (+) Patient Environment. Hayden M, ICAAC, 2001, Chicago, IL.
1 Hayden MK, Clin Infect Diseases 2000;31:1058-1065.
2 Noskin G, Infect Control and Hosp Epidemi 1995;16:577-581.
What is the single most important reason What is the single most important reason
for healthcare workers to practice good for healthcare workers to practice good
hand hygiene? hand hygiene?
1. To remove visible soiling from hands 1. To remove visible soiling from hands
2. To prevent transfer of bacteria from the 2. To prevent transfer of bacteria from the
home to the hospital home to the hospital
3. To prevent transfer of bacteria from the 3. To prevent transfer of bacteria from the
hospital to the home hospital to the home
4. To prevent infections that patients 4. To prevent infections that patients
acquire in the hospital acquire in the hospital
How often do you clean your hands How often do you clean your hands
after touching a PATIENT’S INTACT after touching a PATIENT’S INTACT
SKIN (for example, when measuring a SKIN (for example, when measuring a
pulse or blood pressure)? pulse or blood pressure)?
1. Always 1. Always
2. Often 2. Often
3. Sometimes 3. Sometimes
4. Never 4. Never
2
Estimate how often YOU clean
your hands after touching a Now, estimate how often YOUR CO-
patient or a contaminated WORKERS clean their hands after
surface in the hospital? touching a patient or a contaminated
surface in the hospital?
1. 25% 1. 25%
2. 50% 2. 50%
3. 75% 3. 75%
4. 90%
4. 90%
5. 100%
5. 100%
Which method do you use to

clean your hands at work? Which hand hygiene method
is best at killing bacteria?
1. Plain soap and water
2. Antimicrobial soap and water 1. Plain soap and water
3. Alcohol-based handrub 2. Antimicrobial soap and water
3. Alcohol-based handrub
Which of the following hand

Which hand hygiene method hygiene agents is LEAST drying to
is best at killing bacteria? your skin?
1. Plain soap and water 1. Plain soap and water

2. Antimicrobial soap and water 2. Antimicrobial soap and water
3. Alcohol-based handrub 3. Alcohol-based handrub
3
Which of the following hand It is acceptable for healthcare
hygiene agents is LEAST drying to workers to supply their own
your skin? lotions to relieve dryness of hands
in the hospital.
1. Plain soap and water 1. Strongly agree
2. Antimicrobial soap and water 2. Agree
3. Alcohol-based handrub 3. Don’t know
4. Disagree
5. Strongly disagree
It is acceptable for healthcare How much time would an ICU

workers to supply their own nurse save during an 8 hour shift
lotions to relieve dryness of hands by using an alcohol-based handrub
in the hospital. instead of soap and water?
1. Strongly agree
1. 15 minutes
2. Agree
2. 30 minutes
3. Don’t know
3. 1 hour
4. Disagree
4. 2.5 hours
How much time would an ICU Healthcare-associated

nurse save during an 8 hour shift organisms are commonly
by using an alcohol-based handrub resistant to alcohol.
instead of soap and water?
1. Strongly agree
1. 15 minutes 2. Agree
2. 30 minutes 3. Don’t know
3. 1 hour 4. Disagree
4. 2.5 hours 5. Strongly disagree
* Based on 12 opportunities/hour, handwashing time=60 seconds,

alcohol-based handrub time=20 seconds
4
When a healthcare worker touches a
Healthcare-associated patient who is COLONIZED, but not
organisms are commonly infected with resistant organisms (e.g.,
resistant to alcohol. MRSA or VRE) the HCW’s hands are a
source for spreading resistant
1. Strongly agree organisms to other patients.
2. Agree 1. Strongly agree
3. Don’t know 2. Agree
4. Disagree 3. Don’t know
5. Strongly disagree 4. Disagree
When a healthcare worker touches a A co-worker who examines a patient

patient who is COLONIZED, but not with VRE, then borrows my pen
infected with resistant organisms (e.g., without cleaning his/her hands is likely
MRSA or VRE) the HCW’s hands are a
source for spreading resistant to contaminate my pen with VRE.
organisms to other patients.
1. Strongly agree
1. Strongly agree
2. Agree
2. Agree
3. Don’t know
3. Don’t know
4. Disagree
4. Disagree
A co-worker who examines a patient How often do you clean your hands
with VRE, then borrows my pen after touching an ENVIRONMENTAL
without cleaning his/her hands is likely SURFACE near a patient (for
to contaminate my pen with VRE. example, a countertop or bedrail)?
1. Strongly agree 1. Always
2. Agree 2. Often
3. Don’t know 3. Sometimes
4. Disagree 4. Never
5
How often do you clean your hands Use of artificial nails by
after touching an ENVIRONMENTAL healthcare workers poses no
SURFACE near a patient (for risk to patients.
example, a countertop or bedrail)?
1. Strongly agree
1. Always
2. Agree
2. Often
3. Don’t know
3. Sometimes
4. Disagree
4. Never
Can a Fashion Statement

Use of artificial nails by Harm the Patient?
healthcare workers poses no 40
35 Natural (n=31)
risk to patients. Artificial (n=27)

% Recovery of gram
Polished (n=31)
30
negative bacteria
1. Strongly agree 20
ARTIFICIAL
2. Agree 10
10
5
3. Don’t know POLISHED
NATURAL
0
4. Disagree p<0.05
5. Strongly disagree Avoid wearing artificial nails, keep natural nails

<1/4 inch if caring for high risk patients (ICU, OR)
Edel et. al, Nursing Research 1998: 47;54-59
Glove use for all patient care Glove use for all patient care
contacts is a useful strategy for contacts is a useful strategy for
reducing risk of transmission of reducing risk of transmission of
organisms. organisms.
1. Strongly agree 1. Strongly agree
2. Agree 2. Agree
3. Don’t know 3. Don’t know
4. Disagree 4. Disagree
5. Strongly disagree 5. Strongly disagree
6
Insert graph showing annual,
At your hospital, what percentage monthly, or quarterly trend in
of [insert organism name] isolates antimicrobial (e.g. MRSA)
are resistant to [insert drug prevalence, or number of isolates
name]? at Hospital X below
Prevalence of MRSA at Hospital X
1. <5%
100
2. 15% 80
MRSA
Prevalence (%)
3. 20% 60
40 40
35
40 30
4. 30% 7
20
20 5 10
5. >50% 0
Jun- Jul- Aug- Sep- Oct- Nov- Dec- Jan-
01 01 01 01 01 01 01 02
Hand Hygiene Options at

Insert facility-specific data on Hospital X
HCW hand hygiene adherence
below Insert Wet hands, apply Insert
soap and rub for
HCW Hand Hygiene Adherence at Hospital X photo of >15 seconds.
Rinse, dry & turn
photo of
100 liquid off faucet with
paper towel.
alcohol
MD
80 RN soap handrub
Adherence (%)
SAMPLE Other
from from
60
40 Apply to palm; rub
35
40 30 35
20
25 Hospital hands until dry Hospital
20 X X
0
4th Qtr 01 1st Qtr 02 ~ Use soap and water for visibly soiled hands ~
~ Do not wash off alcohol handrub ~
What is the Story on

Moisturizers and Lotions?
ONLY USE facility-approved and
supplied lotions
Because: Insert
 Some lotions may make medicated photo of
soaps less effective
lotion
 Some lotions cause breakdown of latex
gloves
from
 Lotions can become contaminated with
Hospital
bacteria if dispensers are refilled X
~ Do not refill lotion bottles ~
7
25-Oct-15
Aseptic Processing - Overview

• Certain pharmaceutical products must be
sterile
Aseptic Processing, – injections, ophthalmic preparations, irrigations
Bioburden, Env. Monitoring solutions, haemodialysis solutions
• Two categories of sterile products

– those that can be sterilized in final container
(terminally sterilized)
– those that cannot be terminally sterilized and must
be aseptically prepared
1 Manufacture of sterile medicines – Advanced workshop for SFDA GMP 2

inspectors - Nanjing, November 2009
Aseptic Processing - Overview Aseptic Processing - Overview

Objective
Aseptic processing • To review specific issues relating to the manufacture
of aseptically prepared products:
• Objective is to maintain the sterility of a product, – Manufacturing environment
assembled from sterile components • Clean areas
• Personnel
– Preparation and filtration of solutions
• Operating conditions so as to prevent microbial
contamination – Pre-filtration bioburden
– Filter integrity/validation
– Equipment/container preparation and sterilization
– Filling Process
– Validation of aseptic processes
3 4
What is a particle?
A solid or liquid object which, for the purposes of
classification of air cleanliness, falls within a
cumulative distribution that is based upon a threshold
(lower limit) size in the range from 0.1 μm to 5.0 μm
ISO 14644-1
Particle sizes
Sand: 80 – 2000 µm
Human hair: 100 µm
Meal: 5 – 10 µm
Bacteria: 0.2 – 2 µm
Cigarette smoke: 0.01 – 1 µm
Virus: 0.003 – 0.05 µm
1
25-Oct-15
Why the concern about particles?
For security & quality reasons during production:
Pharmaceutical industry: particles in vaccines cause occlusion of blood
vessels
Medical devices: poor adhesion of coating in stents creates emboli
Medical films: contamination prevents complete image recovery
Electronic industry: particles on circuit boards create false contacts
A PARTICLE WILL BECOME A CONTAMINANT!
THE CONCENTRATION OF PARTICLES INDICATE THE LEVEL OF
CONTAMINATION IN A CRITICAL AREA…..
Sources of human particles
Standards for particle counting
In a cleanroom the human being is the biggest contaminator
Dead skin cells are discarded continuously
Exhalation carries a mixture of gases • ISO 14644
General standards for all cleanrooms
Particle limits for sizes/classes
• EU cGMP
Industry specific (pharmaceutical)
More stringent levels
At rest v. operational
ISO 14644‐1 air borne particle
Manufacturing Environment
cleanliness classes for clean rooms
Classification of Clean Areas
– Comparison of classifications
WHO GMP US 209E US Customary ISO/TC (209) EEC GMP
ISO 14644
Grade A M 3.5 Class 100 ISO 5 Grade A
Grade B M 3.5 Class 100 ISO 5 Grade B
Grade C M 5.5 Class 10 000 ISO 7 Grade C
Grade D M 6.5 Class 100 000 ISO 8 Grade D
12
2
25-Oct-15
Manufacturing Environment Manufacturing Environment

Classification of Clean Areas
Four grades of clean areas:
– Classified in terms of airborne particles • Grade D (equivalent to Class 100,000, ISO 8):
Grade At rest In operation – Clean area for carrying out less critical stages in manufacture of
maximum permitted number of particles/m3 aseptically prepared products eg. handling of components after washing.
0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm >5µ
A 3 500 0 3 500 0 • Grade C (equivalent to Class 10,000, ISO 7):
B 3 500 0 350 000 2 000 – Clean area for carrying out less critical stages in manufacture of
C 350 000 2 000 3 500 000 20 000 aseptically prepared products eg. preparation of solutions to be filtered.
D 3 500 000 20 000 not defined not defined
• Grade B (equivalent to Class 100, ISO 5):
“At rest” - production equipment installed and operating – Background environment for Grade A zone, eg. cleanroom in which
laminar flow workstation is housed.
“In operation” - Installed equipment functioning in defined
operating mode and specified number of personnel present
13 14

• Grade A (equivalent to Class 100 (US Federal • Limits for viable particles (microbiological contamination)
Standard 209E), ISO 5 (ISO 14644-1):
– Local zone for high risk operations eg. product filling,
stopper bowls, open vials, handling sterile materials, Grade Air sample Settle plates (90mm Contact plates Glove print
aseptic connections, transfer of partially stoppered (CFU/m3) diameter) (55mm (5 fingers)
(CFU/4hours) diameter) (CFU/glove)
containers to be lyophilized. (CFU/plate)
– Conditions usually provided by laminar air flow A <3 <3 <3 <3
workstation. B 10 5 5 5
C 100 50 25 -
• Each grade of cleanroom has specifications for D 200 100 50 -
viable and non-viable particles Table 3
– Non-viable particles are defined by the air classification – These are average values
(See Table 2) – Individual settle plates may be exposed for less than 4 hours
• Values are for guidance only - not intended to represent specifications


Environmental Monitoring Environmental Monitoring - Physical
• Physical • Particulate matter
– Particulate matter – Particles significant because they can contaminate and also
– Differential pressures carry organisms
– Critical environment should be measured not more than
– Air changes, airflow patterns 30cm from worksite, within airflow and during filling/closing
– Clean up time/recovery operations
– Temperature and relative humidity – Preferably a remote probe that monitors continuously
– Airflow velocity – Difficulties when process itself generates particles (e.g.
powder filling)
– Appropriate alert and action limits should be set and
corrective actions defined if limits exceeded
17 18
3
25-Oct-15

Environmental Monitoring - Physical Environmental Monitoring - Physical
• Differential pressures • Air Changes/Airflow patterns
– Positive pressure differential of 10-15 Pascals should be – Air flow over critical areas should be uni-directional (laminar
maintained between adjacent rooms of different flow) at a velocity sufficient to sweep particles away from
classification (with door closed) filling/closing area
– Most critical area should have the highest pressure
– Pressures should be continuously monitored and frequently
recorded. • Clean up time/recovery
– Particulate levels for the Grade A “at rest” state should be
– Alarms should sound if pressures deviate
achieved after a short “clean-up” period of 20 minutes after
– Any deviations should be investigated and effect on completion of operations (guidance value)
environmental quality determined
– Particle counts for Grade A “in operation” state should be

maintained whenever product or open container is exposed
19 20

Environmental Monitoring - Physical Personnel
• Minimum number of personnel in clean areas
• Temperature and Relative Humidity – especially during aseptic processing
– Ambient temperature and humidity should not be • Inspections and controls from outside
uncomfortably high (could cause operators to
generate particles) (18°C) • Training to all including cleaning and maintenance staff
– initial and regular
• Airflow velocity – manufacturing, hygiene, microbiology
– should be formally validated and authorized to enter aseptic area
– Laminar airflow work station air speed of approx
0.45m/s ± 20% at working position (guidance value)
• Special cases
– supervision in case of outside staff
– decontamination procedures (e.g. staff who worked with animal
tissue materials)
21 22

Personnel (2) Personnel (3)
• High standards of hygiene and cleanliness
• Clothing of appropriate quality:
– should not enter clean rooms if ill or with open wounds
– Grade D
• hair, beard, moustache covered
• Periodic health checks • protective clothing and shoes
• No shedding of particles, movement slow and controlled
– Grade C
• No introduction of microbiological hazards • hair, beard, moustache covered
• No outdoor clothing brought into clean areas, should be clad • single or 2-piece suit (covering wrists, high neck),
shoes/overshoes
in factory clothing • no fibres/particles to be shed
• Changing and washing procedure
• No watches, jewellery and cosmetics – Grade A and B
• headgear, beard and moustache covered, masks, gloves
• Eye checks if involved in visual inspection
• not shedding fibres, and retain particles shed by operators

4
25-Oct-15
Manufacturing Environment Aseptic Processing

Personnel (4) • In aseptic processing, each component is individually
sterilized, or several components are combined with
• Outdoor clothing not in change rooms leading to Grade B and C rooms resulting mixture sterilized.
• Change at every working session, or once a day – Most common is preparation of a solution which is filtered through a
sterilizing filter then filled into sterile containers (e.g active and
• Change gloves and masks at every working session excipients dissolved in Water for Injection)
• Frequent disinfection of gloves during operations – May involve aseptic compounding of previously sterilized components
which is filled into sterile containers
• Washing of garments – separate laundry facility
– According to validated procedures (washing and sterilization)
– May involve filling of previously sterilized powder
• sterilized by dry heat/irradiation
• Regular microbiological monitoring of operators
• produced from a sterile filtered solution which is then aseptically crystallized
and precipitated
– requires more handling and manipulation with higher potential for contamination
during processing
25 26
Aseptic Processing Aseptic Processing

• Solutions to be sterile filtered prepared in a Grade C environment • Filters should not shed particles (e.g, don’t use asbestos filters)
• Same filter should not be used for more than one day unless validated
• If not to be filtered, preparation should be prepared in a Grade A • If bulk product is stored in sealed vessels, pressure release outlets
environment with Grade B background (e.g. ointments, creams, should have hydrophobic microbial retentive air filters
suspensions and emulsions)
• Time limits should be established for each phase of processing, e.g.
• Prepared solutions filtered through a sterile 0.22μm (or less) – maximum period between start of compounding and sterilization (filtration)
membrane filter into a previously sterilized container – maximum permitted holding time of bulk if held after filtration prior to filling
– filters remove bacteria and moulds – product exposure on processing line
– do not remove all viruses or mycoplasmas – storage of sterilized containers/components
– total time for product filtration to prevent organisms from penetrating filter
• Filtration should be carried out under positive pressure
• Filling of solution may be followed by lyophilization (freeze drying)
• Complementing filtration process with some form of heat treatment – stoppers partially seated, product transferred to lyophilizer (Grade A/B
conditions)
• Double filter or second filter at point of fill advisable – Release of air/nitrogen into lyophilizer chamber at completion of process
should be through sterilizing filter
27 28

Prefiltation Bioburden (2)
Prefiltration Bioburden (natural microbial load)
• No defined “maximum” limit but the limit should not exceed the
• Limits should be stated and testing carried out on each batch
validated retention capability of the filter
• Frequency may be reduced after satisfactory history is established

– and biobuden testing performed on components
• Bioburden controls should also be included in “in-process”
controls
– particularly when product supports microbial growth and/or
• Should include action and alert limits and action taken if limits are
manufacturing process involves use of culture media
exceeded
• Excessive bioburden can have adverse effect on the quality of the

• Limits should reasonably reflect bioburden routinely achieved
product and cause excessive levels of endotoxins/pyrogens
29 30
5
25-Oct-15

Filter integrity Filter Validaton
• Filters of 0.22μm or less should be used for filtration • Filter must be validated to demonstrate ability to
of liquids and gasses (if applicable) remove bacteria
– filters for gasses that may be used for purging or – most common method is to show that filter can retain a
overlaying of filled containers or to release vacuum in microbiological challenge of 107 CFU of Brevundimonas
lyphilization chamber diminuta per cm2 of the filter surface
• filter intergrity shoud be verified before filtration and – a bioburden isolate may be more appropriate for filter
confirmed after filtration retention studies than Brevundimonas diminuta
– bubble point – Challenge concentration is intended to provide a margin
– pressure hold of safety well beyond what would be expected in
– forward flow production
• methods are defined by filter manufacturers and limits – preferably the microbial challenge is added to the fully
formulated product which is then passed through the
determined during filter validation
filter
31 Manufacture of sterile medicines – Advanced workshop for SFDA GMP 32 Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009 inspectors - Nanjing, November 2009

Equipment/container preparation and sterilization
Filter validation (2)
– if the product is bactericidal, product should be passed • All equipment (including lyophilizers) and product containers/ closures
through the filter first followed by modified product should be sterilized using validated cycles
containing the microbial challenge (after removing any – same requirements apply for equipment sterilization that apply to terminally
bactericidal activity remaining on the filter) sterilized product
– filter validation should be carried out under worst case – equipment wrapped and loaded to facilitate air removal
conditions e.g. maximum allowed filtration time and – particular attention to filters, housings and tubing
maximum pressure
– integrity testing specification for routine filtration • Heat tunnels used for sterilization/depyrogenation of glass vials/bottles
should correlate with that identified during filter
– usually high temperature for short period of time
validation
– tunnel supplied with HEPA filtered air


Equipment/container preparation and sterilization (2)
Process Validation
• Not possible to define a sterility assurance level for aseptic processing
• equipment should be designed to be easily assembled and • Process is validated by simulating the manufacturing process using
disassembled, cleaned, sanitized and/or sterilized microbiological growth medium (media fill)
– equipment should be appropriately cleaned - O-rings and gaskets should be – Process simulation includes formulation (compounding), filtration and filling with suitable
removed to prevent build up of dirt or residues media using the same processes involved in manufacture of the product
• rinse water should be WFI grade – modifications must be made for different dosage formats e.g. lyophilized products,
ointments, sterile bulks, eye drops, biological products
• equipment should be left dry unless sterilized immediately after
cleaning (to prevent build up of pyrogens) • Media fill program should include worst case activities
– Factors associated with longest permitted run (e.g. operator fatigue)
• washing of glass containers and rubber stoppers should be validated
– Representative number, type & complexity of normal interventions, non-routine
for endotoxin removal
interventions & events (e.g. maintenance, stoppages, etc)
• should be defined storage period between sterilization and use (period – Lyophilisation
should be justified)
– Aseptic equipment assembly
– No of personnel and their activities, shift changes, breaks, gown changes
– Aseptic equipment connections/disconnections
– Aseptic sample collections
– Line speed and configuration
35 36
6
25-Oct-15
Aseptic Processing (Process Validation) Aseptic Processing

Duration
• Environmental conditions
– Depends on type of operation
– Representative of actual production conditions (no. of personnel, activity
– For conventional operations should include the total filling time levels etc) - no special precautions (not including adjustment of HVAC)
Size • Media
– 5000 - 10000 generally acceptable or batch size if <5000 – Anaerobic media should be considered under certain circumstances
– For manually intensive processes larger numbers should be filled – Should be tested for growth promoting properties (including factory
isolates)
– Lower numbers can be filled for isolators
• Incubation, Examination
Frequency and Number
– In the range 20-35ºC.
– Three initial, consecutive per shift
– If two temperatures are used, lower temperature first
– Subsequently semi-annual per shift and process – Inspection by qualified personnel.
– All personnel should participate at least annually – All integral units should be incubated. Justify any units not incubated.
– Changes should be assessed and revalidation carried out as required – Units removed (and not incubated) should be consistent with routine
Line Speed practices
– Speed depends on type of process – Batch reconciliation
37 38

– Media fills should be observed by QC and contaminated units
• Interpretation of Results reconcilable with time and activity being simulated (Video may help)
– When filling fewer than 5000 units: – Ideally - no contamination. Any contamination be investigated.
• no contaminated units should be detected
– Any organisms isolated should be identified to species level (genotypic
• One (1) contaminated unit is considered cause for revalidation,
identification)
following an investigation
– Invalidation of a media fill run should be rare
– When filling from 5000-10000 units
• One (1) contaminated unit should result in an investigation, Batch Record Review
including consideration of a repeat media fill – Process and environmental control activities should be included in
• Two (2) contaminated units are considered cause for batch records and reviewed as part of batch release
revalidation, following investigation  In-process and laboratory control results
 Environmental and personnel monitoring data
 Output from support systems(HEPA/HVAC, WFI, steam generator)
– When filling more than 10000 units
 Equipment function (batch alarm reports, filter integrity)
• One (1) contaminated unit should result in an investigation  Interventions, Deviations, Stoppages - duration and associated time
• Two (2) contaminated units are considered cause for  Written instructions regarding need for line clearances
revalidation, following investigation  Disruptions to power supply
39 40

• Blow-Fill-Seal (BFS) • Issues relating to Aseptic Bulk Processing
– Located in a Grade D environment
– Critial zone should meet Grade A (microbiological) • Applies to products which can not be filtered at point of fill and
requirements (particle count requirements may be difficult to require aseptic processing throughout entire manufacturing process.
meet in operation)
– Operators meet Grade C garment requirements • Entire aseptic process should be subject to process simulation
studies under worst case conditions (maximum duration of "open"
– Validation of extrusion process should demonstrate destruction
operations, maximum no of operators)
of endotoxin and spore challenges in the polymeric material
– Final inspection should be capable of detecting leakers
• Process simulations should incorporate storage & transport of bulk.
• Multiple uses of the same bulk with storage in between should also
be included in process simulations
• Assurance of bulk vessel integrity for specified holding times.
41 42
7
25-Oct-15
Aseptic Processing Useful Publications

• Bulk Processing (2) • PIC/S Recommendation on the Validation of Aseptic
Process simulation for formulation stage should be Processes
performed at least twice per year. • FDA Guidance for Industry- Sterile Drug Products Produced
by Aseptic Processing - Current Good Manufacturing
Process
Cellular therapies, cell derived products etc • ISO 13408 Aseptic Processing of Health Care Products
• products released before results of sterility tests known – Part 1: General Requirements
(also TPNs, radioactive preps, cytotoxics) – Part 2: Filtration
• should be manufactured in a closed system – Part 3: Lyophilization
• Additional testing – Part 4: Clean-In-Place Technologies
– sterility testing of intermediates – Part 5: Sterilization-In-Place
– microscopic examination (e.g. gram stain) – Part 6: Isolator Systems
– endotoxin testing
43 44 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

8
2015‐10‐25
HYGIENE CONTROL IN THE BREWING INDUSTRY What is a particle?

WITH REFERENCE TO AIR PARTICLE CONTROL
Environmental monitoring in A solid or liquid object which, for the purposes of classification of air cleanliness, falls within
a cumulative distribution that is based upon a threshold (lower limit) size in the range from
the Food and Beverage Industry 0.1 μm to 5.0 μm
ISO 14644-1
South African Breweries
January 2012
Stephen Marrs
IEPSA
Why the concern about particles?
For security & quality reasons during production:
Pharmaceutical industry: particles in vaccines cause

occlusion of blood vessels
Medical devices: poor adhesion of coating in stents

creates emboli
Medical films: contamination prevents complete image

recovery
Electronic industry: particles on circuit boards create false

contacts
A PARTICLE WILL BECOME A CONTAMINANT!

THE CONCENTRATION OF PARTICLES INDICATE THE
LEVEL OF CONTAMINATION IN A CRITICAL AREA…..
1
2015‐10‐25
Particle sizes Sources of particles?
Diameter in micrometer (µm) In a cleanroom the human being

is the biggest contaminator:
Sand: 80 – 2000 µm Dead skin cells are discarded

Human hair: 100 µm continuously
Meal: 5 – 10 µm
Bacteria: 0,2 – 2 µm Exhalation carries a mixture of
Cigarette smoke: 0.01 – 1 µm gases
Virus: 0.003 – 0,05 µm
In critical areas operators need
to ware cleanroom clothing
Emission of particles Sources of human particles
Emission of particles >0,5 µm per minute

Normal Clothes Cleanroom clothes
No movement 100.000
Light movement 500.000
Going to sit 1.000.000
Walking 5.000.000 100.000
Running 7.500.000 1.000.000
Running fast 10.000.000
ISO 14644-1 air borne particle

Standards for particle counting cleanliness classes for clean rooms
• ISO 14644
General standards for all cleanrooms
Particle limits for sizes/classes
• EU cGMP
Industry specific (pharmaceutical)
More stringent levels
At rest v. operational
2
2015‐10‐25
Good practices for particle counting
The cleanroom layout:
the layout has to match the operational situation

the equipment will have a fixed position
overview of the different critical areas
the areas will get a proper identification: (alphanumeric) name
also the sample locations will be identified on the CR layout
both the areas and the sample location names can be set
on the counter
operators need to be trained to use the designated sample
locations to make representative samples
How many sample locations in a clean zone? Position of the Isokinetic probe
ISO 14644 – 1 has a simple formula:
√A = x, Designed to sample air with a nominal velocity of 0,5 meters per sec
A = surface in m² Pointing into the airflow at the sample location
x = minimum number of sample locations acc. to ISO Vertically upward if direction of the airflow is unknown
14644-1 Environmental measurements: at a height of 1,5 meter and upward
Sample locations are spread equidistant, In a process measurement: in the work field of the operator to monitor
at the highest risk for contamination the risk for contamination
A simple example for a “cleanzone” of 16 m²: Check the probe on deformations and cleanliness:
-4 locations ISO 5 @ ≥ 0.5 micron -a stainless steel probe is more robust
-Class limit is 3520 particles/m³ -autoclavable
After revision 14644: 8 sample locations!!
How does a particle counter work?
The zero count filter

There are three basic elements in all particle counting systems:
Will hold particles bigger than 0,2 µm 1) The sensor
Easy tool to check the proper working of the counter: The sensor is the device that detects particles using
-less than 1 count in 5 samples of a minute a light scattering method of detection.
-repeat the test on a regular basis, once per week 2) The sample delivery system
Second function of the filter: to purge the counter The sample is delivered to the sensor by the use of
-if you go from a ISO 9 Class to a ISO 5 a vacuum system.
do a purge action to start with a clean optical system 3) The counting electronics
The particle counts are processed and displayed
on a screen.
3
2015‐10‐25
Optical System Cross-Section
Biotest solutions
Handheld Particle Counter
• Easy and safe operation

• Individual Channels 0,3µm - 10µm
•
•
10.000 records stored in memory
Windows CE & USB connection
Thank you
• Ideally suited for monitoring Processes
and filters
4
25-Oct-15
Microbiological Testing
Objectives
• To review microbiological environmental and
quality control testing
Microbiological Control Tests
– Microbiological Environmental Monitoring
– Container integrity testing
– Pre-sterilization bioburden testing
– Media fill medium growth promotion testing
– Sterility Testing
– Other microbiological laboratory issues

Environmental Monitoring Environmental Monitoring

Limits for Viable Particles Methods
• Surface monitoring
Grade Air sample Settle plates (90mm Contact plates Glove print
– Product contact surfaces, floors, walls, and equipment should be
(CFU/m3) diameter) (55mm (5 fingers)
(CFU/4hours) diameter) (CFU/glove) tested on a regular basis
(CFU/plate) – Touch plates - used for flat surfaces
A <3 <3 <3 <3 • sample area of 25cm2
B 10 5 5 5 • medium protrudes above sides
C 100 50 25 - • medium contains neutralisers
D 200 100 50 -
– Surface Swabs - used for irregular surfaces
Table 3 • area approx 25cm2 is swabbed
– These are average values • qualitative or quantitative
– Individual settle plates may be exposed for less than 4 hours
• Values are for guidance only - not intended to represent specifications Surface monitoring should be performed at conclusion of aseptic processing (to
minimise risk of contaminating critical surfaces during production)
• Levels (limits) of detection of microbiological contamination should be
established for alert and action purposes and for monitoring trends of air
• swabs and contact plates can be used
quality in the facility
3 4

Methods Sampling Locations
– Active Air Monitoring
– Should be based on risk of microbiological contamination
• impaction, centrifugal and membrane (or gelatin) samplers
• a certain volume of air is sampled (volume and location should be
meaningful) – Should be clustered around areas where product or
• instruments should be calibrated components are exposed e.g.
• at filling heads on filling lines
• loading of product into lyophilizers
– Passive Air Monitoring
• stopper bowls
• Settle plates exposed for 30-60 minutes (longer may result in agar
drying out) and replaced for duration of filling • where aseptic connections are made
• Media should be capable of growing a range of bacteria and • where there are high levels of operator activity (but without
moulds (e.g. Soybean Casein Digest Agar (SCDA)/Trypticase Soy impacting on production)
Agar (TSA)
• Should consider use of medium specific for moulds if shown to be – Lower grade areas are monitored less frequently and trends
a problem in the environment monitored
• Only give qualitative or semi-quantitative results
• Data generated considered in combination with active air
sampling results
5 6
1
25-Oct-15

Levels and Trends
Personnel
• Limits in Code of GMP are for guidance only
• For each session - gloves
should be monitored (but not
immediately after sanitizing!) • Manufacturers should set alert and action limits
• Periodic sampling for other • Individual results should be considered

locations on gown
• There should be written procedures (SOPs) for data review and action
• Clean room operators should to be taken if limits are exceeded
be regularly validated to
demonstrate that they do not • Trend Reports
contaminate gowns during – Short and long term reports on environmental and personnel monitoring
gowning up (gowning
– Results of EM should be included in Batch Records
qualification)
– Significant changes in microbial flora should be considered
7 8

Disinfectants Water
• Suitablility, efficacy, limitations of disinfectants and procedures
• microbiological quality of water very important
should be assessed
– minimum contact time established • Should be an extensive, comprehensive water testing programme
• Disinfectants in Grade A/B areas should be sterile, supplied in • Feed water, pre-treatment, reverse osmosis (RO), deionized (DI),
sterile containers and used for a defined period purified/highly purified and water for injection (WFI) should be
tested
• Should be shown to be effective against facility microbial flora
• Alert and Action limits set by manufacturer (with action to be
• Should be sporicidal (if spores found in the environment) and for
taken if limits are exceeded)
“spraying in” of components and equipment
• WHO recommendations (next slide)
• Disinfection SOPs should include sufficient detail to enable
reproducibility • For purified/highly purified water and WFI, limits defined in
– preparation, work sequence, contact time
pharmacopoeia
– purified <100CFU/mL
• Organisms identified from adverse trends should be tested for
– Highly purified and WFI 10CFU/100mL (but is usually kept at high
their sensitivity to the disinfectants used
temperatures)
9 10

Water
Suggested Microbial Limits (CFU/mL) • Water should also be tested for presence of coliforms and/or
for facility water pseudomonads if appropriate (may cause biofilm)
Sampling Location Target Alert Action • Water used for parenterals should be tested for pyrogens
– limit is not more than 0.25 EU/mL
Raw water 200 300 500 • Water should be tested using R2A agar (low nutrient for the
recovery of water borne organisms) incubated for at least 5 days
Post multimedia filter 100 300 500
at 30-35°C
Post softener 100 300 500 • Sampling procedures should follow those used in production
Post activated carbon filter 50 300 500

Feed to RO 20 200 500
Compressed Air/Nitrogen/CO2
• Should be tested for non-viables and viables
RO permeate 10 50 100 • Pressure reduction orifices should be used to provide a steady
stream of air, validation of media should be ensured with
Point of use 1 10 100
consideration of validation
2
25-Oct-15
Container Integrity Testing Bioburden/IPC Testing

• Integrity of container/closure system • Should be written procedures for pre-sterilization
– is intitally validated by filling container with sterile growth bioburden, in-process control and raw material
medium then inserting container in broth containing 106 testing
CFU/mL of suitable microorganism
– containers sealed under a vacuum should be periodically • method should be validated for the recovery of
tested to demonstrate that vacuum is maintained over shelf low numbers of organisms
life
– procedures in place to detect faulty containers during
• use of anaerobic medium should be considered if
manufacture shown to be present in environment
– operators involved in visual inspection should have frequent • target, alert and action limits should be
breaks and regular eye-sight tests
documented and include action taken if limits
exceeded
Growth Promotion Testing Growth Promotion Testing

• Media used for microbiolgical testing should be
tested for its ability to support microbial growth Media
– The inoculated media should be capable of showing growth
– media used for media fills should be able to support the
within 3 days of incubation
growth of a wide range of microorganisms (bacteria and
moulds) – Media used in environmental monitoring should also be tested
for its growth promoting properties. Validation of recovery of
– Soybean Casein Digest Medium is usually used. An anaerobic
organisms under test conditions should be carried out to
medium may also be substituted occasionally if environmental
demonstrate neutralization of disinfectant residuals (media
monitoring indicates presence
should contain neutralisers).
– After the media fill has been completed, it is important to
– Media purchased externally should also be tested
demonstrate that the media would have been able to support
the growth of organisms if they had been present – Media used for media fills and environmental monitoring
should be pre-incubated to demonstrate “sterility” prior to use
– containers with media should be inoculated with 10-100 CFU
of organims such as Bacillus subtilis, Staphylococcus aureus, – Media should have a validated shelf life
Candida albicans, Aspergillus niger. Environmental isolates
should also be included
Sterility Testing Sterility Testing

• Sterility test is a quality control test used as part • Facilities
of product release for product required to be – Sterility testing should be carried out under the same
sterile conditions as aseptic manufacture
– Has significant statistical limitations - will really only detect • In a Grade A laminar air flow cabinet in a Grade B
gross contamination background (may also be carried out in an isolator)
• Air supply through HEPA filters, pressures should be
• Sampling monitored and alarmed
– No of containers and volume to be tested defined in • Access to area should be through airlocks
Pharmacopoeia
• Operators should be appropriately gowned is sterile
– Samples from aseptically manufactured product should be garments
taken from beginning, middle and end of batch fill and also
• Operators should be appropriately trained and validated
after interventions and stoppages
• Appropriate cleaning, sanitisation and disinfection
– Samples from terminally sterilized product should be taken
procedures should be in place
from previously identified cool spots within load
• Environmental monitoring should be conducted
– Sampling should be sufficient to allow for retests if needed
3
25-Oct-15

• Methods are defined in Pharmacopoeia • Media
– membrane filtration is the preferred method if product is – should be tested for growth promoting qualities prior to use
filterable (low number of organisms)
– direction innoculation is alternative – should have batch number and shelf life assigned
• Media types • Incubation Period
– Soybean Casein Digest medium (SCD), (also knows as – At least 14 days incubation
Trypticase Soy Broth(TSB)) and Fluid Thioglycollate medium – 20-25°C for SCD/TSB, 30-35°C for FTM
(FTM) is usually used (to detect aerobic and anaerobic – Test containers should be inspected at intervals
organisms)
– temperatures should be monitored and temperature
– validation studies should demonstrate that the media are monitoring devices should be calibrated
capable of supporting growth of a range of low numbers of
organisms in the presence of product. May need to – if product produces suspension, flocculation or deposit in
incorporate inactivators media, suitable portions (2-5%) should be transferred to fresh
media, after 14 days, and incubated for a futher 7 days
• growth should be evident after 3 days (bacteria), 5 days (moulds)
– media may be purchased or made in-house using validated
sterilization procedures

• Negative Contols
– media should be incubated for 14 days prior to use, either a • Positive Controls
portion or 100% of batch (may be done concurrently with test) • should be performed on all new products and when any changes
– negative product controls - items similar in type and are made.
packaging to actual product under test should be included in • Should be repeated annually
each test session – Stasis test recommended particularly for product with
• facilitate interpretation of test results antibiotics or preservatives or slow release tested by direct
• negative control contamination rate should be calculated and innoculation
recorded • demonstrates that media can support growth at the end of the
incubation period and has not been affected by product
• Positive Test Controls
– bactiostasis/fungistasis test • Results
• should demonstrate that media are capable of supporting growth – Any growth should be identified (genotypic)
of a range of low numbers of organisms in the presence of – Automated/Semi-automated systems used for identification
product. May need to incorporate inactivators should be periodically verified using reference strains
– growth should be evident after 3 days (bacteria), 5 days
(moulds)

• Interpretation and Repeat Tests • Interpretation and Repeat Testing
– No contaminated units should be found • When conditions (a), (b) or (c) apply the test should be
aborted
– A test may only be repeated when it can be
demonstrated that the test was invalid for causes • If a stasis test performed at the end of the test shows no
growth of challenge organisms, this also invalidates the
unrelated to the product being examined
test
– European Pharmacopoeia criteria • For conditions (d) to apply must demonstrate that the
• (a) the data of the micro monitoring of the sterility test orgamisms isolated from the sterility test is identical to an
facility show a fault isolate from materials (e.g. media) and/or the environment
• (b) a review of the testing procedure used during the test – must use genotypic identification methods
in question reveals a fault
– Repeat test is carried out with same number of samples
• (c) microbial growth is found in negative controls as first test
• (d) after determination of the identity of the
– Any contamination detected in repeat test, product does
microorganisms isolated from the test, the growth of this
species or these species may be ascribed unequivocally to not comply
faults with respect to the material and/or technique used in
conducting the sterility test procedure
4
25-Oct-15
Other Microbiological Laboratory Issues Other Microbiological Laboratory Issues

Reference Culture Collections
• Reference cultures may be used for Reference Cultures (2)
• Quality contol of media • Laboratory must have a system for preserving and
• Test method validation maintaining reference cultures with their original
• Control of test reagents characteristics.
• Must remain genetically stable to retain characteristics for • Laboratory should:
which they have been selected. – maintain suitable reference cultures for QC of culture media
and test reagents and for test method validation;
• Cultures of microorganisms tend to undergo
– ensure reference cultures are traceable to a recognised culture
variation/genetic change that can affect characteristics of a collection eg. ATCC, NCTC;
culture - unsuitable for further use.
– ensure reference cultures are uniquely identified within
• Probability of variation/genetic change increases with laboratory, with traceability to recognised culture collection.
frequency of repeated subculture of reference culture –
working culture must be no more than 5 generations
removed from original source culture.
Reference Cultures (3) QC of Culture Media

• Lab should have documented procedures:
• Media other than sterility testing media and media fill media
– that maintain hierarchical control of reference cultures (ie.
must be subject to quality contol
master, stock & working cultures);
– for purchase, preservation, maintenance, identification and
• quantitative or semi-quantitative method/s to assess growth
frequency of subculturing of reference cultures; promotion/fertility
– that prevent use of working cultures as replacements for • use of positive and negative controls for selective and/or
depleted stock and/or master cultures. dirrerential culture media
• Maintain records for each reference culture: • different levels of QC required dependent on whether
– identity, source and history and date of receipt of master culture is
culture; – manufactured in house (every batch should be tested)
– resuscitation, preservation, maintenance and storage
– purchased ready to use (supplier tests media with testing
conditions for master, stock and working cultures;
periodically verified in house)
– results of purity and identification tests for master and/or
stock cultures; and
– dates of preparation of stock and working cultures.
QC of Culture Media (2) Sterilization processes for Culture Media

• Laboratory should: • sterilzation process for culture media should be validated
– have documented procedures for preparation, QC, release and and monitored using same procedures as for sterilization of
storage of culture media; product
– have validated shelf life of culture media under normal storage
conditions;
– maintain records of preparation and QC of individual batches
Equipment Calibration and Checks
of culture media; • Laboratory equipment (e.g. pipettes, balances, incubators,
– ensure that records of microbiological QC performance testing refrigerators, thermometers, autoclaves, laminar flow
are traceable to batch preparation records; and workstations etc) should be calibrated and recalibrated and
– that microbiological QC performance test results are assessed routinely monitored (where appropriate)
against acceptance/rejection criteria.
Personnel
• Should be appropriately trained and authorized to perform
testing
5
25-Oct-15
Testing of Biological Indicators Endotoxin Testing (2)

• if tested in-house the method should include a heat-shock • LAL (Limulus Amebocyte Lysate) test is used for detecting
step (this verifies that the indicators do actually contain endotoxin (previously a rabbit test)
spores and not vegetative organisms) – based on clotting reaction of horseshoe crab blood to
• BIs should occasionally be tested in house to verify the endotoxin
suppliers count • Types of LAL test
– Gel Clot
– Turbidimetric
Endotoxin Testing
– Colorimetric
• Parenteral products should be free from endotoxin
• Endotoxin is a lipopolysaccharide present in the cell wall of
gram negative bacteria which can cause fever if introduced • Equipment used in test must be endotoxin free
into the body
• Raw materials, WFI used in manufacture and some finished • Validation of accuracy and reliability of the method for each
product must be tested for endotoxin product is essential
Endotoxin Testing (4)

Endotoxin Testing (3)
Gel Clot Method Turbidimetric Method
• Original method
• A kinetic method
• The official “referee test”
• The specimen is incubated
with LAL and either the
• The specimen is incubated rate of increase in turbidity
with LAL of a known or the time taken to reach
senstivity. Formation of a gel a particular turbidity is
clot is positive for endotoxin. measured
spectrophotometrically
and compared to a
standard curve.
Endotoxin Testing (5) Endotoxin Testing (6)
Colorimetric Method Gel Clot Chromogenic Chromogenic Turbidimetric

Endpoint Kinetic
Semi- Quantitative Quantitative Quantitative
• Endotoxin catalyzes the
quantitative
activation of a proenzyme in Simple Least Requires Requires Requires
LAL which will cleave a expensive, spectrophotometer incubating plate or incubating plate or
colorless substrate to Requires 37 degree tube reader tube reader
or plate reader
produce a colored bath
endproduct which can be Manually read and Can be automated, Can be automated, Can be automated,
measured recorded allows electronic allows electronic allows electronic
spectrophotmetrically and data storage data storage data storage
compared to a standard
curve. Sensitive down Sensitive down Sensitive down Sensitive down
to 0.03 EU/ml to 0.1 EU/ml to .005 EU/ml to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
• Can be kinetic or endpoint
6
25-Oct-15
Questions?
37 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

7
ORA.007, Version 1.1
DATE: 04-25-2014
Pharmaceutical
Microbiology
Manual
2014
The purpose of this Pharmaceutical Microbiology Manual (PMM) is to
collectively clarify, standardize, and communicate useful analytical procedures
that are not specifically addressed in the microbiology methods chapters in the
United States Pharmacopeia. In addition, some sections of this manual can
serve as a technical reference when conducting microbiological inspections of
drug, biotechnology and medical device manufacturers. The contents of this
PMM were collaboration between ORA and CDER in order to maximize the
efficiency of our analytical results to support CDER’s goal to assure the safety
and reliability of commercially distributed medical products.
DATE: 04-25-2014
Table of Contents
i. Introduction…………………………………………………………………………..…………iv
ii. Pharmaceutical Microbiology Editorial Board……………………………………................v
1. Antimicrobial Effectiveness Testing…………………………………………………..…1
A. Media
B. Growth Promotion of the Media
C. Suitability of the Counting Method in the Presence of Product
D. Test Organisms
E. Preparation of the Inoculum
F. Procedure
G. Interpretation
2. Microbial Examination of Non-Sterile Products………………………………………...5
A. Product Storage and Handling
B. Gowning Requirements
C. Growth Promotion and Inhibitory Properties of the Media
D. Suitability of the Test Method
E. Test Procedure
F. Interpretation of the Results
3. Sterility Testing…………………………………………………………………………….9
A. Method Suitability Test
B. Sample Analysis
C. Preparation for the Analysis
D. Clean Room Activities
E. Method of Analysis
F. Analysis of Purified Cotton, Gauze, Sutures and Surgical Dressings
G. Control Systems
H. Sub-Culturing Primary Media
I. Preservation of Isolates
J. Worksheet Notations
K. Preservation of Revival of Cultures
4. Investigating USP Sterility Testing Failure…………………………………………….24
5. Bacterial Endotoxin Testing…………………………………………………………….26
A. Gel Clot Method
B. Kinetic Assays
C. Medical Devices
D. Endotoxin References
6. Particulate Matter………………………………………………………………………...33
A. Light Obscuration Particle Count Test
B. Microscopic Particle Count Test
7. Bioburden Estimation for Medical Devices……………………………………………46
8. Environmental Monitoring……………………………………………………………….48
A. Materials/Equipment
B. Sampling Preparation
C. EM Sampling Procedure
D. Recommended Environmental Monitoring Sites
E. Analysis Preparation conducted by FDA field laboratory
F. Analytical Procedure
9. Rapid Screening Methods………………………………………………………………56
ii
This document is uncontrolled when printed: 10/28/2014
For the most current and official copy, check the Master List
DATE: 04-25-2014
A. Screening Protocol for Direct Staining on Products with Appearance of
Visible Contamination
10. Inspectional Guidance………………………………………………………….............60
A. Microbiological Issues for Inspection of Pharmaceutical Laboratories
B. Microbiological Issues for Inspection of Pharmaceutical Manufacturing
Facilities
C. Sample Data Review – All Negative Results
D. Sample Data Review – Microbial Growth Indicated
E. How to Investigate Microbiological OOS test result(s)
F. Laboratory Facility and Analytical Review
G. Manufacturing Facility Review
H. Inspectional Elements of CP 7356.002
Appendix A: Literature and References……………………………………………………….70
iii
DATE: 04-25-2014
i. Introduction
The Pharmaceutical Microbiology Manual (PMM) evolved from the Sterility Analytical
Manual and is a supplement to the United States Pharmacopeia (USP) for pharmaceutical
microbiology testing, including antimicrobial effectiveness testing, microbial examination of
non-sterile products, sterility testing, bacterial endotoxin testing, particulate matter, device
bioburden and environmental monitoring testing. The goal of this manual is to provide an
ORA/CDER harmonized framework on the knowledge, methods and tools needed, and to
apply the appropriate scientific standards required to assess the safety and efficacy of
medical products within FDA testing laboratories. The PMM has expanded to include
some rapid screening techniques along with a new section that covers inspectional
guidance for microbiologists that conduct team inspections.
This manual was developed by members of the Pharmaceutical Microbiology Workgroup

and includes individuals with specialized experience and training.
The instructions in this document are guidelines for FDA analysts. When available,
analysts should use procedures and worksheets that are standardized and harmonized
across all ORA field labs, along with the PMM, when performing analyses related to
product testing of pharmaceuticals and medical devices. When changes or deviations are
necessary, documentation should be completed per the laboratory’s Quality Management
System. Generally, these changes should originate from situations such as new products,
unusual products, or unique situations.
This manual was written to reduce compendia method ambiguity and increase
standardization between FDA field laboratories. By providing clearer instructions to FDA
ORA labs, greater transparency can be provided to both industry and the public.
However, it should be emphasized that this manual is a supplement, and does not replace
any information in USP or applicable FDA official guidance references. The PMM does
not relieve any person or laboratory from the responsibility of ensuring that the methods
being employed from the manual are fit for use, and that all testing is validated and/or
verified by the user.
The PMM will continually be revised as newer products, platforms and technologies
emerge or any significant scientific gaps are identified with product testing.
Reference to any commercial materials, equipment, or process in the PMM does not in
any way constitute approval, endorsement, or recommendation by the U.S. Food and
Drug Administration.
U.S. FDA, Office of Regulatory Affairs

Office of Regulatory Science
Medical Products and Tobacco Scientific Staff
iv
DATE: 04-25-2014
ii. Pharmaceutical Microbiology Editorial Board

Editor-In-Chief: Dennis Guilfoyle (ret), Northeast Regional Laboratory
Senior Editor: Angele Smith, Office of Regulatory Science
Rhonda Alexander, Southeast Regional Laboratory

Jasna Braut-Taormina, Northeast Regional Laboratory
Jennifer Brzezinski, Forensic Chemistry Center
Jennifer Gogley, Pacific Regional Laboratory – South West
Andrew Gonzales, Denver Laboratory
Lawrence James, Northeast Regional Laboratory
Marilyn Khanna, Office of Regulatory Science
David Lau, San Francisco Laboratory
Philip McLaughlin, Winchester Engineering and Analytical Center
Zachary Miller, Denver Laboratory
George Salem, Office of Regulatory Science
Tammara Stephens, Southeast Regional Laboratory
Selen Stromgren, Office of Regulatory Science
Vilasini Suktankar, Southeast Regional Laboratory
Evelyn Wong, Pacific Regional Laboratory – South West
Jonathon Yenovkian, San Francisco Laboratory
Special Appreciation:
Radhakrishna Tirumalai, United States Pharmacopeia
Rick Friedman, Center for Drug Evaluation and Research
Diane Raccasi, Office of Enforcement and Import Operations
v
DATE: 04-25-2014
Chapter 1: Antimicrobial Effectiveness Testing

Antimicrobial Effectiveness testing is described in USP <51>. Previously this chapter was
known as “Preservative Effectiveness Testing”. Detailed procedure for the performance of
the test can be found in USP <51>.
A. Media
For the cultivation of the test organisms, select agar medium that is favorable to
the rigorous growth of the respective stock culture. The recommended media are
Soybean Casein Digest Agar/Broth and Sabouraud’s Dextrose Agar/Broth. Add a
suitable inactivator (neutralizer) for the specific antimicrobial properties in the
product to the broth and/or agar media used for the test procedure if required.
B. Growth Promotion of the Media

Media used for testing needs to be tested for growth promotion by inoculating the
medium with appropriate microorganisms. It is preferable that test microorganisms
be chosen for growth promotion testing (Section D).
Solid media tested for growth promotion is to be set up using pour plate method in
order to determine a microbial plate count (CFU) which must be ≥ 70% of the
microorganism inoculum’s calculated value.
C. Suitability of the Counting Method in the Presence of

Product
For all product types, follow current USP methodology in chapter <51>, with the
following additional instructions.
Prior to the Antimicrobial Effectiveness testing, determine if any antimicrobial

properties exist by performing a Suitability testing utilizing microorganisms used for
product testing (section D). Should the Suitability Test fail, the results of Suitability
test are invalid and will need to be repeated with proper method modification to
neutralize the inhibiting property.
If multiple samples of the same product from the same manufacturer (same
amount and form) are collected, one sample may be used for method suitability for
all the samples collected.
D. Test Organisms
All cultures must be no more than 5 passages removed from the original stock
culture.
Candida albicans (ATCC No. 10231)

Aspergillus brasiliensis (ATCC No. 16404) (formerly Aspergillus niger)
Escherichia coli (ATCC No. 8739)
Pseudomonas aeruginosa (ATCC No. 9027)
Staphylococcus aureus (ATCC No. 6538)
Page 1 of 81
DATE: 04-25-2014
E. Preparation of Inoculum
Preparatory to the test, inoculate the surface of the appropriate agar medium from
a recently grown stock culture of each of the above test microorganisms. Use
Soybean-Casein Digest medium for Escherichia coli ATCC 8739, Pseudomonas
aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 and incubate at
32.5 ± 2.5° C for 3 – 5 days. Use Sabouraud Dextrose medium for Candida
albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 and incubate at
22.5 ± 2.5° C for 3 – 5 days for Candida albicans and 3 - 7 days for Aspergillus
brasiliensis.
Harvest the cultures by washing the growth with sterile saline to obtain a microbial
count of about 1x108 CFU/mL (see Microbial Enumeration Tests <61> and Tests
for Specified Microorganisms <62>). For the A. brasiliensis ATCC 16404 culture,
use sterile saline containing 0.05% polysorbate 80.
Alternatively, cultures may be grown in a liquid medium, i.e. Soybean Casein

Digest Broth or Sabouraud’s Dextrose Broth, (except for the A. brasiliensis ATCC
16404 culture) and harvested by centrifugation, washing and suspending in sterile
saline to obtain a count of about 1 X 108 colony forming units (CFU) per mL.
The estimate of inoculum concentration may be obtained by turbidimetric

procedures for the challenge microorganisms and later confirmed by plate count.
Refrigerate the suspension if not used within 2 hours at 2-8° C.
Determine the number of CFU/mL in each suspension using the appropriate media
and recovery incubation times to confirm the CFU/mL estimate.
Use bacterial and yeast suspensions within 24 hr. of harvest. The mold
preparation may be stored under refrigeration (2-8° C) for up to 7 days.
Note: Alternative commercially available standardized cultures may be used in lieu

of in-house prepared cultures.
F. Procedure
The procedure requires that the test be conducted with a suitable volume of
product. It is advisable to begin with at least 20 mL of product. Use the original
product containers whenever possible or five sterile, capped bacteriological
containers of suitable size into which a suitable volume of product has been
transferred. If the diluted product exhibits antimicrobial properties, specific
neutralizers may need to be incorporated into the diluents or the recovery media.
For purposes of testing, products have been divided into four categories:
Category 1 – Injections, other parenteral including emulsions, otic products,

sterile nasal products, and ophthalmic products made with aqueous bases
or vehicles.
Page 2 of 81
DATE: 04-25-2014
Category 2 – Topically used products made with aqueous bases or

vehicles, non-sterile nasal products, and emulsions, including those applied
to mucous membranes.
Category 3 – Oral products other than antacids, made with aqueous bases
or vehicles.
Category 4 – Antacids made with aqueous bases or vehicles.
Inoculate each container with one of the prepared and standardized inoculums and
mix. The volume of the suspension inoculums used is 0.5% to 1.0% of the volume
of the product. The concentration of the test organisms added to the product for
Categories 1, 2 and 3 is such that concentration of the test preparation immediately
after inoculation is between 1x105 and 1x106 colony forming organisms (CFU) per
mL of product. For category 4 products (antacids) the final concentration of the
test organisms is between 1x103 and 1x104 CFU/mL of product.
Immediately determine the concentration of viable organisms in each inoculum

suspension and calculate the initial concentration of CFU/mL by the plate count
method (see Microbial Enumeration Tests <61> and Tests for Specified
Microorganisms <62>).
Incubate the inoculated containers between 22.5 ±2.5°C in a controlled

environment (incubator) and sample the container at specified intervals. The
container sampling intervals include: Category 1 products are sampled at 7, 14,
and 28 days and Category 2 – 4 products are sampled at 14 and 28 days. Refer to
table within USP <51>. Record any changes in appearance of the product at these
intervals. Determine the number of viable microorganisms per mL present at each
of these sampling intervals by the plate count method utilizing media with suitable
inactivator (neutralizer). Calculate the change in log10 values of the concentration
per mL based on the calculated concentration in CFU/mL present at the start of the
test for each microorganism at the applicable test intervals, and express the
changes in terms of log reductions.
NOTE: The USP does not require a specific volume of product to be added to
each of the five sterile tubes. It is recommended that 20 mL/tube be used to
standardize testing for all FDA laboratories.
NOTE: All plate counts should be performed in duplicate (2 plates per dilution),
and in a dilution series to detect growth inhibited by the preservative system at the
lower dilutions. Carrying the test to the 10-3 dilution would be sufficient in most
cases to overcome preservative inhibition.
G. Interpretation
The criteria for microbial effectiveness are met if the specified criteria are met, see
table below. No increase is defined as not more than 0.5 log10 unit higher than the
previous value measured.
Page 3 of 81
DATE: 04-25-2014
CRITERIA FOR TESTED MICROORGANISMS
Category 1 Products
Bacteria: Not less than 1.0 log reduction from the initial calculated
count at 7 days, not less than 3.0 log reduction from the
initial count at 14 days, and no increase from the 14 day
count at 28 days.
Yeast and Molds: No increase from the initial calculated count at 7, 14, and
28 days.
Category 2 Products
Bacteria: Not less than a 2.0 log reduction from the initial count at 14
days, and no increase from the 14 day count at 28 days.
Yeast and Molds: No increase from the initial calculated count at 14 and 28
days.
Category 3 Products
Bacteria: Not less than a 1.0 log reduction from the initial count at 14
days, and no increase from the 14 day count at 28 days.
Yeast and Molds: No increase from the initial calculated count at 14 and 28
days.
Category 4 Products
Bacteria, Yeast and No increase from the initial calculated count at 14 and 28
Molds: days.
Page 4 of 81
DATE: 04-25-2014
Chapter 2: Microbial Examination of Non-Sterile Products

This section contains supplemental information for the quantitative enumeration of viable
microorganisms and the determination of the absence of specified microorganisms in finished
pharmaceutical products and raw materials, commonly referred to as Microbial Limits Testing
(MLT). The detailed procedures for these tests are not addressed in this PMM chapter since
they are found in USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE
PRODUCTS: MICROBIAL ENUMERATION TESTS and <62> MICROBIOLOGICAL
EXAMINATION OF NONSTERILE PRODUCTS: TESTS FOR SPECIFIED
MICROORGANISMS.
Methods for enumeration of microorganisms from pharmaceuticals (as described in USP <61>)
include membrane filtration, conventional plate count (including pour-plate method, surface-
spread method), and the Most-Probable-Number (MPN). USP Chapter <62> describes specific
enrichment procedures depending on the target specified microorganism that must be absent,
as required by a product monograph. Products which are insoluble or immiscible in water must
be appropriately treated to obtain a suspension suitable for the test procedures.
It is important to note that even though the USP delineates methods for the recovery and
identification of specified microorganisms based on monograph requirements, it is still the goal
of the regulatory microbiologist to screen for any other microorganisms that may also be present
in the product(s) and report these microorganisms on worksheets. In many cases, these may
be opportunistic or emerging pathogens not targeted for recovery by USP <62>. Alternative
methods, or the use of additional general enrichment agar plates or broth without selective
properties, may better suit the screening of test samples. The application of these additional
agars or methods may need to be considered based on the target population of the drug or
product under analysis and may require a dialogue with the laboratory supervisor for additional
instructions.
A. Product Storage and Handling

1. Samples are to be held under the same storage conditions required by the package
label or insert.
2. Prior to product testing, the exterior of the unit container should be disinfected before
transfer to the work station or HEPA filtered laminar flow hood. If the product
container is not hermetically sealed do not soak the product container in a
disinfection solution which may allow the ingress of bactericidal solution into the
product.
3. The work area for opening the unit container should be either a HEPA filtered
laminar flow hood or an alternate controlled environment to safeguard the exposure
of open media and product to either environmental or personnel contamination.
4. If the sample is an aqueous based product, the unit(s) should be shaken prior to
transfer to work area to maximize microbial dispersement.
5. All subsequent manipulation of test tubes with product or sub-culturing can be
conducted on the laboratory work bench or within a Biological Safety Cabinet (BSC)
if filamentous fungi are suspected.
B. Gowning Requirements
Page 5 of 81
DATE: 04-25-2014
1. When conducting the testing, the analyst should wear a clean lab coat, sterile
sleeves and sterile gloves. Gloves should be frequently disinfected especially
between opening and handling sample (product) units.
2. Depending on the type of Laminar Flow Hood or equipment barriers in a particular
laboratory, it might be beneficial to also wear a surgical mask and hair net.
C. Growth Promotion and Inhibitory Properties of the Media

Test each batch of ready-prepared medium and each batch of medium prepared either
from dehydrated medium or from ingredients following USP <61> and <62>. Each
chapter provides guidance on test strains to be used for each type of media, refer to
Table 1 of USP <61> and <62>. Ensure that seed-lot cultures used are not more than
five passages removed from the original master seed-lot. Test strains suspensions
should be used within 2 hours, or within 24 hours if refrigerated between 2ºC and 8ºC.
Spore suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) refrigerated between
2ºC and 8ºC may be kept for a validated period of time. Additionally, all bacterial and
spore suspensions should be prepared to yield ≤100 cfu. Growth promotion (and
suitability test) plates and tubes should not be incubated in the same incubators used for
product testing. If this cannot be avoided because of limited space, it is preferable to
store the “spiked samples” in the lower half of the incubator below the sample inoculated
plates and tubes.
D. Suitability of the Test Method

Suitability demonstrates that the products tested do not exhibit inhibitory effects on the
growth of microorganisms under the conditions of the tests. Although the intent is to
perform the suitability test before performing the analysis of the product, it is acceptable
to run the product test and the suitability test concurrently. However, it should be noted
that if the suitability test is run concurrently with the product test and the suitability test
should fail, the results of the product test are invalid and the suitability test as well as the
product test will need to be repeated with proper method modification to neutralize the
inhibiting property.
Neutralizing agents may be used to neutralize the activity of antimicrobial agents in

products, see USP <61> Table 2 for a list of potential neutralizing agents/methods. The
appropriate neutralizing agent should be added preferably before sterilization of the
media. Include a blank control with neutralizer and without product to demonstrate
efficacy and absence of toxicity for microorganisms.
USP <61> and <62> describe the suitability tests necessary for each analysis. The
correct inoculum of not more than 100 CFU is required as are specific incubation
temperatures and durations. Ensure that seed-lot cultures used are not more than five
passages removed from the original master seed-lot. Test strains suspensions should
be used within 2 hours, or within 24 hours if refrigerated between 2ºC and 8ºC. Spore
suspensions (A. brasiliensis, B. subtilis, C. sporogenes) refrigerated between 2ºC and
8ºC may be kept for a validated period of time. USP <61> and <62> require a control
which is without test material to be included in the suitability test. The following viable
ATCC derived cultures may be used. Please be aware that under the revised
harmonized Microbial Limits chapters <61> and <62> users are allowed alternative
sources of the below listed strains. The organisms below are recommended for FDA
use in order to have a consistent and standard worksheet format:
Page 6 of 81
DATE: 04-25-2014
Staphylococcus aureus (ATCC 6538)

Escherichia coli (ATCC 8739)
Pseudomonas aeruginosa (ATCC 9027)
Bacillus subtilis (ATCC 6633)
Candida albicans (ATCC 10231)
Aspergillus brasiliensis (ATCC 16404)
Salmonella enterica (ATCC 14028)
Clostridium sporogenes (ATCC 11437)
USP <61> and <62> each contain the acceptance criteria for their respective suitability
test. For USP <61> the Results and Interpretation section requires the inoculated
product to have a mean count of any of the test organisms not differing by a factor
greater than 2 from the control which was without test material. USP <62> requires the
specified microorganisms to be detected with the indicated reactions.
E. Test Procedure
Prepare the sample in a manner to achieve a uniform solution or suspension. This is
critical because microbial contamination is not evenly dispersed throughout a lot or
sample of product. Use conventional mechanical and shaking methods to the extent
that original numbers and types of microorganisms are not altered in the product.
Use the following general procedures to prepare and handle samples.
1. Analyze samples as soon as possible after receipt. Inspect each unit visually for
integrity of primary container and note any irregularities. Do not use the product
container if it has been compromised or damaged without supervisor approval.
Testing of a compromised or damaged container should be evaluated on a case by
case basis. Discuss with supervisor if compromised unit containers need to be
tested for forensic purposes (i.e. product tampering).
2. Identify units to be tested with Analyst's initials, date, subsample number, and
sample number.
3. Cleanse outer surfaces of sample containers with sterile wipes using a validated
effective antimicrobial agent. Place on a disinfected tray or surface in a properly
disinfected laminar flow hood or biosafety cabinet. Allow containers to dry.
4. Aseptically open containers and perform weighing procedures in a laminar flow hood
or biological safety cabinet if possible.
5. Appropriate environmental controls such as air exposure plates should be used in

accordance with local quality procedures.
6. Appropriate negative controls should be run concurrently with the sample.
F. Interpretation of the Results

Regarding USP <61> the acceptance criterion for microbiological quality as it pertains to
quantitative analyses has an allowable variability of the final colony forming units
Page 7 of 81
DATE: 04-25-2014
(CFUs). There is a two-fold tolerance in the final results. For example, if the monograph
requires a 100 cfu/ml limit, the acceptable upper limit for these results would be 200
cfu/ml. Additional information is included in the “Interpretation of the Results” section of
USP <61> that should be read and understood when reviewing quantitative test results.
Page 8 of 81
DATE: 04-25-2014
Chapter 3: Sterility Testing
A. Method Suitability Test

For all product types, follow current USP methodology in <71>, with the following
additional instructions.
Prior to or concurrently with the sterility test, determine if any bacteriostatic or fungistatic
residue has been retained on the filter membrane. The Method Suitability Test can be
run concurrently with the sterility test per USP. However, it should be noted that if the
Method Suitability Test is run concurrently with the product test and the Method
Suitability Test should fail, the results of the product test are invalid and the Method
Suitability Test as well as the product test will need to be repeated with proper method
modification to neutralize the inhibiting property.
Units selected for suitability testing should be subjected to the same disinfection
procedure utilized in the sample analysis.
In all cases, even if the product does not include a preservative, the product itself may
have growth inhibiting properties. All products should undergo a prescribed Method
Suitability test.
If multiple samples of the same product from the same manufacturer (same dosage and
form) are collected, one sample may be used for method suitability for all the samples
collected.
1. When to run Method Suitability:

a. Run the test prior to conducting the sterility test in accordance with USP
requirements under the following conditions:
i. If insufficient information about the product exists to judge its probable

growth inhibiting activity.
ii. In all cases, when there is sufficient analytical time available, i.e., survey
type samples.
b. Run the test concurrently with product sterility tests when time is critical and
problems associated with Part I above have been resolved. However, it should
be noted that if the Method Suitability Test is run concurrently with the product
test and the Method Suitability Test should fail, the results of the product test are
invalid and the Method Suitability Test as well as the product test will need to be
repeated with proper method modification to neutralize the inhibiting property.
c. If an insufficient amount of product is collected and the analysis is critical the

suitability test can be conducted at the end of the 14 day incubation period. Be
sure to use best judgment and maximum neutralization approach when initially
conducting the Sterility test. If the suitability results indicate inhibition then the
results, if negative, are invalid. However, if the product test results indication
microbial presence and the suitability test show inhibition the results are still
valid.
Page 9 of 81
DATE: 04-25-2014
2. Method Suitability Test Procedures

Method Suitability, and any other positive control tests which require the use of
viable microorganisms, should be performed outside the clean room or isolator,
in a biosafety cabinet or equivalent,
a. Membrane filtration
i. Pass product fluid through filter membrane. Rinse the membrane with
three 100 ml portions (or more if necessary or required) of specified rinse
fluid. Do not exceed a washing cycle of five times 100mL per filter. This
step hopefully will neutralize and remove any antimicrobial residue on the
filter membrane.
ii. Add specified test organisms in specified numbers (less than 100 cfu) into
the last 100 ml rinse fluid used.
iii. Filter the fluid and divide the filter membrane between the specified
media. If conducting the sterility test using a closed canister system, rinse
each canister with the inoculated rinse fluid.
iv. If the available number of test vessels is insufficient for a complete

challenge test for each individual microorganism then the test organisms
may be composited as necessary. However, confirmation of growth for
the composited microorganisms will need to be confirmed by isolation,
Gram stain, and genus/species identification after the completion of
incubation.
v. Confirm composited microorganisms by Gram stain, microscopic

examination, and biochemical identification after the completion of
incubation.
vi. See step c. below for additional considerations.
b. Direct inoculation:
For direct inoculation, add the test organisms to separate containers of
product/culture media if sufficient product is available. See step c. below for
additional considerations.
c. The following test procedures apply to Direct Inoculation and Membrane

Filtration:
i. Inoculate the same microorganism using the same medium without the
product as a positive control.
ii. For bacteria and fungi, incubate tubes/bottles according to USP

requirements. Ensure that seed-lot cultures used are not more than five
passages removed from the original master seed-lot. Test strains
suspensions should be used within 2 hours, or within 24 hours if
refrigerated between 2ºC and 8ºC. Spore suspensions (A. brasiliensis, B.
Page 10 of 81
DATE: 04-25-2014
subtilis, C. sporogenes) refrigerated between 2ºC and 8ºC may be kept

for a validated period of time. Additionally, all bacterial and spore
suspensions should be prepared to yield ≤100cfu.
iii. If growth comparable to that of the positive control vessel without product
is obtained, then you may proceed with the sterility test. If visible growth
is not obtained, the antimicrobial activity of the product has not been
eliminated under the conditions of the test. Modify the test conditions and
repeat the Method Suitability test.
iv. If there is not enough product material to perform method suitability test
using all the specified number of organisms for the test, select the
organisms most appropriate for the product based on available
information. If insufficient information is available for making a judgment,
perform the test using ATCC-derived organisms (or acceptable alternative
sources, if necessary) in the following order:
a. Clostridium sporogenes, ATCC 11437

b. Candida albicans, ATCC 10231
c. Bacillus subtilis subsp. spizizenii, ATCC 6633 (Formerly
Bacillus subtilis)
d. Pseudomonas aeruginosa, ATCC 9027
e. Aspergillus brasiliensis, ATCC 16404 (Formerly Aspergillus
niger)
f. Staphylococcus aureus, ATCC 6538
Note: refer to ATCC documentation for the most current organism names.
d. If product is found to exhibit growth inhibiting activity when determined

concurrently with product testing, the sterility test must be repeated using an
additive (or increase media volume) to modify the conditions in order to eliminate
the antimicrobial activity. One may need to search the literature or have the CSO
contact the manufacturer and request a copy of their sterility test methodology.
e. Cultures used for the method suitability test can be purchased commercially, pre-
counted and ready to use, or can be prepared and maintained locally.
B. Sample Analysis
1. Sample Containers
a. Open the sample package on a laboratory bench disinfected with a sporicidal /
viricidal antimicrobial agent such as 2% glutaraldehyde or equivalent solution
Refer to the AOAC and available literature for choosing suitable antimicrobial
agents for use in your facility.
b. Count the number of units received. Compare this number with the number of
units collected.
c. Inside the clean room preparation area located outside the certified class 100
areas (if available), remove all outer containers from sample units that will be
Page 11 of 81
DATE: 04-25-2014
tested without compromising the sterile integrity of the product. Remove sample
units and place them on a tray or cart disinfected with an effective antimicrobial
agent.
Note: One or more units can be sacrificed for sample exploration if the number of
the units received is sufficient.
d. Examine all units visually for container closure integrity, for the presence of any
foreign matter and other defects present in the product. Note findings on
analyst's worksheet.
e. If foreign matter is observed within the primary container, discuss with supervisor
the employment of ORS procedure Document ORA-LAB.015 entitled “Screening
Protocol for Direct Staining on Products with Appearance of Visible
Contamination” (see QMiS for Procedure).
2. Sample Identification
If sample units are not identified by the collector, the analyst should identify unit with
sample #, initials, date, and sub sample # as appropriate for sample traceability.
Otherwise, date and initial each unit.
3. Unit Container Disinfection

Cleanse the exterior of all product primary containers using antimicrobial agents
meeting requirements described under step 1. These suggested disinfection
procedures can be performed on commonly encountered units as follows:
a. Ampoules can be wiped with lint free sterile towel/wipes saturated with
disinfectant. Ampoules may be soaked in disinfectant for 1 hour.
b. Vials should only be wiped with disinfectant. Vials should not be soaked due
to the possibility of migration of disinfectant under the closure and into the
product.
c. Laminated Tyvek package: Disinfect polyethylene/plastic laminate with sterile

towel/wipes soaked in disinfectant. Tyvek portion lightly scrubbed with sterile
particle free dry wipe and air cleanse in a HEPA filtered laminar flow hood for
several hours before testing.
d. Paper Package: Disinfect with UV light if possible. Scrub with sterile particle
free dry wipes and air cleanse as above.
1. Depending on the clean room design, immediately move the sample to the clean
room on a disinfected designated stainless steel cart or place it inside the clean
room pass thru for final preparation. If conducting the sterility test in an isolator
place the sample on a designated stainless steel cart. Allow the sample to react
with the disinfectant for 1 hour before further handling.
2. Number of units and/or amount of product tested:

Follow the current edition of the USP to determine the correct number of units to
be tested and the amount of product to be analyzed from each unit. It is
Page 12 of 81
DATE: 04-25-2014
preferable to test the entire contents of each unit if possible. Follow laboratory
policy if it requires testing more units than the USP requires.
If the number of units collected is less than the USP requirements, discuss with
the laboratory supervisor before proceeding. Samples collected in a for-cause
situation may be analyzed with a number of units less than the USP
requirements.
C. Preparation for the Analysis

1. Media Preparation:
Follow current USP when preparing media used for sample analysis. Commercially
purchased media may also be used for the analysis. Both prepared and purchased
media must meet the requirements of the USP growth promotion test of aerobes,
anaerobes and fungi. The most common media used are:
a. Fluid Thioglycollate medium (FTM) This medium should be prepared in a

suitable container to provide a surface to depth ratio so that not more than
the upper half of the medium has undergone a color change indicative of
oxygen uptake at the end of the incubation period. If more than the upper
third of the medium has acquired a pink color, the medium may be restored
once by heating until the pink color disappears. Care should be taken to
prevent the ingress of non-sterile air during cooling.
b. Soybean Casein medium (SCD medium)
c. Alternative Thioglycollate medium NOTE: This type of media must be

incubated under anaerobic conditions.
d. Media for Penicillin and Cephalosporin containing drugs. Add sufficient

quantity of sterile Beta-lactamase to the media to inactivate the effect of
these antibiotics.
e. Diluting and rinsing fluids. These fluid rinses must be filtered before
sterilization to avoid clogging of the filter membrane during testing.
2. Media storage
For laboratory prepared media, do not use medium for longer storage period than
has been validated.
For commercially purchased media, follow the manufacturer’s recommended storage

requirements and expiration date.
3. Media qualification:
Perform the following tests on the prepared media before use:
a. Sterility: The media batch may be used if the sterilization cycle is validated
and monitored with the use of a biological indicator, and the batch passes
other quality control testing. Also, if possible incubate a portion of the media
at the specified temperature for 14 days.
Page 13 of 81
DATE: 04-25-2014
b. Growth promotion test; follow the current USP using recommended strains of
organisms (Table 1, USP <71>). Do not use cultures that are more than five
passages removed from the original master seed lot. Commercially prepared
and certified pre-counted cultures of the recommended organisms can also
be used. Test strains suspensions should be used within 2 hours, or within 24
hours if refrigerated between 2ºC and 8ºC. Spore suspensions (A.
brasiliensis, B. subtilis, and C. sporogenes) refrigerated between 2ºC and 8ºC
may be kept for a validated period of time. Additionally, all bacterial and
spore suspensions should be prepared to yield ≤100cfu.
4. Equipment Preparation
Analytical equipment and tools used in sterility analysis and suitability should be
cleaned and sterilized using a validated sterilization procedure. Commercially
purchased equipment and tools should be labeled sterile and accompanied by a
certificate of analysis for sterility.
D. Clean Room Activities

1. Gowning
Personnel are critical to the maintenance of asepsis in the controlled environment.
Thorough training in aseptic techniques is required. Personnel must maintain high
standards each time they deal with sterile product.
a. Personnel gowning qualification should be performed by any analyst that

enters the aseptic clean room to ensure proper gowning techniques are
followed. Personnel gowning qualification should consist of:
i. Training of gowning techniques by a qualified trainer.
ii. Observation of trainee by trainer while gowning.
iii. General growth media touch plates utilized to analyze if the trainee
gowned correctly without contaminating the sterile outer gown, sterile
gloves and sterile head cover.
Some consideration should be taken before entering the clean room (see
below). Follow applicable specific procedures for the facility.
Proper gowning immediately prior to entry the clean room is
required of all personnel without exception.
Remove street clothes when possible and wear clean clothes the day of
the analysis. . Non-linting clean room scrubs that cover as much skin as
possible is the ideal inner-suit to wear before gowning up for an aseptic
clean room.
Remove jewelry and makeup.
Scrub hand (and arm when possible) before gowning. Take a shower the
day before analysis if possible to reduce skin shedding.
Non shedding sterile uniform components should be used all the time.
Use aseptic gowning procedure to don sterile uniform components. The
usual order of gowning should be hair cover, shoe cover, mask, sterile
Page 14 of 81
DATE: 04-25-2014
hood, sterile boots, and sterile coverall.

Care should be taken to choose gowning that does not expose any skin
to the aseptic clean room environment.
Sterile non powdered gloves should be used at all times. A new pair of
non-powdered sterile gloves is to be put on as the last uniform
component.
70% sterile alcohol can be used to sanitize the gloves if possible. Note
that alcohol is not a sterilant.
If possible post the gowning procedures in the gowning room or area to
help individuals follow the correct order of gowning.
Should an analyst find it necessary to leave the room, he/she should
discard all gowning components and put on new ones upon re-entry.
If an individual scheduled to enter the clean room for analysis feels sick or
is sunburned, he/she should talk to his/her supervisor to postpone entry
into the clean room. Analysts that have also undergone any surgeries or
procedures (tattoos) that compromise the skin should also postpone
entry into the clean room until they have fully healed.
2. Sample Preparation
Repeat disinfection procedure using filtered sterilized 70% alcohol immediately prior
to placing product primary containers in a working certified laminar flow hood. Allow
all disinfected containers to completely air dry (recommended for at least ½ hour) in
the laminar flow hood prior to opening for analysis. Alternatively if conducting the
testing in an isolator, place the disinfected items into the isolator and proceed with
the local procedures for the proper decontamination of the interior of the isolator.
3. Room Cleaning After Analysis

Remove inoculated tubes of media and all controls from the analytical area by
putting them in the pass-thru or on a stainless steel cart used for transporting
materials in and out of the clean room.
After analysis, all sample containers, wrapping paper, used equipment and tools
are to be removed from the clean room before the analyst exits.
Sample containers used in the analysis should be replaced in original outer
containers for storage as part of the reserve sample.
An inventory sheet should be filled to account for the amount of material
available for the next analysis, if required by local procedures.
Disinfect working area before exiting the clean room.
4. Clean room disinfection and surface monitoring should be done on a routine

bases. The frequency is to be determined by the local lab policy.
E. Method of Analysis
1. Filtration
Follow the current edition of the USP for the amount of sample to be tested.
2. Direct Inoculation
Follow the current edition of USP for the amount of sample and media to be used.
For example: Use 200 ml of each medium when analyzing solid form products. If the
Page 15 of 81
DATE: 04-25-2014
membrane filter method is unsuitable, certain liquids may be tested by direct

inoculation method.
3. Devices
All devices except devices with only the pathways labeled as sterile are to be tested
by direct inoculation method.
4. Incubation of Sterility Test Media

a. Incubate Fluid Thioglycollate (THIO) at 32.5 ± 2.5oC. Do not shake or swirl
test media during incubation or during examination to minimize aeration of
this broth.
b. Incubate Soybean-Casein Digest Broth (SCD) at 22.5 ± 2.5oC. Gentle

swirling, on occasion (3-5 days) is acceptable to increase aeration of media.
c. Incubation period for THIO and SCD:
i. Not less than 14 days except for products sterilized using ionizing
radiation. If tubes are not read on day 14 due to holiday or weekend
then record the results, even if positive, on the first available day to
observe the tubes.
ii. Additional incubation time may be warranted if the analyst is made

aware of sterilization processes other than heat or filtration (e.g. 30
days (at minimum) for products sterilized using ionizing radiation).
F. Analysis of Purified Cotton, Gauze, Sutures and Surgical

Dressings
The USP method for analysis of purified cotton, gauze and surgical dressings does not
require testing the entire unit. The USP method for the analysis of purified cotton, gauze
and surgical dressings calls for portions no larger than 500 mg, from the innermost part
of the unit, to be tested in each medium. The entire unit shall be tested for individually
packaged single-use articles.
1. Gauze, Purified Cotton, Sutures and Surgical Dressings

a. Using media containers as large as quart jars analyze entire unit of product.
b. If unit is too large for the container, analyze as much of unit as can be placed
in container and covered by the medium.
Due to the limited availability of media and glassware that occurs when a large
number of samples are collected for analysis, it would be expedient to perform
compositing of device samples. This would allow the samples to be completed in
a timely manner.
2. Compositing of Medical Devices
Page 16 of 81
DATE: 04-25-2014
a. Devices may be tested in composites (2-4 units/composite) as long as all

units are completely immersed in the medium and all composite units are of
the same lot number.
b. Perform the Method Suitability Test to ensure that the growth of

microorganisms is not inhibited by the number of units used in a composite.
Compositing cannot be performed if the sample does not pass the Method
Suitability Test.
G. Control Systems
The objective of a control system is to ensure the sterility, within designated limits, of all
tems, media, rinsing fluids, and equipment used in a sterility test. The control systems
which will accompany all sterility analyses are outlined below.
1. System Control
A "system control" is used to demonstrate maintenance of sample integrity during all
analytical manipulations. Any piece of equipment that comes in contact with the
product under analysis, along with any manipulations by the analysts, must be
controlled. Thus, all equipment, fluids, and culture media for the "system control"
must be handled in a manner which duplicates, as closely as possible, the
manipulations of the actual sample being analyzed. All materials used as system
controls must be sterilized by the analyzing laboratory. However, the method of
sterilization need not be the same as for the product, but must render the material
sterile.
The first choice for the system control is the actual product, if enough test units are
available. When complex medical devices must be sacrificed in order to design a
suitable sterility test, consider using them for a system control after cleaning,
repacking and sterilizing.
When there are viable alternatives, a product unit should not be sacrificed for use as
a system control if this will reduce the number of units available for sterility testing
below USP requirements or FDA policy requirements, except as provided in the
preceding paragraph. If using a product unit would reduce the subsamples
examined below the number required by USP or FDA policy, the analyzing
aboratory should prepare a control from other material than a unit of the sample
product whenever possible.
a. Membrane Filtration: The filter funnel furthest from the vacuum source
connection on each manifold used in the test is used for the system control.
Alternatively if a closed canister system is used to conduct the sterility test a
canister set from the same lot used during the analysis should be used for the
system control.
i. Filterable Materials (liquids, soluble solids, etc.)

Use material similar to product under test. Control material must be of
the same volume, and similarly packaged as test product. Filter-sterilized
and autoclaved Peptone water (USP Fluid A) may be useful for this
purpose in many cases.
Page 17 of 81
DATE: 04-25-2014
ii. Devices with sterile Fluid Pathway

Use tubing or other containers similarly fitted with needles, valves,
connectors, etc., as product under test. Use USP Fluid D to flush lumens.
b. Materials tested by direct inoculation (devices, insoluble solids, and other

non-filterable materials)
Use materials similar in size, shape, and texture, and similarly packaged as
product under test. Duplicate as nearly as possible pertinent, unusual
features that may reflect on the credibility of the sterility test.
In designing "system controls" for sterility testing, care must be taken to

duplicate the sample product for most aspects, as nearly as possible. Be
novel and innovative to meet this requirement, and make the system control
meaningful.
2. Equipment Controls
All items listed below will be controlled individually. One item from each autoclave lot
of equipment is tested in each medium used in the test. Therefore, for a sample
tested in THIO and SCD, one item from each sterilizer load (oven or autoclave) is
tested in each medium giving a total of two controls for each forceps, syringe, etc.,
used in the test.
Forceps
Syringes
Scissors
Scalpels
Swabs
Pipettes
Membranes (dry, directly from the package). If membranes are sterilized
in place, this control may be omitted.
Hemostats
Other special items that may be required by a specific test.
3. Media and Rinse Fluid Controls

a. Uninoculated media of the autoclave load(s) as media used in the sample is
controlled.
b. Portions of each rinse fluid which is used in sample test by membrane

filtration are transferred to media (i.e., 10 ml into THIO and SCD).
If rinse fluids are drawn from bulk containers during sterility tests, individual
controls may be omitted. Controls for these materials are accomplished as
part of the "system control" for each manifold. This will also include
membrane cutters, and other items that contact the product but cannot be
individually controlled.
4. Environmental Controls
Page 18 of 81
DATE: 04-25-2014
a. Open Media Controls

Tubes of each medium (THIO and SCD) used in the sterility analysis are
exposed to the immediate environment of the test (e.g., laminar flow hood) for
the duration of the test.
b. Agar Settling Plates

Glass or plastic Petri dishes containing an effective non-selective medium
(based on test requirements) are exposed in the hood for a period not to
exceed one hour during the analysis. If the analysis exceeds one hour, use
fresh plates for each subsequent hour. Dishes of medium are sterilized by
the analyzing laboratory before use.
Plates should be incubated for 48 hours at 35 C, and an additional 5 days at

25oC in order to detect mold contamination.
c. Controls within an Isolator

When conducting the sterility test within an isolator if the isolator has been
designed to allow for a connection to an air sampler and particle counter this
sampling may be performed for the duration of the sample analysis in lieu of
the environmental samples described above. If the isolator is unable to
accommodate an air sampler and/or particle counter or the instruments are
unavailable the environmental controls described in section a. and b. should
be used.
5. Personnel Monitoring
Personnel monitoring may be performed after analysts conclude sterility testing and
prior to exiting the aseptic clean room. The analyst shall use general media touch
plates to monitor the sterile condition of their clean room attire and to ensure aseptic
techniques were followed.
For example, a minimum of five touch plates should be used for the following
personnel gowning sites:
RH glove finger tips.
LH glove finger tips.
Chest
Left Forearm
Right Forearm
General media touch plates will be incubated for 2 days at 35ºC.
NOTE: The numerical values for personnel monitoring limits and specifications are
established on the basis of a review of actual findings within the facility. All isolates
are to be identified to ensure that the analyst did not contaminate the sample.
Analysts should be sanitizing their gloves throughout the sterility analysis and
changing gloves when needed. However, changing gloves prior to performing
personnel monitoring is unacceptable.
H. Sub-culturing Primary Media

Daily observations of primary test media (THIO and SCD) containing product should be
performed without unnecessary disturbance. All handling of positive tubes, streaked
Page 19 of 81
DATE: 04-25-2014
plates, or subsequent inoculations of additional media will be done outside the clean
room. These culture transfers are to be performed within a HEPA filtered biosafety
cabinet or equivalent outside the clean room which has been cleansed with an effective
antimicrobial agent. The analyst should be gowned with at least sterile gloves, sterile
sleeves and a mask to minimize any possible cross contamination.
1. Record on Analyst's worksheets the day the primary isolation media, Fluid
Thioglycollate Broth (THIO), or Soybean-Casein Digest Broth (SCD) is "positive,"
and inform supervisor. Streak tubes on the day they first appear positive and again
at 14 days to determine the presence of other possible slow-growing (i.e., fungi)
microorganisms.
2. Within a HEPA filtered biosafety cabinet or equivalent outside the clean room, streak
positive tubes onto Modified Soybean-Casein Digest Medium [SCD broth + 1.5%
agar] (Modified SCDA).
a. Fluid Thioglycollate Broth: Streak two plates; incubate one aerobically, and
one anaerobically, each at 32.5 ± 2.5oC. NOTE: It is suggested to pipet an
aliquot of media from close to the bottom of the tube to maximize the
recovery of strict anaerobes.
b. Soybean-Casein Digest Broth: Streak one plate; incubate aerobically at 22.5

± 2.5oC.
3. All streaked plates are incubated for a period at least as long as the time required for
growth in original isolation media (THIO or SCD). However, incubate plates no
onger than seven days.
4. Pick a single colony, representative of each colony type, as follows:*

a. Plates Streaked with Primary Fluid Thioglycollate Broth
i. Anaerobic Modified SCDA Plate:

Using growth from the single colony pick, inoculate duplicate Modified
SCDA slants (as in step ii below), and one SCDA slant. Incubate one
aerobically and one anaerobically. Note the oxygen requirements of the
pure culture isolate from this test, and preserve the anaerobic slant
culture by lyophilizing or ultra-freezing. Determine the Gram stain
reaction of the pure culture isolate from the USP SCDA slant incubated
anaerobically.
* If discrete colonies are not present due to overgrowth on isolation

plates, pick representative colonies from such plates and re-streak to
another plate. Alternatively, transfer a pick back to a new sterile tube of
original isolation broth, and when growth develops, re-streak to obtain
discrete colonies.
ii. Aerobic Modified SCDA Plate:
Page 20 of 81
DATE: 04-25-2014
Following the protocol above for Anaerobic Modified SCDA Plate, using
growth from the single colony pick, inoculate duplicate Modified SCDA
slants, and one SCDA slant. Incubate one Modified SCDA slant
aerobically and one anaerobically. Note the oxygen requirements of the
pure culture isolate from this test, and if indicated, preserve the culture
from the aerobic modified slant. Determine the Gram reaction from the
USP SCDA slant.
b. Soybean Casein Digest Broth

From the aerobic Modified SCDA plate, or from the anaerobic plate if aerobic
growth fails to develop, pick a single colony representative of each type to
duplicate Modified SCDA slants. Incubate one Modified SCDA slant
aerobically and one anaerobically. Report the oxygen requirements of the
aerobic or anaerobic slant culture. Determine the Gram stain reaction of the
culture isolate from the sugar-free USP SCDA agar slant.
5. Identify each isolate as to oxygen requirement, gram reaction, presence or absence

of spores, and whether the organism is a yeast or mold. If possible, the isolate can
be further identified to genus and species using a rapid identification kit such as
VITEK or API. Additionally DNA sequencing may be used when necessary. Follow
the subculture chart in the WS section.
strict anaerobe
strict aerobe
facultative anaerobe
facultative aerobe
gram-positive organism
gram-negative organism
yeast or mold
spore forming organism
I. Preservation of Isolates
Preserve by lyophilizing (if possible) or ultra-freezing one representative culture of each
identified isolate. Identify each isolate with sample number, subsample number, initials,
and date. Also identify the pick number if more than one isolate was picked from a
single plate.
If by observing colony morphology, Gram stain reactions, and other growth

characteristics, it appears that all isolates generated from the foregoing protocol are the
same organism, only one culture should be preserved from each subsample. If more
than one type of organism is demonstrated from one or both primary isolation media,
based on oxygen requirements, Gram reaction and colonial morphology, preserve each
type by lyophilizing (if possible) or ultra-freezing.
1. Molds
Special procedures should be discussed with the supervisor for isolating molds by
the preceding protocol. If filamentous fungi are suspected, streak positive tubes
within a HEPA filtered biosafety hood or equivalent to minimize air-borne spreading
Page 21 of 81
DATE: 04-25-2014
of conidia (i.e., spores). This will minimize contamination of work environment and
risk to analyst.
2. Subculture of original test tubes to another set of tubes containing the same
medium:
In case the product reacts with the media and shows turbidity due to the nature of
the product, subculture the original tubes after 14 days of incubation. Incubate both
original and subculture vessels for not less than 4 days.
J. Worksheet Notations
1. Recording Dates
a. Form FD 431, Front Page
Block 4, Date Received- Enter in this space the date the sample was
received from the sample custodian. This date must be the same as that
entered in the FACT system screen for sample received.
All laboratory data, observations and findings resulting from the analysis of a
sample will be recorded using worksheets, continuation sheets and
attachments as described in the ON LINE ORA LAB MANUAL (LPM), Vol. III,
Sec. 3 (Recording of Results- Analyst Worksheets). These include;
The analyst worksheet (FD-431)

The general continuation sheet (FD-431a)
All preprinted and computer generated worksheets
b. Form FD 431a, General Continuation Sheet

On modification of this form resulting in a pre-printed worksheet used for daily
observations of sample tests and controls, the following information should be
included:
In the space provided at the top of the Form enter the date primary test media
were inoculated with product, which also indicate the date when incubation
began.
Describe whether each test was a subsample or a composite.
At space provided of each column for notation of "Daily Observations," enter

the date observations were made, and validate with analyst's initials.
For the days that observations were not made, the following entries will be
made:
Weekend - W
Holiday - H
Leave - L
Other - O (describe)
2. Product Turbidity in Primary Test Media
Page 22 of 81
DATE: 04-25-2014
When a direct inoculation protocol is used for sample products that cause turbidity of
the medium upon inoculation, the following systems for recording daily observations
of incubating media should be used.
a. Record "T" for any subsample which is turbid due to product-medium mixture.
b. On the daily observation page, indicate the meaning of "T" as: "T = product
induced turbidity”.
c. At the end of the initial 14 days of incubation, transfer portions of the medium
(not less than 1 ml) to fresh vessel of the same medium and then incubate
the original and transferred vessels for not less than 4 days. Note: Follow the
current edition of the USP for any changes concerning subculturing and
incubation of turbid samples.
d. Examine original product inoculated media and the subculture media for
growth daily when possible for not less than 4 days of incubation and record
the results on a new daily observation continuation sheet.
K. Preservation and Revival of Cultures

The most common methods for preserving cultures are Lyophilization and Ultra-
freezing. ATCC recommends the following procedure for the rehydration of
Freeze-Dried cultures. In all cases follow any specific manufacturer’s instructions
if provided.
1. Opening Ampoules
Score the ampoule once briskly with a sharp file about one inch from the tip.
Disinfect the ampoule with alcohol-dampened gauze. Wrap gauze around ampoule
and break at the scored area. Care should be taken not to have the gauze too wet
or alcohol could be sucked into the culture when the vacuum is broken.
2. Opening Vials
Care should be taken so that the freeze-dried material does not aerosolize and
contaminate the working area.
3. Rehydrating the freeze-dried culture

a. Rehydrate the culture using the medium and incubation temperature
specified for that organism.
b. Aseptically add 0.3 to 0.4 mL of the specified medium to the freeze-dried

culture with a sterile Pasteur pipette.
c. Mix well and transfer the mixture to a test tube containing 5-6 mL of the
recommended broth.
d. Cultures in stoppered vials should be rehydrated with 0.5 mL of the

appropriate broth, mixed and transferred to 5mL of the recommended broth.
Page 23 of 81
DATE: 04-25-2014
e. Mold cultures should be rehydrated with sterile water since fungi must be
soaked for at least 30 minutes before being transferred to an agar surface.
f. Incubate at the appropriate temperature.
g. Most cultures grow in a few days. However, some may exhibit a prolonged
lag phase and should be given twice the normal incubation before discarding
as not viable.
Page 24 of 81
DATE: 04-25-2014
Chapter 4: Investigating USP Sterility Testing Failure

INTRODUCTION:
When microbial growth is detected in a pharmaceutical or medical device
product, the product is considered non-sterile, pending an investigation.
Because of the public health importance of a non-sterility finding, preliminary
results should be reported by your laboratory management, without delay, to
ORS and the appropriate Center (e.g., Office of Compliance/OCTEC).
Concurrently, a laboratory review should be conducted to answer the following
question: Was the result true product contamination or was there a clear
laboratory error that caused contamination of the sample during the analysis?
The Out-of-Specification (OOS) investigation will review and document that the
test results are based on sound laboratory operation.
INVESTIGATIONS:
Whenever a sterility positive occurs, lab supervisors are responsible for starting
the investigation immediately. Four factors should be evaluated in the basic
investigation:
1. Equipment:
Determine whether equipment malfunctioned or was not operated

properly. If a malfunction occurred, determine whether it was likely to
cause the contamination. Determine if any checklists or logs indicate that
the ISO 5 device was in good state of repair at the time of the sterility test.
Be aware of the most likely failure modes in the equipment (e.g., laminar
flow hood, glovebox, or isolator) used.
2. Adherence to Analytical Method:
Determine whether there were any anomalies or deviations from the

analytical method. Adherence to method should be verified at the time of
analysis, and any major breach of sterility test procedure should also be
documented at that time. If any method breaches occurred, determine
whether it was likely to cause the contamination. Be aware of any possible
weaknesses in the test method (e.g., kit, manifold, etc.) used.
3. Analyst:
Evaluate the analyst’s qualifications, including proficiency, training record,

and experience. Also note whether the sterility testing practice of the
analyst was observed during this or a recent analysis.
4. Cleanroom and ISO 5 (Class 100) Environmental Conditions:
Page 25 of 81
DATE: 04-25-2014
Determine if disinfection/decontamination of the ISO 5 device was

properly done.
Determine whether there was adverse environmental data. Note that a

negative control failure, on its own, is not necessarily cause for
invalidating a result. If a negative control was contaminated, consider
whether the microbe identified is similar to, or the same as, the sterility
test isolate and also consider whether there are other adverse
environmental trends.
It is advisable to summarize this review process in a standard report, and maintain a

sufficient record to reflect that these areas were investigated. In addition to the four
considerations listed above, overall cleanroom design is also an important
consideration. There are differences in the construction, configuration, and material
flow of FDA field laboratories cleanrooms. There may be differences in size, number of
rooms, shape, air handling system, pass through autoclaves, gowning room
accommodations (sink, HEPA filtration, adequate space, bench, etc.). Proper practices
and conditions should be assured by mitigating contamination hazards potentially
presented by layout and other design provisions. These include appropriate procedures
for room and material disinfection, proper cleanroom uniforms (disposable or reusable),
sample preparation area, etc. These and other factors can play a major role in
preventing sample contamination during product handling and testing. Such cleanroom
risks can be prevented by the design of ISO 5 testing equipment. Equipment that
provides barrier protection can mitigate risks of the surrounding cleanroom environment.
If an investigation finds that the conduct of the analysis included errors or events that
caused the test specimens to be contaminated by the lab environment, the Sterility Test
result would be invalid and the substandard laboratory practice should be corrected to
prevent this problem from recurring. For more information on how to judge
investigational findings to make this evaluation, see Section XI.C1.and 2 of FDA’s
guidance on Sterile Drug Products Produced by Aseptic Processing for principles and
expectations for investigating a sterility positive.
http://www.fda.gov/downloads/Drugs/.../Guidances/ucm070342.pdf
Also see the current version of USP <71>, which provides some guidance on
investigations under Interpretation of Data.
CONCLUSION:
This suggested list of areas and conditions to review should not be considered as
comprehensive. Additional areas of review may need to be added based on
some of the unique features or procedures employed by individual FDA ORA
laboratories.
Page 26 of 81
DATE: 04-25-2014
Chapter 5: Bacterial Endotoxin Testing

This chapter of the Sterility Analytical Manual is intended to supplement the methodology
procedures found in the USP <85> BACTERIAL ENDOTOXINS TEST.
The scope of the initial portion of this section includes the Gel Clot Method. The organization of
this chapter will follow the format of the USP chapter.
Kinetic assays are addressed in the second part of this section. Kinetic assays involve a
change in color or turbidity (depending on the assay reagents used) over time. The time for
change is inversely proportional to the concentration of endotoxin in the solution, i.e. more rapid
change at higher endotoxin concentrations. An instrument is used to read the changes. These
assays are rapid and sensitive, allowing large numbers of samples to be screened quickly.
However, the gel-clot method is the reference method per USP and should be used if there are
any doubts or disputes, unless otherwise indicated in the monograph for the product tested. In
case of both procedures being performed, the gel clot method is considered the reference
method and overrides any kinetic assay results.
New microbiologists should review the references at the end of this chapter.
A. Gel Clot Method

1. Reference Standard and Control Endotoxins
The potency of the control standard endotoxin (CSE) with respect to the reference
standard endotoxin (RSE) is determined by the CSE manufacturer. This information
s found in the associated package insert and need not be repeated
NOTE: Follow manufacturers’ recommendations in the storage, reconstitution, and

preparation of CSEs, lysates, and other LAL reagents. In case of a dispute, final
decision is based on results obtained with the USP Endotoxin RS.
The field laboratories are encouraged to mix the CSE for at least 5 minutes and at
east 1 minute between dilutions.
2. Preparatory testing
Run appropriate negative controls with each sample tested. This assures that the
equipment and solutions used in the test contain no extraneous endotoxin. For
example, a beaker full of pyrogen- free test tubes stored for an extended period
should be screened with a negative control for each sample tested. Record these
results on the worksheets.
When using commercially purchased pyrogen-free water for product dilutions be sure
to transfer a working volume from the original stock container to an individual
pyrogen-free test tube or flask in order to minimize back contamination. Run a
negative control for the working volume for each sample run.
NOTE: Pyrogen free pipettes, micropipettor tips, test tubes, and other accessories
are commercially available. Include appropriate negative controls to assure they do
not contain extraneous endotoxin.
Page 27 of 81
DATE: 04-25-2014
3. a. Test for Confirmation of Labeled LAL Reagent Sensitivity

Prior to use in the test, prepare one single dilution series. Inoculate four replicates
from each tube. Multiple dilution series are not required.
The labeled LAL reagent sensitivity must be confirmed before a new LAL lot is
introduced into the laboratory. Prepare control standard endotoxin having at least
four concentrations equivalent to 2 λ, λ, 0.5 λ, and 0.25 λ in quadruplicates. The
geometric mean of the endpoints must be within the limits of labeled claim. The
acceptable variation is one half (0.5 λ) to two times (2 λ) the labeled sensitivity (λ).
3. b. Inhibition or Enhancement Test

The suitability of the test results for bacterial endotoxin require an adequate
demonstration that specimens of the article or of solutions to which the test is to be
applied do NOT of themselves inhibit or enhance the reaction or otherwise interfere
with the test.
USP states to perform this test “on aliquots of the specimen… in which there is no
detectable endotoxin”. However, this characteristic of the product cannot be
ascertained prior to the analysis because the specimens are unknown samples.
Because of this limitation, any positive result below the 0.5 lambda level may not be
an enhancement trait of the product, but instead a positive reaction due to
contamination in the sample. The evidence for this conclusion should be obvious
with the results of the assay tubes containing product only.
A large percent of small volume parenterals appear to be inhibitory to the LAL gel-
clot method because of low pH, or some excipient / active component of the product.
In order to expedite the neutralization of this interfering trait, determine the lowest
product dilution overcoming the interference but still within the Maximum Valid
Dilution. The detailed description of this protocol is delineated in LIB No. 2433 (July
25, 1980), “A condensed procedure for diluting product in determining compatibility
with the Limulus Amebocyte Lysate test for endotoxin”. In addition, the use of
neutralizers such as sodium laurel sulfate or pyrospersetm has also been described
(see references).
NOTE: Contact LAL manufacturers for recommendation of commercially available

neutralizing buffer to be used with their LAL kits.
4. Test Procedure
The storage and mixing of samples prior to analysis may affect recovery of endotoxin
contamination. Sample (product) bottles should be vigorously shaken prior to
analysis, preferably on a vortex (see reference for supporting evidence for this step).
A minimum of 30 seconds to 1 min on the vortex is recommended for each product
unit.
5. Endotoxin Calculation
Calculate endotoxin concentration per the USP Bacterial Endotoxins test chapter.
Note: Adjust the final endotoxin value taking into account the volume of the rinse
solution used in the extraction procedure.
6. Compositing Samples
Page 28 of 81
DATE: 04-25-2014
The Bacterial Endotoxin test <85> does not directly address the issue of combining
product units (compositing). The risk of unit composites is that one unit (vial,
ampoule, etc) may have bacterial endotoxin contamination at a higher level but the
dilution of this one unit with endotoxin-free units of product may reduce the
detectable level of endotoxin below the sensitivity of the lysate or dilute the level of
endotoxin below the acceptable monograph level. Therefore, when using a
composite format for screening drug products for endotoxin it is important to
adjust the MVD calculation to account for this reduced lysate sensitivity.
Secondly, when compositing is performed for product screening, if a positive
result is detected a repeat test is acceptable under the conditions stated by the
Interpretation section of the USP chapter.
It would be advisable when performing the repeat test from a composite mixture that,
if remaining product is available and had been opened aseptically under controlled
conditions, the repeat test be performed on the original individual units. It is strongly
advised that the individual units be adequately shaken to assure that the endotoxin is
re-suspended back into solution before taking the sample test aliquot. If any of the
original individual units fail the USP test at this point, the compendium does
not allow any additional repeat testing unless the test can be proven not to be
suitable as defined by the USP chapter.
7. Frequently asked questions: (derived from the FDA Industry Guidance for Pyrogen
and Bacterial Endotoxin testing)
Question 1: Can FINISHED product units (vials, ampoules, pre-filled syringes, etc)
be "Pooled" into a composite and screened for bacterial endotoxin?
Response 1:
Yes. With some exceptions (see below), finished drug product units may be pooled
into a composite sample and assayed for bacterial endotoxins. The composite
sample may be represented by the entire unit or partial aliquots (equal volumes) of
finished product containers from one manufactured lot of aqueous-based
pharmaceuticals. Pooling would generally be accepted for small-volume parenterals
(those with volumes of 100 mL or less) as long as the MVD is adjusted to a
proportional, lower value because of the potential for diluting a unit containing
harmful levels of endotoxins with other units containing lower, less harmful, levels of
endotoxins. This “adjusted MVD” is obtained by dividing the MVD computed for an
individual sample by the total number of samples to be pooled. FDA suggests
pooling no more than three units per composite in keeping with the concept of testing
representative beginning, middle, and end finished product containers. If this
reduction in MVD results in an inability to overcome product-related assay
interference because of an insufficient dilution, then the samples should be tested
individually.
Finished medical devices may also be pooled into a composite sample and assayed
for bacterial endotoxins. Testing for medical devices should be conducted using
rinsing/eluting and sampling techniques as described in ISO 10993-1 and ISO
10993-12, as also used for inhibition/enhancement. Sampling can be adjusted for
special situations. After a suitable eluate/extract pool is obtained from a finished
Page 29 of 81
DATE: 04-25-2014
production lot, this pooled extract should be kept under conditions appropriate for
stability until it is tested in duplicate.
FDA recommends that pooled samples be a composite of aseptically removed

aliquots (after at least 30 seconds of vigorous mixing) from each of the product
containers. In this way, the original, individual containers will be available for possible
retesting in the event the pooled sample displays an OOS result.
Some product types should not be pooled. Two examples are drug products that
have an initial low MVD (see discussion above of “adjusted MVD”) and products that
are manufactured as a suspension, because sample aliquot homogeneity may
present significant interference issues.
Question 2: Can INTERMEDIATE (IN-PROCESS) sample aliquots be "pooled" into a

composite and screened for bacterial endotoxin?
Response 2:
FDA does not recommend pooling in-process samples from different in-process
stages of the manufacturing process because it may be difficult to ensure the
homogeneity of these materials.
Question 3: Retesting when test failure occurs:
Response 3:
When conflicting results occur within a test run, the analyst should consult USP
Chapter <85>, Gel Clot Limits Test, Interpretation, for guidance on repeat testing. As
specified in Chapter <85>, if the test failure occurred at less than the maximum valid
dilution (MVD), the test should be repeated using a greater dilution not exceeding the
MVD. A record of this failure should be included in the laboratory results. If a test is
performed at the MVD and an out-of-specification (OOS) test result occurs that
cannot be attributed to testing error, continue product dilution until the actual
endotoxin concentration can be calculated. These results should be recorded on
your worksheets.
B. Kinetic Assays
Kinetic assays are quantitative assays used for the detection of bacterial endotoxins.
Kinetic assays may utilize turbidimetric or chromogenic formats.
This section provides procedural information that can be applied to the QCL
chromogenic assay. The Kinetic QCL Chromogenic Assay may be purchased as a kit.
A certificate of analysis is provided with each kit along with testing procedures, control
standard endotoxin, Limulus Amebocyte Lysate (LAL), and pyrogen free water. Other
materials such as pyrogen free pipettes, micropipettor tips, test tubes, and 96-well
microplates may be purchased from various vendors.
1. Kinetic QCL Assays: The kinetic QCL software program is designed to run the
following assays.
i. Initial qualification of the testing analyst
Page 30 of 81
DATE: 04-25-2014
ii. RSE/CSE assesses potency of control standard endotoxin (CSE) in terms

of reference standard endotoxin (RSE)
iii. Inhibition or Enhancement Test
iv. Sample Test
The initial qualification assay verifies the proficiency of the analyst operating
the Kinetic QCL program and equipment. The initial qualification assay may
also be used to qualify each new lot of kinetic QCL test kits.
The RSE/CSE assay may be used to compare the potency of the CSE with
the concentration of the RSE. Normally, the RSE/CSE assay does not need
to be performed, unless there is reason to believe the values in the
manufacturer’s certificate of analysis (COA) are not correct.
The Inhibition or Enhancement assay must be run on all samples having
positive test results, and on all sample tests that exhibit inhibition.
The routine assay program is designed to test unknown samples for bacterial
endotoxins. Samples collected for LAL analysis should be run using the
routine assay program, after taking the other three programs into
consideration.
2. Procedure
Perform the assay according to the instructions that enclosed with the LAL test kit.
Additional instructions may be found in references 8 and 9 listed below.
C. Medical Devices
The analytical approach for testing medical devices is sparsely covered in USP. A
collaborated method prepared by an FDA field laboratory is available for extraction of
endotoxin from devices. Modification of testing may be necessary depending on the
product configuration. Analytical validation of the final version should be conducted by
the responsible laboratory. The protocol is summarized below for convenience:
1. Extraction and analysis of Endotoxin from Medical Devices:

This section applies to sterile disposable syringes and cartridges, transfusion and
infusion assemblies, implants, etc.
a. Preparation of 1% SLS solution
Prepare a 1% stock solution by placing one (1) gram of sodium lauryl sulfate
(SLS) into a depyrogenated glass flask and add 99 ml of pyrogen free water.
Allow the SLS to fully dissolve. This should be followed by filtration through a 10,
000 MW depyrogenation membrane filter into a pyrogen free glass or plastic
container.
b. Equipment needed: Ultrasonic bath with a range of 150 to 440 watts.
c. Extraction procedure
i. Dilute 2mL of 1% SLS stock solution to 20mL (0.1%) using LAL reagent
water in a 20 x 150 mm screw-cap tube.
Page 31 of 81
DATE: 04-25-2014
ii. Dilute 1.5mL of the 0.1% SLS solution to 15 mL (0.01%) using LAL
reagent water in a 20 x 150 mm screw-cap tube.
iii. Prepare the appropriate number of tubes (one tube for each device)
and one as a negative / system control. Preheat in a waterbath to
40°C.
iv. Aseptically remove the device from its packaging and cut it diagonally
into pieces less than 5mm in length. Metal pieces such as needles and
luer-locks should be tested whole.
v. Place all pieces into the 20 x 150 mm tube containing 15 mL of

preheated (40°C) 0.01% SLS rinse solution.
vi. Vortex the tubes for 30 – 60 seconds or until all pieces of the device are
immersed in the rinse solution.
vii. Sonicate the test containers for 60 minutes (wattage range 150 – 480
watts) at 40°C. Do not sonicate more tubes than can be vortexed within
15 minutes of completion of the sonication. Make sure the water in the
sonicator covers the rinse solution in the 20 x 150 mm tubes. Do not
allow the water in the sonicator to exceed 50°C.
viii. Vortex the tubes for 2 minutes. Remove a portion of the eluate (5 – 10
mL) for LAL testing. If the eluates are not tested immediately for
endotoxin, they should be refrigerated. All eluates must be tested within
24 hours of extraction. Prior to analysis vortex at least one minute.
ix. Screen all eluates for endotoxin content using 10-fold dilutions to the
10-3 dilution. Prepare dilutions using pyrogen free water in pyrogen
free tubes. Positive and negative controls must be run simultaneously
with all LAL tests.
x. Quantitate the eluate by making 1:2, 1:4, 1:6, 1:8 dilutions of the last
positive 10-fold dilution in step 8.
D. Endotoxin References
1. United States Pharmacopeia (USP) Chapter <85> Bacteria Endotoxins Test. Official
December 1, 2012
2. The U.S. Food and Drug Administration, Guidance for Industry, Pyrogen and
Endotoxins Testing: Questions and Answers, June 2012
3. Guilfoyle, D.E., Yager, J.F. and S.L. Carito. 1989. The effect of refrigeration and
mixing on detection of endotoxin in parenteral drugs using the Limulus Amebocyte
Lysate (LAL) test. J. Parenteral Science & Technology. Vol 43, No 4, p. 183-187
4. Novitsky, T.J., Schmidt-Gengebach, J. and J.F. Remillard. 1986. Factors affecting

recovery of endotoxin adsorbed to container surfaces. J. Parenteral Science &
Technology. Vol 40, No 6, P. 284-286
Page 32 of 81
DATE: 04-25-2014
5. Twohy, C.W., Duran, A.P. and J.T. Peeler. 1986. Extraction of bacterial endotoxin
from medical devices. J. Parenteral Science & Technology. Vol 40, No 6, p. 287-291
6. Guilfoyle, D.E. and T.E. Munson. 1982. Procedures for improving detection of
endotoxin in products found incompatible for direct analysis with Limulus amebocyte
lysate, in Endotoxins and Their Detection with the Limulus Amebocyte Lysate Test.,
Watson, S.W., Levin, J., and Novitsky, T.J. (eds), Alan R. Liss Inc., NY, p. 79-90
7. Guilfoyle, D.E., Munson, T. and J.P. Schrade. 1981. Use of Pyrospheretm for
Reducing Product Interference in the Limulus Amebocyte Lysate Test, Laboratory
Information Bulletin, U.S. Food and Drug Administration, Washington D.C., No. 2503
(115), p. 1-8
8. Tepedino, A., Guilfoyle, D.E. and Munson, T. 1980. A Condensed Procedure for
Diluting Product in Determining Compatibility (Inhibition / Enhancement test) with the
Limulus Amebocyte Lysate Test for Endotoxin, Laboratory Information Bulletin, U.S.
Food and Drug Administration, Washington D.C., No. 2433, p. 1-17
Page 33 of 81
DATE: 04-25-2014
Chapter 6: Particulate Matter
This chapter is intended to supplement the methodology procedures found in the USP <788>
PARTICULATE MATTER IN INJECTIONS and USP <789> PARTICULATE MATTER IN
OPHTHALMIC SOLUTIONS 1. Particulate matter consists of mobile, randomly-sourced
extraneous substances, other than gas bubbles, that cannot be quantitated by chemical
analysis due to the small amount of material that it represents and heterogeneous composition.
Injectable solutions, including solutions constituted from sterile solids intended for parenteral
use, is essentially free from particulate matter observable on visual inspection. The tests
described herein are physical tests performed for the purpose of enumerating sub-visible
extraneous particles within specific size ranges.
All large-volume injections for single-dose infusion and those small-volume injections for which
the monographs or product specifications specify such requirements are subject to the
particulate matter limits set forth for the test being applied, unless otherwise specified in the
individual monograph or product specification.
Not all injection formulations can be examined for particles using the light obscuration method.
Any product that is not a pure solution having clarity and viscosity approximating those of water
may provide erroneous data when analyzed by the light obscuration counting method. Refer to
specific monographs when a question of test applicability occurs. The microscope counting
method may be used to analyze such materials. In some instances, the viscosity of a material
to be tested may be sufficiently high so as to preclude its analysis by either method. In this
event, a quantitative dilution with an appropriate diluent may be made to decrease viscosity, as
necessary, to allow the analysis to be performed.
A. Light Obscuration Particle Count Test

The test applies to large-volume injections labeled as containing more than 100 mL and
single-dose or multiple-dose small-volume injections labeled as containing 100 mL or
less that are either in solution or in solution constituted from sterile solids, where a test
for particulate matter is specified in the individual monograph or drug product
specification. It counts suspended particles that are solid or liquid. Products for which
the labeling in the individual monograph specifies that the product is to be used with a
final filter are exempt from these requirements.
1. Test Apparatus
The apparatus is an electronic, liquid-borne particle counting system that uses a light-
obscuration sensor with a suitable sample feeding device. A variety of suitable devices
of this type are commercially available. It is the responsibility of those performing the test
to ensure that the operating parameters of the instrument are appropriate to the required
accuracy and precision of the test result and that adequate training is provided for those
responsible for the technical performance of the test. It is the responsibility of the user to
apply various methods of standardization applicable to the specific instrument.
Critical operational criteria consist of the following:
a. Sensor Concentration Limits

Use an instrument that has a concentration limit (the maximum number of
1
Text in portions of this document chapter was selected from the current United States Pharmacopeia (USP).
Page 34 of 81
DATE: 04-25-2014
particles per ml) identified by the manufacturer that is greater than the
concentration of particles in the test specimen to be counted. The vendor-
certified concentration limit for a sensor is specified as that count level at which
coincidence counts due to simultaneous presence of two or more particles in the
sensor view volume comprise less than 10% of the counts collected for 10
micron particles.
b. Sensor Dynamic Range

The dynamic range of the instrument used (range of sizes of particles that can be
accurately sized and counted) must include the smallest particle size to be
enumerated in the test articles.
2. Instrument Standardization
The following discussion of instrument standardization emphasizes performance
criteria rather than specific methods for calibrating or standardizing a given
instrument system. This approach is particularly evident in the description of
calibration, where allowance must be made for manual methods as well as those
based of firmware, software, or the use of electronic testing instruments.
Appropriate user validation of software and firmware systems is essential to
performance of the test according to requirements. Since different brands of
instruments may be used in the test, the user is responsible for ensuring that the
counter used is operated according to the manufacturer's specific instructions. The
principles to ensure that instruments operate within acceptable ranges are defined
below.
The following information for instrument standardization helps ensure that the
sample volume accuracy, sample flow rate, particle size response curve, sensor
resolution and count accuracy are appropriate to performance of the test.
a. Sample Volume Accuracy

Since the particle count from a sample aliquot varies directly with the volume of
fluid sampled, the sampling accuracy must be known to be within a certain range.
i. For sample volume determination, determine the dead (tare)
volume in the sample feeder with Water for Injection or distilled
water that has been passed through a filter having a porosity of
1.2 microns or finer.
ii. Transfer a volume of water that is greater than the sample volume
to a container and weigh.
iii. Withdraw through the sample feeding device a volume that is

appropriate for the specific sampler, and again weigh the
container.
iv. Determine the sample volume by subtracting the tare volume from
the combined sample plus tare volumes.
v. Verify that the value obtained is within ± 5% of the appropriate

sample volume for the test.
b. Sample Flow Rate

Page 35 of 81
DATE: 04-25-2014
Verify that the flow rate is within the manufacturer's specifications for the sensor
used. This may be accomplished by using a calibrated stop watch to measure
the time required for the instrument to withdraw and count a specific sample
volume (i.e. the time between beginning and ending of the count cycle as
denoted by instrument indicator lights or other means). Perform the Test
Procedure at the same flow rate as that selected for calibration of the instrument.
c. Calibration and Sensor Resolution

There are three methods of choice depending on the type of system used:
Manual, Automated and Electronic methods. Please refer to the current USP for
guidance.
i. Prepare the suspension and blank using the USP Particle Count
RS.
Set the instrument to count at 10 and 25 microns (the apparatus is

calibrated using dispersions of spherical particles of known sizes
between 10µm and 25µm), according to USP <788>.
ii. Mix the blank by inverting 25 times within 10 seconds and de-gas
the mixture by sonicating for 30 seconds or by allowing to stand.
iii. Remove the closure from the container and gently stir the
contents by hand-swirling or by mechanical means, taking care
not to introduce air bubbles or contamination. Stir continuously
throughout the analysis. Note: Electronic Particle Analyzers have
a mixing stand attached to the instrument for this purpose.
iv. Withdraw directly from the container three consecutive volumes of

not less than 5 mL each.
v. Obtain particle counts and discard the data from the first portion.
NOTE: Complete procedure within five minutes.
vi. Repeat procedure, using the suspension in place of the blank.
vii. From the averages of the counts resulting from the analysis of the
two portions of the suspension at 10 microns and from the
analysis of the two portions of the blank at 10 microns, calculate
the number of particles in each ml taken as follows: Subtract the
average particle count of the blank from the average particle count
of the suspension, then divide the result by the average volume of
the 4 portions tested. This can be summarized in the following
formula:
(Ps - Pb)/V
in which Ps is the average particle count obtained from the
suspension, Pb is the average particle count obtained from the
blank, and V is the average volume, in ml, of the 4 portions tested.
viii. Repeat the calculations, using the results obtained at 15 microns.
Page 36 of 81
DATE: 04-25-2014
ix. Interpretation: The instrument meets the requirements for Particle

Counting Accuracy if the count obtained at 10 microns and the
ratio of the counts obtained at 10 microns to those obtained at 25
microns conform to the values that accompany the USP Particle
RS.
x. If the instrument does not meet the requirements for Particle

Counting Accuracy, recalibrate with the remaining suspension and
blank.
xi. If the results of the second test are within the limits given above,
the instrument meets the requirements of the Particle Counting
Accuracy Test. If on the second attempt the system does not
meet the requirements of the test, determine and correct the
source of the failures and retest the instrument.
3. Test Environment
a. Perform the test in an environment that does not contribute any significant
amount of particulate matter. Note: Glassware from Qorpack come in particle
free packages which is a suitable particulate free vehicle for compositing
liquid samples.
b. Specimens must be cleaned to the extent that any level of extraneous

particles added has a negligible effect on the outcome of the test.
c. The test specimen, glassware, closures and other required equipment

preferably are prepared in an environment protected by high efficiency
particulate air (HEPA) filters.
d. It is preferable to wear non-shedding garments and powder-free gloves

throughout the preparation of samples.
e. Cleanse glassware, closures and other required equipment preferably by

immersing and scrubbing in warm water, nonionic detergent solution. Rinse
in flowing tap water and then rinse again in flowing filtered water. Organic
solvents may also be used to facilitate cleaning. Note: Particle free glassware
is available which reduces the chance of contamination with environmental
particles.
f. Finally, rinse the equipment in filtered water using a hand-held pressure

nozzle with final filter or other appropriate water source, such as distilled
water passed through a capsule filter. The filter used should have a porosity
of 1.2 microns or finer.
g. To collect background counts:

i. Use a clean vessel of the type and volume representative of that
to be used in the test.
ii. Place a 50 mL volume of particle free water in the vessel, and

agitate the sample in the cleaned glassware by inversion or
Page 37 of 81
DATE: 04-25-2014
swirling. [NOTE: A smaller volume, consistent with the article to

be counted, can be used.]
iii. De-gas by sonicating for 30 seconds or by allowing to stand.
iv. Swirl the vessel containing the water sample by hand or agitate by
mechanical means to suspend particles.
v. Withdraw and obtain the particle counts for five consecutive

samples of not less than 5 mL each.
vi. If more than 25 particles of 10 micron size or greater size are

observed in the combined 25 mL sample, the environment is not
suitable for particulate analysis. The particle free water and
glassware have not been properly prepared. Reexamine the
procedure followed, equipment used and the environment in which
the test was performed. Make any changes needed until a
satisfactory preparatory test result is attained.
4. Test Procedure
For containers having volumes of less than 25 mL, test a solution pool of 10 or more
units to obtain a volume of no less than 25 mL. Single units of small-volume injections
may be tested individually if the individual unit volume is 25 mL or greater.
Prepare the test specimens in the following sequence:

a. Agitate the contents of the sample by inverting the container 20 times.
b. Remove outer closures, sealing bands, and any loose or shedding paper
labels.
c. Rinse the exterior of containers with particle free water as described under
Test Environment, and dry. Take care to protect the contents of the
containers from environmental contamination.
d. Allow the container to stand for 2 minutes or sonicate in order to remove gas
bubbles.
e. Withdraw the contents of the containers in the normal or customary manner

of use, or as instructed in the package labeling. Containers with removable
stoppers may be sampled directly by removing the closure. Optional: If test
specimens are being pooled, remove the closure and empty the contents into
a clean container.
5. Determination of Particle Counts

a. Method for Small Volume Parenterals
NOTE: Because of the small volume of some products, it may be necessary to
agitate the solution more vigorously in order to suspend the particles completely
and homogeneously.
Page 38 of 81
DATE: 04-25-2014
i. Obtain and combine in a cleaned container the contents of 10 or

more units to obtain a volume of not less than 25 mL.
ii. De-gas by sonicating for 30 seconds or by allowing to stand until

the solution is free from air bubbles.
iii. Gently stir the contents of the container by hand-swirling or by

mechanical means, taking care not to introduce air bubbles or
contamination.
iv. Withdraw not less than 4 aliquot portions, each not less than 5 mL
in volume, into the light obscuration counter sensor.
v. Obtain the particle counts, and discard the data from the first
portion.
NOTE: For some products, a pool of 15 or more units may be necessary to

achieve a pool volume sufficient for three 5 mL sample aliquots. Smaller sample
aliquots (i.e., less than 5 mL) can be used if the assay result obtained with the
smaller aliquots is validated to give an assessment of batch suitability equivalent
to that obtained with the 5 mL aliquots specified above.
b. Method for Large Volume Parenterals where the contents of each unit are 25
mL or more, Individual units are tested
i. Mix 1 unit following the same procedure as described above;

Removal of closure and agitation is same as described above;
Withdraw not less than 3 aliquot portions, each not less than 5 mL
in volume, into the light obscuration counter sensor.
ii. Obtain particle counts, discarding the data from the first portion.
c. Dry or Lyophilized Product for Parenteral Use

i. Open the container, taking care not to contaminate the opening or
cover.
ii. Constitute as directed in the product label with a suitable volume

of particle free water, or with the appropriate particle free solvent if
water is not suitable.
iii. Replace closure, and manually agitate the container to dissolve

the drug.
iv. Allow to stand until the drug is completely dissolved.
v. Prior to analysis, gently stir the contents of the containers by

hand-swirling or by mechanical means, taking care not to
introduce air bubbles or contamination.
Page 39 of 81
DATE: 04-25-2014
vi. Pool or test individually the appropriate number of units. Withdraw

no less than 4 aliquots, each not less than 5 mL in volume, into
the light obscuration counter sensor.
vii. Obtain the particle counts, and discard the data from the first
aliquot.
d. Solid Drugs Packaged with Diluents

For products packaged in containers that are constructed to hold the drug
product and a solvent in separate compartments, mix each unit as directed in the
labeling, activating and agitating each unit so as to ensure thorough mixing of the
separate components. Analyze the solutions as described under methods for
large or small volume parenterals depending on container volume.
.
e. Multiple-dose Containers
For products labeled Pharmacy Bulk Packages, proceed for each unit as directed
under method for small volume parenterals, calculating the results on the basis of
a sample volume that is equal to the maximum dose stated in the labeling. For
the calculations below, consider a maximum-dose volume to be the equivalent of
the contents of one full container.
6. Calculations
a. Pooled Samples (Small-volume Injections)
Average the counts from the 2 or more aliquot portions analyzed. Calculate the
number of particles in each container by multiplying the average particle count
obtained from the portion by the volume of the pooled sample in mL, and divide
by the product of the volume of each portion analyzed and the number of
containers pooled. This calculation is summarized by the formula:
PVt/Van
in which P is the average particle count obtained from the portion analyzed, Vt is
the volume of pooled sample, in mL, Va is the volume, in mL of each portion
analyzed, and n is the number of containers pooled.
b. Individual Samples (Small-volume Injections)
To calculate the number of particles in each container, average the counts

obtained for the 5 mL or greater aliquot portions from each separate unit
analyzed, multiply by the volume in mL of the unit tested, and divide this result by
the volume in mL of each portion analyzed. This calculation is summarized by
the formula:
PV/Va
in which P is the average particle count obtained from the portions analyzed, V is
the volume, in mL, of the tested unit, and Va is the volume, in mL, of each portion
analyzed.
c. Individual Unit Samples (Large Volume Injections)

To calculate the number of particles in each mL, average the counts obtained for
the two or more 5 mL aliquot portions taken from the solution unit, then divide by
the volume in mL of the portion taken. This calculation is summarized by the
formula:
Page 40 of 81
DATE: 04-25-2014
P/V
in which P is the average particle count for an individual 5 mL or greater sample
volume, and V is the volume, in mL, of the portion taken.'
7. Interpretation
The injection meets the requirements of the test if the calculated number of particles
present in each discrete unit tested or in each pooled sample tested does not exceed
the appropriate value listed in Table 1. If the average number of particles exceeds
the limit, test the article by the Microscopic Particle Count Test.
Table 1. Light Obscuration Test Particle Counts

≥10 microns ≥25 microns
Small –volume 6000 per 600 per container
Injections: container
Large-volume 25 per mL 3 per mL
Injections:
8. Particulate Matter in Ophthalmic Solutions

Every ophthalmic solution for which the monograph or drug product specification
includes a test for particulate matter is subject to the particulate matter limits set forth
for the test being applied, unless otherwise specified in the individual monograph or
specification. When higher limits are appropriate, they will be specified in the
individual monograph or specification. Ophthalmic preparations that are
suspensions, emulsions, or gels are exempt from these requirements, as are medical
devices. Refer to the specific monograph when a question of test applicability
occurs.
Light obscuration and microscopic procedures for the determination of particulate

matter in ophthalmic solutions are identical to those for injections; therefore, where
appropriate, USP <788> Particulate Matter in Injections is cross-referenced.
a. Light Obscuration Particle Count Test

This test applies to ophthalmic solutions, including solutions constituted from
sterile solids, for which a test for Particulate matter is specified in the individual
monograph. The test counts suspended particles that are solid or liquid.
i. Test Apparatus, Instrument Standardization, Test Environment,
Test Procedure, and Calibrations: Proceed as directed for Light
Obscuration Particle Count Test under USP <788> Particulate
Matter in Injections.
ii. Interpretation: if the average number of particles exceeds the limit,

test the article by the Microscope Particle Count Test.
Table 2. Light Obscuration Test Particle Count

≥10 microns ≥25 microns
Number of particles: 50 per mL 5 per mL
NOTE: Any product that is not a pure solution having clarity and viscosity
approximating those of water may provide erroneous data when analyzed by the light
Page 41 of 81
DATE: 04-25-2014
obscuration counting method. Such materials may be analyzed by the microscope

counting method.
B. Microscopic Particle Count Test

The microscope particulate matter test may be applied to both large-volume and
small volume parenteral injections and to ophthalmic solution products as well.
This test enumerates essentially solid particulate matter >/- 10 um in these
products, after collection, rinsing and drying on a micro-porous membrane filter.
Since a wide range of aliquots may be utilized, particle counts may be
determined on a per-volume or per-container basis without dilution or
extrapolation.
In the performance of the membrane microscope assay, one estimates the size
of retained solids viewed at 100x magnification. Tabulating them into specific size
categories. In this process, one may encounter materials on the membrane
surface that do not appear solid or substantial, showing little or no surface relief
such as a “stain” or discontinuity on the membrane. Chapter <788> advises not
to attempt to size or enumerate such semi-solid particles, due to historical
comment from LVP terminal sterilization manufacturers that encountered stain-
like brown residues after heat sterilization of Dextrose solutions.
However, if not sampling a carbohydrate solution or similarly-performing

formulation, recognizing the presence of such materials may be an indication that
further development research is warranted to count or investigate must be based
upon product formulation experience. Interpretation of microscopical
enumeration may be aided by testing a sample of the solution by the LO particle
count or a validated, alternate method.
The Test Apparatus is described in <788>. Additional comments are:
Use a compound binocular microscope that corrects for changes in interpupillary

distance by maintaining a constant tube length.
The objective must be of 10X nominal magnification, a planar achromat or better

in quality, with a minimum 0.25 numerical aperture.
The objective must be compatible with an episcopic illuminator attachment.
The eyepieces must be matched. In addition, one eyepiece must be designed to

accept and focus an eyepiece grarticule. The microscope must have a
mechanical stage capable of holding and traversing the entire filtration area of a
25 –mm or 47 mm membrane filter.
Page 42 of 81
DATE: 04-25-2014
Two illuminators are required. Both illuminators must be of sufficient output to

provide a bright and even source of illumination and may be equipped with blue
daylight filters to decrease operator fatigue during use.
1. Stage Micrometer - Graduated in 10-um increments, utilized each day-of-

use. For initial calibration, use a stage micrometer that is certified by NIST to
verify the USP graticule installation. Thereafter, for daily
calibration/verification, one may utilize a commercial stage micrometer
graduated in 10-um increments to verify proper setup.
2. Filtration Apparatus - Use a filter funnel suitable for the volume to be tested,
generally having an inner diameter of about 16 mm for 25-mm membranes or
about 37 mm for 47 mm membranes. The funnel is made of plastic, glass or
stainless steel. Use a filter support made of stainless steel screen or sintered
glass as the filtration diffuser. A solvent dispenser capable of delivering
solvents filtered through a membrane filter at a large range of pressures from
10 psi to 80 psi.
Membranes-As describes by <788>; however, finer pore size selections will

have smoother surfaces, facilitating the microscopical examination; however,
may impede more viscous sample fluid during the assay.
3. Equipment:
Use a suitable binocular microscope, a filter assembly for retaining particulate
matter, and a membrane filter for examination.
The microscope is adjusted to 100 +/- 10 magnifications and is equipped with

an ocular micrometer calibrated with an objective micrometer, a mechanical
stage capable of holding and traversing the entire filtration area of the
membrane filter, and two suitable illuminators to provide episcopic illumination
in addition to oblique illumination.
Figure 1:
Page 43 of 81
DATE: 04-25-2014
The ocular micrometer is a circular diameter graticule (Figure 1) and consists

of a large circle divided by crosshairs into quadrants, transparent and black
reference circles 10 um and 25 um in diameter at 100 magnifications, and a
linear scale graduated in 10-um increments. It is calibrated using a stage
micrometer that is certified by either a domestic or international standard
institution. A relative error of the linear scale of the graticule within +/- 2% is
acceptable. The large circle is designated the graticule field of view (GFOV).
Figure 1: Circular diameter graticule. The large circle divided by crosshairs

into quadrants is designated the graticule field of view (GFOV). Transparent
and black circles having 10-um and 25-um diameters at 100X are provided as
comparison scales for particle sizing.
Two illuminators are required. One is an episcopic bright field illuminator

internal to the microscope; the other is an external, focusable auxiliary
illuminator that can be adjusted to give reflected oblique illumination at an
angle of 10-20 degrees.
The filter assembly for retaining particulate matter consists of a filter holder
made of glass or other suitable material, and is equipped with a vacuum
source and a suitable membrane filter.
The membrane filter is of suitable size, black or dark gray in color, non-
gridded or gridded and 1.0 um or finer in nominal pore size.
4. General Precautions:
Page 44 of 81
DATE: 04-25-2014
The test is carried out under conditions limiting particulate matter, preferably
in a laminar flow cabinet.
Very carefully wash the glassware and filter assembly used, except for the
membrane filter, with a warm detergent solution, and rinse with abundant
amounts of water to remove all traces of detergent. Immediately before use,
rinse both sides of the membrane filter and the equipment from top to bottom,
outside and then inside with particle free water. In order to check that the
environment is suitable for the test, that the glassware and the membrane
filter are properly cleaned, and that the water to be used is particle-free, the
following test is carried out. Determine the particulate matter of a 50-ml
volume of particle-free water according to the method described below. If
more than 20 particles of 10um or larger in size or if more than five particles
25 um or larger in size are present within the filtration area, the precautions
taken for the test are not sufficient. The preparatory steps must be repeated
until the environment, glassware, membrane filter and water are suitable for
the test.
Mix the contents of the samples by slowly inverting the container 20 times
successively. If necessary, cautiously remove the sealing closure. Clean the
outer surfaces of the container opening using as jet of particle-free water, and
remove the closure, avoiding any contamination of the contents.
For large volume parenterals, single units are tested. For small volume
parenterals less than 25 ml in volume, the contents of 10 or more units are
combined in a cleaned container; the test solution may be prepared by jixing
the contents of a suitable number of vials and diluting 25 ml with particle-free
water or with an appropriate particle-free solvent when particle-free water is
not suitable. Small volume parenterals having a volume of 25 ml or more may
be tested individually.
Powders for parenteral use are constituted with particle-free water or with an
appropriate particle-free solvent when particle-free water is not suitable.
The number of test specimens must be adequate to provide a statistically

sound assessment.
For large volume parenterals or for small volume parenterals having a volume
of 25 ml or more, fewer than 10 units may be tested, using an appropriate
sampling plan.
Wet the inside of the filter holder fitted with the membrane filter with several
ml of particle-free water. Transfer to the filtration funnel the total volume of a
solution pool or of a single unit, and apply a vacuum. If needed, add stepwise
a portion of the solution until the entire volume is filtered. After the last
Page 45 of 81
DATE: 04-25-2014
addition of solution, begin rinsing the inner walls of the filter holder by using a
jet of particle-free water. Maintain the vacuum until the surface of the
membrane filter is free from liquid. Place the membrane filter in a Petri dish,
and allow the membrane filter to air-dry with the cover slightly ajar. After the
membrane filter has been dried, place the Petri dish on the stage of the
microscope, scan the entire membrane filter under the reflected light form the
illumination device, and count the number of particles that are equal to or
greater than 10um and the number of particles that are equal to or greater
than 25um. Alternatively, partial membrane filter count and determination of
the total filter count by calculation is allowed. Calculate the mean number of
particles for the preparation to be examined.
The particle sizing process with the use of the circular diameter graticule is
carried out by estimating the equivalent diameter of the particle in comparison
with the 10um and 25 um reference circles on the graticule. Thereby the
particles are not moved from their initial locations within the graticule field of
view and are not superimposed on the reference circles for comparison. The
inner diameter of the transparent graticule reference circles is used to size
white and transparent particles, while dark particles are sized by using the
outer diameter of the black opaque graticule reference circles.
In performing the Microscopic Particle Count Test, do no attempt to size or

enumerate amorphous, semiliquid, or otherwise morphologically indistinct
materials that have the appearance of a s stain or discoloration on the
membrane filter. These materials show little or no surface relief and present a
gelatinous or film-like appearance. In such cases, the interpretation of
enumeration may be aided by testing a sample of the solution by the Light
Obscuration Particle Count Test.
6. Evaluation:
For preparations supplied in container with a nominal volume of more than
100 ml, apply the criteria for Test 2.A.
For preparations supplied in containers with a nominal volume of less than

100 ml, apply the criteria for Test 2.B
For preparations supplied in containers with a nominal volume of 100 ml,

apply the criteria for Test 2.B (Note: Test 2.A is used in the Japanese
Pharmacopeia).
Test 2.A: (Solutions for parenteral infusion or solutions for injection supplied
in containers with a nominal content of more than 100 ml)-The preparation
complies with the test if the average number of particles present in the units
tested does not exceed 12 per/ml equal to or greater than 10 um and does
not exceed 2 per/ml equal to or greater than 25 um.
Page 46 of 81
DATE: 04-25-2014
Test 2.B (Solutions for parenteral infusion or solutions for injection supplied in
containers with a nominal content of less than 100 ml)-The preparation
complies with the test if the average number of particles present in the units
does not exceed 3000 per container equal to or greater than 10 um and does
not exceed 300 per container equal to or greater than 25 um.
DATE: 04-25-2014
Chapter 7: Bioburden Estimation for Medical Devices

According to FDA Compliance program 7382.845, Inspections of Medical Device
Manufacturers, Part IV, - “Bioburden testing is to be performed in accordance with the guidance
provided in ISO 11737-1, Sterilization of medical devices – Microbiological methods – Part I:
Estimation of population of microorganisms on products. The methodology used for estimating
the bioburden is to be validated. Twenty units are to be tested.” 1
The term “bioburden” is commonly used to describe the population of microorganisms present
on unsterilized material or products. The bioburden quantity and types of bioburden organisms
present can impact the sterilization process of the material or product. It is important to develop
procedures which provide accurate, precise, and reproducible measurement of the bioburden
population associated with the material or product. There are several approaches to remove
microorganisms from a medical device. Some examples of these recovery methods include:
filtration followed by plating; ultrasonics/shaking followed by filtration then placed on an agar
medium; Stomaching/rinsing/flushing followed by filtration and plated on an agar medium; if all
else fails perform a direct swabbing or contact plate.
The bioburden estimation of a medical device generally consists of four distinct stages:
1. Collection of microorganisms from the medical device. (See Annex A and B)

2. Enumeration of the collection sample containing recovered microorganisms.
3. Bioburden characterization.
4. Application of the correction factor(s) determined during bioburden recovery studies in
order to calculate the bioburden estimate from the raw presterilization count.
It is not possible to define a single microbial collection technique because of the wide variety of
materials used in health care products. Furthermore, the selection of conditions for enumeration
will be influenced by the types of microbial contamination which may be anticipated.
The current method “ANSI/AAMI/ISO 11737-1:2006/(R)2011 sterilization of health care products

– Microbiological methods – Part 1: Determination of the population of microorganisms on
product” has the latest revisions and provides a great deal of information that will guide an
analyst to the method needed for a particular type of product.
Annex A contains a decision tree “that addresses designing a bioburden method based on the
nature of the product being tested and includes guidance for choosing such things as agitation
techniques or filtration versus direct plating.”2 Annex A also addresses the procedures
(repetitive recovery method, product inoculation method) available for the validation of the
method for determining bioburden.
Annex B has a comprehensive list of the different removal techniques that can be employed and
alternatives for samples where removal of microorganisms by elution is not used.
Annex C has a more in-depth explanation of the validation of the repetitive recovery and product
inoculation methods.
References:
Page 48 of 81
DATE: 04-25-2014
1. FDA Compliance Program 7382.845 Inspections of Medical Device Manufacturers,

February 2, 2011.
2. ANSI/AAMI/ISO 11737-1:2006/(R) 2011, Sterilization of health care products –
Microbiological methods – Part 1: Determination of the population of microorganisms
on product.
3. PDA Technical Report No. 21, Bioburden Recovery Validation. 1990
Page 49 of 81
DATE: 04-25-2014
Chapter 8: Environmental Monitoring

FDA field microbiologists are requested to assist CSOs during on-site inspection of
pharmaceutical manufacturers of drugs made under controlled environmental conditions.
Occasionally they may be required to perform environmental monitoring (EM) sampling of those
facilities to assess the microbiological bioburden of critical surfaces. This chapter describes a
suggested procedure for conducting this activity. This should only serve as a guide with some
modifications depending on the specific facility or special instructions from FDA management.
Be sure to confirm with ORS which FDA field laboratory was designated to receive the EM
samples. These procedures allow for a qualitative and quantitative assessment of
environmental monitoring samples for the microbial presence in critical processing or laboratory
area(s) being monitored.
A. Materials/Equipment
1. Sampling Materials
a. Sterile Dacron or cotton swab with a sterile transport media solution.
b. Alternative sampling system:
c. Sterile sponge with detachable handle
d. *Hycheck surface samplers
e. *RODAC plates (with locking lid)
*Use media containing lecithin and tween neutralizers
f. Sterile Whirl-pak® bags.
g. Sterile water for irrigation or sterile saline held in screw cap containers.
h. Dey/Engley (D/E) neutralizing broth
i. Sterile 70% alcohol spray bottle or wipes
j. Black magic marker (permanent, fine point).
k. Digital Camera
2. Testing Equipment and Materials

a. Biological Safety Cabinet (BSC) with HEPA filtration
b. Laminar Flow Hood (LFH) with HEPA filtration
c. 10% Bleach or appropriate disinfectant/sporicide
d. Sterile 70% ethanol (ETOH) or Isopropyl Alcohol (IPA)
e. Sterile Sleeves
f. Sterile Gloves
g. Hair Net
h. Lab Coat
i. Beard Cover and/or Mask
j. Incubator set at 32.5ºC ± 2.5ºC
k. Incubator set at 22.5ºC± 2.5ºC
l. Modified Letheen Broth (MLB)
m. Modified Letheen Agar (MLA)
n. Sabouraud Dextrose Broth
o. Sabouraud Dextrose Agar
p. Malt Extract Agar w/chlorotetracycline
q. TSA w/5% Sheep Blood Agar
Page 50 of 81
DATE: 04-25-2014
r. MacConkey Agar
s. RODAC plates
t. Hycheck slides
u. Soybean Casein Digest Agar (TSA)
v. Soybean Casein Digest Broth (with neutralizers)
w. Neutralizers (i.e. lecithin, tween, etc...)
B. Sampling Preparation
Don appropriate personal protective equipment (PPE) as follows:
Note: When on an inspection, it’s best to use the firms PPE when/if provided. The firm’s
procedures/guidelines should be adhered to when entering classified areas.
a. Sterile Disposable Lab Coat or Gown.

b. Safety Goggles
c. Hair net, mask and/or beard cover
d. Sterile Gloves
e. Shoe Covers
1. Sampling Equipment Controls

a. Aseptic Technique Control: Place one (1) sterile Dacron or cotton swab into
sterile water and place back into its sterile transport media solution. Place this
negative control into a sterile Whirl-pak® bag.
Note: There is no surface contact for this control.
b. Swab/Sponge Sterility Control: Place an intact unused swab (or sponge) unit
into a sterile Whirl-pak® bag.
c. RODAC/Hycheck Sterility Control: Place an unused RODAC plate and/or

Hycheck plate within a sterile Whirl-pak® bag.
d. Whirl-pak® bag Sterility Control: Include one unopened Whirl-pak® bag as a

closed control.
e. Glove Sterility Control: If the sampler uses FDA sterile gloves then have an
intact unit containing gloves placed into a sterile plastic bag and sent as a
control.
f. Include any other sterile equipment used during EM sampling (i.e. sterile
specimen cup, sterile media, etc.).
C. EM Sampling Procedure
It is recommended that the investigative team bring equipment for both qualitative and
quantitative EM methods. Qualitative methods utilizing sponges/ swabs are used for
hard to reach areas. RODACs or Hychecks are employed for the quantitative method to
Page 51 of 81
DATE: 04-25-2014
enumerate microbes on open flat work surfaces. See suggested sampling locations
listed in section E of this procedure.
1. Disinfect gloved hand with a suitable sanitizing agent (i.e. sterile 70% alcohol).
a. Repeat this step between each EM sample.
b. Allow gloves to air dry so no alcohol is dripping from gloves.
c. Some swab/sponge sampling packages include a secondary set of sterile

gloves. In these instances, the secondary glove can be aseptically used on
top of the primary gloves to expedite the sampling process. The secondary
pair of gloves will need to be disposed of aseptically after use. If the primary
gloves touch the secondary gloves outer surface, then a suitable sanitizing
agent must be used on the primary gloves. Allow primary gloves to air dry
after sanitizing.
d. When sampling a Class 100 scenario. For example,

i. Verify current certification of LFH/BSC
ii. Allow LFH/BSC to run approximately 10 minutes before initiating

sampling
iii. Wipe down all outer sampling containers with a suitable sanitizing
agent before placement in the LFH/BSC
iv. Do not open sampling materials outside the LFH/BSC
2. Qualitative Swabbing
a. Open a sterile swab (or sponge). Dampen with wetting agent (sterile water,
saline, or D/E neutralizing broth) and squeeze off excess by pressing against
the inside of the container holding the wetting agent.
b. Apply swab (or sponge with handle) to surface (or equipment) being
monitored with firm application pressure. Be sure to emphasize that the swab
contact should be firmly pressed against the surfaces being sampled.
c. When sampling (monitoring) flat surfaces allow the swab (or sponge with
handle) to firmly rub an area of approximately 24 to 30 cm2.
d. Apply the swab (or sponge) within this contact area in both a horizontal and
vertical direction for approximately 10 seconds (count it out).
e. Replace the swab (or sponge) back into the carrier container (if it came with
one) and place into the sterile Whirl-pak® bag. (Be sure to break off the
handle portion of the sponge applicator stick.)
3. Quantitative RODAC/Hycheck Sampling
Page 52 of 81
DATE: 04-25-2014
a. Carefully remove the lid of RODAC plate or loosen the cap on the Hycheck
slide tube. Take care not to touch the agar surface.
Note: Examine agar for contamination and or dehydration
b. Gently but firmly touch the RODAC agar surface against the area being
sampled, exert moderate, even, vertical pressure and then carefully replace
lid. Avoid using rubbing motions with the plate at the sample site as this may
break the agar.
c. When using Hycheck press down on the spike to bend the paddle at the
hinge line gently lowering the slide and press the agar to the surface with firm
and even pressure. Repeat this step using the 2nd agar surface on an area
adjacent to the initial test site. Replace slide in container and close tightly.
4. Place the EM sample into a sterile Whirl-pak® bag and identify the bag immediately
after.
5. Assign a consecutive number to the sample (i.e.1, 2, 3, etc.), in addition include the
date, location of sample site (be specific) and your initials. Record in your
inspectional record book the swab number and the location of the swab site.
6. Later, place all the Whirl-pak® bags in an officially sealed plastic or brown paper bag.
Do not freeze or refrigerate. Freezing can kill the microbes and the cold can induce
a shock to vegetative cells. These microbes were surviving at room temperature
(ambient). Therefore, an ambient temperature should be maintained during
transportation.
7. Place into a suitable mailing container to prevent crushing or physical damage to the
swabs. The container should have some insulation capacity to prevent extreme
temperature (freezing or excessive heat).
8. Ensure contact with the receiving laboratory in advance regarding pending samples
in order for them to have appropriate personnel and materials for sample set up
within 48 hours.
D. Recommended Environmental Monitoring Sites

When on an inspection, do not allow the firm to disinfect the work area prior to sampling.
The facility and the equipment should be sampled during a ready-to-use state as
determined by the firm. The presence of disinfectant on the swab may reduce the
microbial bioburden or increase inhibition during broth incubation. When collecting EM
samples start in locations that are under the greatest control (ISO 5- HEPA filtered
LFH/BSC or Isolator) and sample to lesser controlled areas (areas outside the work
station but still within the room).
1. Swab the frequently utilized surfaces within the controlled work station such as:
Page 53 of 81
DATE: 04-25-2014
a. Center of work surface

b. Fingertips & sleeves of Isolator gloves
c. Storage bins inside work station
d. Shelving inside work station or any other stationary items
e. Equipment control panels including on/off switches of LFH/BSC
f. Flexible plastic curtains used to separate multiple workstations
2. Swab corner crevices inside the HEPA Filtered work station.
3. Swab the handle, squeeze trigger and nozzle of any bottle kept in the clean room
or work station used for spraying (i.e., 70% alcohol, disinfectant solutions, etc.).
4. Swab the underside of the chair in front of the work station. Specifically, on the
front bottom rim where personnel would hold to pull up the chair.
5. Swab tables or benches within the controlled room where product container(s) or
post sterilized product may be held outside of the HEPA filtered workstation.
6. Swab the air in-take grid on each of the HEPA filtered work stations. Usually
located on top of the unit holding the course filters.
7. Swab the exhaust (return) grid for the room air handling system that is connected
to the facility air supply where the product manufacturing or compounding occurs.
8. Swab the light switch and door knob or handles leading into and out of the clean
room.
9. Swab any cardboard boxes, handles of plastic containers, tools (crimpers) or

scissors, key pads on weighing scales or computers kept in the cleanrooms.
10. Swab the exterior cuffs of the used lab coats worn by personnel during
manufacturing or compounding. They may be hanging in the entry (ante)
gowning room.
11. Swab the bottom horizontal window sill within the clean room.
12. Swab any area under open or dislodged ceiling panels.
13. Sample areas of discoloration, stains or water and oil droplets.
14. Use your discretion to swab any other high risk surface locations.
15. Photograph surfaces or equipment that display gross signs of contamination (i.e.,
particulate matter, fungi, discoloration, etc).Try to include a distant picture of the
targeted area along with a focused close up. Be sure to EM sample this location,
as well.
E. Analysis Preparation conducted by FDA field laboratory

Page 54 of 81
DATE: 04-25-2014
1. All processing of swabs must be aseptically performed within a HEPA Filtered

Class II Biological Safety Cabinet (BSC) or HEPA Filtered Laminar Flow Hood
(LFH). It is not necessary to analyze environmental monitoring samples in a
clean room or isolator.
2. All surfaces within the BSC or LFH must be thoroughly disinfected with
appropriate disinfectant (i.e. 10% bleach, etc.) followed by filter sterilized 70%
ethanol or IPA prior to placing swabs under the hood and beginning analysis.
3. In order to assure that the BSC/LFH and media are free of microbial
contamination, standard open and closed controls used for sterility testing should
be performed concurrently with analysis.
4. Don appropriate PPE as follows:
a. Freshly laundered lab coat

b. Sterile disposable sleeves
c. Hair net
d. Sterile gloves
e. Beard Cover and/or Mask
5. Sterile gloves must be decontaminated between the processing of each

individual swab. Sterile gloves and sleeves can be discarded and replaced as
needed.
6. Sample Preparation
a. Examine swab containers for closure integrity to ensure tampering,

leakage, or potential cross contamination has not occurred.
b. Carefully disinfect exterior of each swab container and place into the
sanitized BSC/LFH and allow to air dry.
7. Media Selection
a. Neutralizing additives (i.e. Tween/Polysorbate, Lecithin, etc.) are utilized

to neutralize inhibitory disinfectant residues transferred to the swab during
sampling that might inhibit microbial growth.
b. For a broad spectrum recovery of microorganisms, one should utilize a

nutrient rich general purpose media containing neutralizers (i.e. MLB,
MLA, etc.).
c. When targeting fungal populations only, it is necessary to use an

appropriate fungal media such as Sabouraud Dextrose or Malt Extract
media. It is beneficial to use an antibiotic (i.e. Chlortetracycline) which will
help to selectively inhibit bacterial growth and restrict the size and height
of colonies of more rapidly growing molds.
Page 55 of 81
DATE: 04-25-2014
d. TSA w/5% Sheep Blood Agar is beneficial for cultivating fastidious

microorganisms.
e. MacConkey Agar is used for the isolation and differentiation of gram

negative and enteric organisms.
f. RODAC plates and Hycheck slides are used for the detection and
quantification of microbiological contamination.
F. Analytical Procedure
1. Approximately 100 ml of sterile media (i.e., SDB, MLB, etc.) should be aseptically
added to each plastic bag containing a square sponge swab. Mix or swirl
thoroughly.
2. Approximately 10 ml of sterile media (i.e., SDB, MLB, etc.) should be aseptically

added to each tube containing a swab. Mix or swirl thoroughly.
3. All swabs are incubated at 25ºC- 30ºC for at least 14 days to allow for the
resuscitation of potentially stressed microbes.
4. Hycheck slides and RODAC plates should be directly incubated at 20ºC- 35ºC for
at least 5-7 days. Longer incubation times may be required when contaminants
are suspected to be slow growing. Check plates daily for colony formation to
minimize obscuring visualization of smaller colonies by over growth.
a. Count and record the number of colony forming units for RODAC plates.
All colony types should be picked and re-streaked for purity and
subsequently identified.
b. Count the number of colonies on both sides of the paddle for the Hycheck
slide. Report the colony counts for each side of the paddle and all colony
types should be picked and re-streaked for purity and subsequently
identified.
5. Check all swabs daily for turbidity and subculture for isolation as turbidity is
observed. All sub culturing must be performed under LFH or BSC.
6. Subculture all turbid swabs onto a combination of non-selective media (i.e. MLA,
etc.) and selective/differential media (i.e. MacConkey agar, MEA, etc.). It is
recommended to include TSA w/5% Sheep Blood Agar as one of the differential
media for subculturing.
a. Fungal media should be incubated at 20º to 25º for 5 to 7 days. Possibly

longer but no more than 14 days
b. All other cultural media should be incubated at 30º to 35º for 2 to 3 days.
Page 56 of 81
DATE: 04-25-2014
7. Re-incubate all cultured swabs until the full incubation (14 days) timeframes are
met.
8. Culture all submitted negative controls & diluents in order to confirm that the
equipment or any aseptic techniques were not compromised.
9. Once isolation of the organism is achieved, perform microbial characterization

and identification following USP <1113> Microbial characterization, Identification
and Strain Typing, as guidance. Typically, rapid identification systems (i.e.
VITEK) are employed after primary screening and characterization are
performed. Other identification platforms may be beneficial if acceptable
identification is not obtained through biochemical testing.
Page 57 of 81
DATE: 04-25-2014
Chapter 9: Rapid Screening Methods

A. Screening Protocol for Direct Staining on Products with
Appearance of Visible Contamination
1. Purpose
To standardize and implement a protocol that can rapidly identify microbial
presence in drugs intended to be sterile through direct microscopic staining.
2. Scope/Policy
Any sample received with visible, potentially microbiological matter in a sterile
drug must be given the highest priority for testing, due to the importance of
quickly ascertaining whether the product poses an infection risk to patients. This
procedure requires all ORA laboratories involved in microbiological testing of
sterile pharmaceutical products to conduct microscopic screening of samples that
appear to have visible contamination.
3. Responsibilities
ORA Microbiology Laboratories that routinely test pharmaceutical products using
the USP compendia methods must adopt a rapid screening practice to visually
examine sterile pharmaceutical products that appear to have macroscopic
contamination by using a direct microscopic staining protocol to determine if
microbial contamination is present. It is the responsibility of the analyst to note
any modifications to this procedure in the worksheet.
4. Background
FDA analysis of marketed drugs purporting to be sterile and containing visible
foreign matter has revealed microbial contamination, which has been linked in
some cases to outbreaks of patient infections and fatal outcomes. Because
microbiologically contaminated products pose a grave health hazard to patients
and the consequences of exposure to contaminated product can be mitigated
once healthcare providers and their patients are alerted, it is important for FDA to
obtain analytical information as early as possible when microbial contamination is
suspected.
This requires the FDA laboratory to undertake special measures upon receipt of
a sample with visible, potentially microbiological contamination. Accordingly,
CDER and ORA have met and agreed on recommendations for the immediate
conduct of a microscopic screening test, under specified circumstances, to
rapidly identify microbiological contamination in products purporting to be sterile.”
The objective of this protocol is institute rapid microscopic screening in ORA
laboratories to expedite provision of the analytical findings to ORS and CDER.
5. Procedure
a. Before Sample is transferred to Class 100 Clean room or Sterility

Test Isolator
i. Remove and count the number of product units from the sample
collection package bag and examine each unit to confirm the
Page 58 of 81
DATE: 04-25-2014
number of units and labeling accuracy (most importantly the

product lot numbers) with the collection report.
ii. Examine each unit (vial, ampoule, pre-filled syringe, IV bag, etc.)
for the integrity of the container/closure system, and record in lab
worksheets whether units are intact. There should be no product
leakage, cracks, existing puncture in the rubber septum or
damage to the unit container that may have allowed microbial
ingress during storage and shipping. Include a description of any
units that are defective or damaged and set them aside. However,
set aside any open samples in case there is potential interest in
additional analysis of these units. Seek management advice with
regard to whether any subsequent analysis should be performed.
If contamination of the product is observed, for example color of
the solution, turbidity, pellicle, sediment, these too should be
noted in your worksheets under “Product Description.”
iii. During step 2, the analyst(s) should wear sterile gloves and use
sterile 70% alcohol and sterile lint free wipes to disinfect the
exterior of the unit to assure that there is no exterior debris
obstructing the view of the product.
iv. Using appropriate lighting, carefully inspect each unit against both
a black and white background. Allow the container to stand for
several minutes to allow diffuse bubbles to disperse and dissipate
out of solution. Shaking of the solution during shipment and
handling can cause bubbles to appear, making it difficult to screen
for particulate matter and microbiological contamination.
v. Some obvious signs of microbial contamination would be: (a)

general turbidity; (b) stringy or thread-like fibers;(c) granular or
particulate clumps floating within the solution or settling to the
bottom; (d) an oil-like layer or pellet on top of the solution; (e) a
film on the interior glass that appears to lift off upon swirling the
container in a gentle manner; or any other anomaly that is beyond
the normal appearance of the product (e.g., crystals, unusual color
tint, etc.).
vi. Set aside those units that contain the potential visible
contaminants noted in step 5 and have their appearance
confirmed by a second analyst or a laboratory supervisor.
Photograph the unit(s) with a digital camera is the best way to
observe and document the contamination. Use a ruler (or another
appropriate object) for scale and be sure to follow the laboratory
procedures manual for documenting this as evidence.
Page 59 of 81
DATE: 04-25-2014
vii. Consult with a supervisor or subject matter expert on how many of

these units to examine with the sterility test and concurrent
staining procedure. Also, consult with the Center on testing needs
whenever there is a potential public health hazard posed by the
product.
b. Samples moved into the Class 100 Clean room or Sterility Test
Isolator
i. Follow the standard disinfection procedure for transferring the
suspect unit(s) into the bioclean room/sterility test glovebox used
for sterility testing.
ii. When using a sterility test isolator, ensure it has been exposed to
a decontamination cycle and that any materials or supplies are
disinfected and enter the isolator through transfer ports.
iii. Immediately after conducting the USP Sterility test, remove an

aliquot of the visually contaminated product(s) and place onto a
sterile glass slide and place in a sterile container within the HEPA
filtered laminar flow hood/glovebox/sterility test isolator. Make
every effort to capture the suspected foreign matter for
microscopic examination and characterization.
iv. Remove the sterile container containing the glass slide to outside
of clean room/glovebox/sterility test isolator and fix the smear to
the glass slide using the standard procedure. Perform a stain (i.e.,
simple stain using crystal violet, or other stain), record the results
and report your finding to your supervisor. Detailed description
should be provided regarding cell morphology, and other notable
features (e.g., presence of fungal hyphae, presence and location
of spores in cell, chain-like cocci). If possible, photograph the
images from the slide with a digital camera. If microbial
contamination appears to be present, communicate the findings to
CDER and ORA/ORS without delay. State clearly if the findings
are still preliminary. Tabulate the results in a standard table or
spreadsheet (see Attachment B) and forward the results to the
appropriate contacts in ORS and CDER (OCTEC and/or Office of
Compliance).
v. Continue with the Sterility test as outlined in the USP and FDA
standard procedures. Observe daily, and report any growth in the
media as soon as possible to ORS and CDER.
6. Attachments
Page 60 of 81
DATE: 04-25-2014
C:\Documents and Medical Product

Settings\marilyn.khanAdverse Event Trackin
Page 61 of 81
DATE: 04-25-2014
Chapter 10: Inspectional Guidance

A. Microbiological Issues for Inspection of Pharmaceutical
Laboratories
1. Finished product testing using USP or Non-compendia method
Sterility, Bacterial endotoxin, Microbial limits test: specified microorganisms and

enumeration, Antimicrobial- effectiveness test, Bioburden determination, water
quality control testing; review all original test results
2. Method Suitability (Sterility), Preparatory test (Bacterial endotoxin), Validation of

method used for bioburden and water analysis
3. Reagents and medium- proper storage, expiration date activity, and growth
promotion
4. Equipment and Instrument- (Steritest, manifold, Automated/Molecular identification

system, Vitek, etc) isolator & bio-decontamination system calibration, maintenance,
validation- IQ, OQ, PQ
5. Sterility testing area design, operational procedures, monitoring, aseptic technique,

gowning procedures, proper sample container disinfection, surface/air monitoring,
HEPA filter certification, etc
6. Method description, modifications, and verification along with recording of sample

results and appropriate review and evaluation by management
7. Qualifications, training and identification of the personnel conducting each step of the
analysis
8. Qualification and training of management to critically review data and interpret its
significance (Risk Assessment)
9. Microbial standards set for raw material, finished product, water bioburden, and EM
for analytical areas
10. Integrity and accuracy of the laboratory information management system (LIMS) for
microbiology data entry, review and approval Selection, handling, and storage of
Biological Indicators (BIs)
11. Private (contract) testing laboratory quality agreements, data review, and associated
problems; Have there been any changes in contract labs and why?
12. Proper use and control for In-vitro diagnostic test kits, Positive and negative controls,
Interpretation and reliability of results
13. Risk assessment of microbiological results for non-sterile products
Page 62 of 81
DATE: 04-25-2014
14. Request a list of the entire laboratory’s Microbiological Data Deviations (OOS/OOL
results) and Corrective Action Preventative Actions (CAPA) ( since last FDA on-site
inspection)
15. Stability Testing – sample storage conditions, missed sampling dates, etc.
B. Microbiological Issues for Inspection of Pharmaceutical

Manufacturing Facilities
1. Product Sterilization or bioburden reduction stage and validation- Aseptic/Filtration,
steam, ETO, radiation, and other chemical processes
2. Depyrogenation- dry heat ovens for glass containers, Wash/rinse for stoppers,
adequacy of validation using spiked endotoxin, Recovery studies before
depryrogenation, Filtration and column applications
3. Environmental monitoring- Types of equipment, calibration, operation and

maintenance; Surface, Air, Personnel and Water; Critical work areas for aseptically
filled products (class 100, isolators, etc); Process simulation (media fills) studies for
process validation, growth promotion, reading turbidity, volume adequacy, surface
contact, surface sanitizer neutralizing media (e.g., TSA w/ Lecithin & Polysorbate
80), sampling technique (observe), sample must represent dynamic/operational
conditions, trending/CAPA, etc.
4. Disinfection and sanitization- agents used (sporicidal?), preparation problems, (over

dilution); Applicator (i.e., mop, spray. Aerosol), time of exposure, areas of contact,
supervision; residues, UV lights, water systems, filling equipment, work surfaces,
process columns, Verification and validation
5. Room design and Equipment- accessibility for disinfection and cleaning; ‘aseptic
filling critical area; HEPA filter certification and maintenance , air flow patterns/smoke
studies, change evaluation/re-certification (rearranging cleanroom, adding
equipment, HVAC, etc.), test during dynamic/operational conditions with maximum
number of personnel in place, personnel equipment traffic, room differential pressure
and temperature; adequacy of primary and secondary barriers
6. Water Purification and delivery system: Vulnerability of Distillation, RO, Deionizers,

cartridge filters, etc; UV lights, dead legs, biofilm; corrosion ( heat exchangers);
Water borne microorganisms (nanobacteria) and endotoxin production; Cold system
problem, disinfection problems
7. Personnel- training procedures for aseptic technique, gowning procedures, Cleaning

and maintenance personnel training for Class 100 room entry; Glove and garment
monitoring and procedures
8. Product Sampling: representation of lot required minimum values per USP <71>:
quantity per container & units per batch, sample storage (time and temp), sampling
Page 63 of 81
DATE: 04-25-2014
port sanitization or sterilization problems; problem with skip lot testing on raw
material.
9. Maintenance records- determine dates, and location of equipment failure or out-of-

service that may have an impact on microbial ingress; looks for signs of roof leaks
and water stains on ceiling panels, the degree of dirt and dust accumulation on
supply and exhaust vents; Ask about new construction, plumbing or air handling
system and the reason for change.
10. Compressed Air Systems—sterile process air, microbial particulate filtration (0.2 µm,
hydrophobic), condensate causing blockage & microbial growth, routine point-of-use
sampling, maintenance, filter integrity test
C. Sample Data Review – All Negative Results

What to review when all the firm’s Sterility and/or Microbial Limits test results indicate no
microbial growth and you need to know whether this is true:
1. Medium- Growth Promotion; pH; Low agar or broth volume in container, Incubator
temp not set correctly; improper medium storage after QC (crystals from freezing,
inadequate mixing prior to dispensing, agar plates dried out during incubation, etc.)
2. Method Suitability testing- Validation for Sterility; Preparatory testing (BET). May
need added neutralizers, product dilution, filtration; water chemical contaminant with
toxin effects in buffers, Not following method (excess of product added to broth
during test but not during suitability testing, etc)
3. Improper tube or agar plate examination- check filter surface on submerged filter
membrane (mold budding); surface film, light hazy growth in Thio broth, microbes
settle to bottom of tube, pinpoint colonies (microaerophilic); medium not inoculated.
Disinfection process adds antimicrobial residue onto/into product during sample
preparation; Gas used for Isolator sterilization with medium inside chamber may
penetrate into liquid broth and/or test product packaging
4. Fastidious microorganisms found in the product bioburden may require special

additives to the medium- Halophilic contaminants in bicarbonate, or high salt
products need medium supplements with essential salts for survival
5. Water test method- membrane filter with a 0.45 micron pore size may miss quantities
of water-borne organisms. Suitability testing necessary. Note: USP <71> only
requires “pore size not greater than 0.45µm”.
6. Inadequate incubation time (14 days)/temp (USP required temps)
7. Possible falsification or incorrect entry onto worksheets or Laboratory LIM system.

Compare LIMs database entries to the analyst’s laboratory notebook; Phrase the
question “When you get a positive test result….” not “If you get a positive test
Page 64 of 81
DATE: 04-25-2014
result...” Inspect the laboratory refrigerator or freezer for evidence of stored sample
isolates. If they lyophilize sample isolates ask to review the spread sheet data
storage directly from the computer screen; hard copies could be obtained later.
Review the Vitek or Micro Id isolate log book for all microorganisms identified and
work backwards to the product lot number, filling rooms, equipment used,
components or raw material used for that lot. This may allow you to find other lots
associated with the contaminated lot.
8. Personnel- review training records and personnel qualifications and experience

Observe analysts during sample collection, preparation, etc., to look for errors which
may inhibit microbial recovery.
9. Visit the microbiology laboratory and look into the refrigerators, incubators, discarded
plates from that day’s work or request speciation log book and determine if microbial
recovery has occurred, but not recorded on official worksheets or entered into LIMs.
D. Sample Data Review – Microbial Growth Indicated

When you encounter inspectional evidence that the firm has manufactured
microbiologically contaminated product, the few suggestions listed below should help
you evaluate and proceed with this information.
1. Documentation- Review and obtain copies of all records for lots indicating
contamination; determine if there are other lots manufactured either before or after
the “bad” lot(s); Review all associated activity and equipment related to the
contaminated lot. There may be common water, mixing tanks. Piping, raw material,
sterilizers, filters, etc that may have been cross contaminated and transferring
microbes to subsequent lots of products.
2. Review current established validation studies for product/component sterilization

(disinfection for non-sterile products); has there been any equipment changed or
modified; has there been a change of personnel or training; any new source material
for equipment (vent filters, gaskets, filter manufacturer, etc.); any processing
changes or room modifications, construction elsewhere in the facilities, etc.
3. Review of Environmental monitoring (EM) procedures and results for manufacturing

and laboratory area- Would the product contaminant grow on the EM medium; was
the product contamination found in the manufacturing area; Growth promotion
potential of contaminant in other medium (i.e., TSB and Thio)
4. Speciation- Record and copy the method of identification (i.e. API, Vitek, etc);
Determine if there were possible secondary contaminants that were not identified or
recorded (check original plates, or isolates); verify accuracy into the LIM system.
5. Determine potential source- Staphylococcus (skin, insect, etc); Pseudomonas (water,

plants, etc) ; yeast and mold (spores) (environmental)
Page 65 of 81
DATE: 04-25-2014
6. Review investigation report- Source of contamination; Corrective action; repeat

testing and release; does it include related lots and ancillary systems? Was the
product rejected or released? If released ask why? Evaluate justification.
E. How to Investigate a Microbiological OOS test result(s)

1. When a firm has an end product test result that indicates a failure (USP test failure,
OOS, etc) the inevitable question is –Were the results laboratory error or a true
process contamination? Below are a few starter questions to help in your FDA review
of the firm’s investigative report.
2. During the subsequent investigation, there are two areas for the review to focus: the
manufacturing site and the laboratory that determined the OOS result. The following
review questions suggest possible variables that may impact the final conclusions.
The investigations may run concurrently between manufacturing and the laboratory.
For ease of review I listed my questions first with manufacturing and secondly with
laboratory data. I divided the manufacturing review into aseptic manufacturing (High
risk) and terminally sterilized products (low risk). Part two covers those questions
that I would ask for a critical review of the microbiological data accumulated for the
Sterility failures, Microbial limits failures, etc.
F. Laboratory Facility and Analytical Review

1. Review QC records for proper/validated sterilization of all equipment and media used
during the sterility test method: manifold/ steritest; rinse fluid, culture media, canister
kits, etc.
2. Review the EM data acquired during sterility testing (i.e., settling plates, RODAC),
simulation system controls, etc. What are the microbial species and its determined
normal habitat (i.e., water, plants, people, etc?)
3. Review training records and qualification of analysts performing the test; interview
and/or observe analysts
4. Review the qualification of the bio-clean room facilities or isolator chamber used
during testing. Are there any leaks in the gloves, improper sanitization of product
container before placement into work station or isolator? Has the isolator been
evaluated for leaks?
5. Review cleaning and sterilization requirements for reusable glassware and

equipment. Poorly cleaned glassware will make sterilization of equipment more
difficult and possibly shelter trapped microbes from the killing effect of the sterilant.
6. Review laboratory areas used for sub-culturing the sterility test medium onto
enrichment plates. Cluttered work space or un-sanitized surfaces may cause plate
contamination.
Page 66 of 81
DATE: 04-25-2014
7. Check the original plates used for isolation for possible pre-existing contamination
(i.e. growth in non-streaked locations on the agar surface, subsurface growth)
8. Check to see if the medium had been recalled or has had past problems with
contamination during manufacturing.
9. It may be necessary to perform a genotype identification on the two isolates (product

source and manufacturing area isolate) if they are the same species.
G. Manufacturing Facility Review

1. Aseptically filled pharmaceuticals
Check environmental monitoring (EM) data taken from production areas and the testing
environment (i.e., S-T-A, settling plates, RODAC, etc) for microbial contamination that
matches the microbe isolated from the finished product sterility test
If no microorganisms are detected, check the adequacy of the EM method used during
manufacturing for proper sensitivity and applicability, for example
a. Are they using proper medium (i.e. non-selective medium)?
b. Have they performed growth promotion?
c. Did they use appropriate incubation time and temperatures?
d. Are they sampling in the appropriate room locations, during dynamic conditions,
longest time between cleanings/sanitation and at frequency to assure reliability of
the results?
e. If they recovered an anaerobic bacterium from the sterility test (Thioglycollate

broth) do they perform EM for anaerobic bacteria?
Have they performed a filter integrity test on the membrane use for the product
sterilization? Review the products pre-filtration bioburden levels to assure that the
concentration of bacteria in the bulk has not exceeded the membrane filtration capacity
that was determined in their validation studies. Have they changed the source or model
for the membrane filter cartridge used in the process?
Has the firm manipulated or excluded some of the data used in the final QC report?
Perhaps raw data was averaged to bring the bioburden count below the alert or action
levels. It can be helpful to request electronic Excel sheet version of data, to allow sorting
(by frequency of organism, location, etc.) and trend analysis; hard copies can be
requested later, if necessary.
Review the media simulation studies. Did the microbial species recovered in past
simulation studies match the microbe(s) recovered from the current product test failure?
Page 67 of 81
DATE: 04-25-2014
Has there been a change or breach in the personnel barrier system to protect the
product? Were there any interventions by maintenance or other staff personal during the
manufacturing of the contaminated lots? Review glove/uniform monitoring results.
Review the Antimicrobial Effectiveness challenge studies for the product. Where there
any changes to the container/closure component source or requirements?
Were there any changes to the disinfection procedure, reagents use, new personnel,
application, equipment (mops, aerosols, etc.) etc.?
2. Terminally sterilized drug product

Check autoclave validation studies for sterilization process- cold spot, heat penetration
(challenged inside dry tubing, connectors/caps/stoppers, largest liquid volume, etc.),
changes in chamber load configuration, etc.)
Check maintenance records for house steam, records for autoclave repair, new
plumbing
Check Biological Indicator (BI) information- improper storage of BIs; changes in the
culture (inoculum level and/or BI organism species) and incubation parameters
Evaluate the heat resistance characteristics of the microbial isolate found in the product
during Sterility testing and determine if it can survive during the process condition ,
review product container/closure integrity data and possible recent supply source
changes to vials or rubber stoppers; Check possible post sterilization package integrity
problem- mostly medical device issue.
H. Inspectional Elements listed in the six (6) Inspectional

Systems covered by the CP 7356.002 that cover ONLY
Microbiological Issues
Using the Inspectional criteria described in the FDA Compliance Program Guidance
Manual Program 7356.002, I selected only key coverage elements listed in five of the six
(6) Inspectional Systems that relate to microbiological issues and in some cases I
included an example for clarification.(Labeling system not included)
1. QUALITY SYSTEM
-Discrepancy and failure investigations related to manufacturing and testing:
documented; evaluated; investigated in a timely manner; includes corrective action
where appropriate.
- Validation: status of required validation/revalidation (e.g., computer, manufacturing

process, laboratory methods).
- Training/qualification of employees in quality control unit functions.
2. FACILITIES AND EQUIPMENT SYSTEM

a. Facilities
Page 68 of 81
DATE: 04-25-2014
- Cleaning and maintenance
- Facility layout and air handling systems for prevention of cross-contamination

(e.g. penicillin, beta-lactams, steroids, hormones, cytotoxics, etc.)
- Specifically designed areas for the manufacturing operations performed by the

firm to prevent contamination or mix-ups
- General air handling systems
- Lighting, potable water, washing and toilet facilities, sewage and refuse
disposal
- Sanitation of the building, use of rodenticides, fungicides, insecticides, cleaning

and sanitizing agents
b. Equipment
- Adequacy of equipment design, size, and location
- Equipment surfaces should not be reactive, additive, or absorptive
- Appropriate use of equipment operations substances, (lubricants, coolants,

refrigerants, etc.) contacting products/containers/etc.
- Cleaning procedures and cleaning validation
- Controls to prevent contamination, particularly with any pesticides or any other

toxic materials, or other drug or non-drug chemicals
- Qualification, calibration and maintenance of storage equipment, such as

refrigerators and freezers for ensuring that standards, raw materials, reagents,
etc. are stored at the proper temperatures
3. MATERIALS SYSTEM
-Representative samples collected, tested or examined using appropriate means
- Testing or validation of supplier's test results for components, containers and closures
- Rejection of any component, container, closure not meeting acceptance requirements.

Investigate fully the firm's procedures for verification of the source of components.
- Appropriate retesting/reexamination of components, containers, closures
- Water and process gas supply, design, maintenance, validation and operation
- Containers and closures should not be additive, reactive, or absorptive to the drug
product
- Documented investigation into any unexpected discrepancy

Page 69 of 81
DATE: 04-25-2014
4. PRODUCTION SYSTEM
Training/qualification of personnel
- Validation and verification of cleaning/sterilization/ depryrogenation of containers and

closures
- Established time limits for completion of phases of production (i.e. microbial growth
potential of product)
- Implementation and documentation of in-process controls, tests, and examinations

(e.g., bioburden determination pH, adequacy of mix,)
- Justification and consistency of in-process specifications and drug product final

specifications
- Prevention of objectionable microorganisms in non-sterile drug products
- Equipment cleaning and use logs
- Process validation, including validation and security of computerized or automated

processes (i.e. simulation studies)
- documented investigation into any unexpected discrepancy
5. LABORATORY CONTROL SYSTEM

Training/qualification of personnel
- Adequacy of staffing for laboratory operations
- Adequacy of equipment and facility for intended use
- Calibration and maintenance programs for analytical instruments and equipment
- Validation and security of computerized or automated processes
- Reference standards; source, purity and assay, and tests to establish equivalency to
current official reference standards as appropriate
- System suitability checks on chromatographic systems (e.g., GC or HPLC)
- Specifications, standards, and representative sampling plans
- Adherence to the written methods of analysis
- Validation/verification of analytical methods
- Control system for implementing changes in laboratory operations
- Required testing is performed on the correct samples
Page 70 of 81
DATE: 04-25-2014
- Complete analytical records from all tests and summaries of results
- Quality and retention of raw data (e.g., chromatograms and spectra)
- Correlation of result summaries to raw data; presence of unused data
- Adherence to an adequate Out of Specification (OOS) procedure which includes timely

completion of the investigation
- Adequate reserve samples; documentation of reserve sample examination
- Stability testing program, including demonstration of stability indicating capability of the

test methods (i.e. container/closure, AET)
SAMPLING
Samples of defective product constitute persuasive evidence that significant CGMP
problems exist. Physical samples may be an integral part of a CGMP inspection where
control deficiencies are observed. Physical samples should be correlated with observed
control deficiencies. Consider consulting your servicing laboratory for guidance on
quantity and type of samples (in-process or finished) to be collected. Documentary
samples may be submitted when the documentation illustrates the deficiencies better
than a physical sample. Districts may elect to collect, but not analyze, physical samples,
or to collect documentary samples to document CGMP deficiencies. Physical sample
analysis is not necessary to document CGMP deficiencies.
When a large number of products have been produced under deficient controls, collect
physical and/or documentary samples of products which have the greatest therapeutic
significance, narrow range of toxicity, or low dosage strength. Include samples of
products of minimal therapeutic significance only when they illustrate highly significant.
Dennis E. Guilfoyle, Ph.D. (ret)

Pharmaceutical Microbiologist
US Food and Drug Administration
Northeast Regional Laboratory
DOCUMENT HISTORY/CHANGE HISTORY:

Version 1.1, 10/06/14:
ii.- updated
Chapter 3. A. 2. a. iv. – revised
Chapter 8. D. 1st paragraph – revised
Chapter 10. H. 5. – author information updated
Page 71 of 81
DATE: 04-25-2014
Appendix A: Literature and Resources
A Comprehensive List of Only Microbiological Regulatory and

Scientific Literature Resources
The scope of this reading material will ONLY include microbiological scientific and regulatory
publications or websites for conventional drugs, biologics and combinatorial products. Some
references to medical device regulations will be included if relevant during an FDA investigation
that covers microbiology.
I. Legal requirements and regulations-

CFR 210 & 211 “cGMPs for finished Pharmaceuticals”
(http://www.fda.gov/cder/dmpq/cgmpregs.htm)
CFR 210 & 211 amended effective Dec, 2008 (several changes that include microbiological
requirements of aseptically filled products)
(http://frwebgate6.access.gpo.gov/cgi-
bin/TEXTgate.cgi?WAISdocID=336828190639+0+1+0&WAISaction=retrieve)
CFR 610 General Biological Product standards” (Not covered during this review)
(http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=610)
CFR 820 “Quality Systems Regulation (Devices, not covered)

(http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=820)
CFR 314.81(b)(3)(ii)- Applications for FDA approval to market a new drug (revised April 1, 2008)
For submission of an alternate microbiological method with a comparability study
(http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm)
CFR 1271 Human cells, tissues, and cellular and tissue-based products
(http://www.fda.gov/BiologicsBloodVaccines/TissueTissueProducts/default.htm)
II. FDA Compliance program Guidance Manual for FDA Staff: Drug
Manufacturing Inspections program 7356.002
(http://www.fda.gov/cder/dmpq/compliance_guide.htm)
(http://www.fda.gov/ora/cpgm/default.htm)
During an inspection this program designated six (6) critical systems for review. They include:
Quality System (always covered during an FDA inspection); Facilities &Equipment; Material;
Production; Packaging and labeling; and Laboratory control systems.
For ease of review, I included the Inspectional section for the
LABORATORY CONTROL SYSTEM

Page 72 of 81
DATE: 04-25-2014
For each of the following, the firm should have written and approved procedures and
documentation. The firm's adherence to written procedures should be verified through
observation whenever possible. These areas are not limited only to finished products, but may
also incorporate components and in-process materials. These areas may indicate deficiencies
not only in this system but also in other systems that would warrant expansion of coverage.
When this system is selected for coverage in addition to the Quality System, all areas listed
below should be covered; however, the depth of coverage may vary depending upon
inspectional findings.
- Training/qualification of personnel
- Adequacy of staffing for laboratory operations
- Adequacy of equipment and facility for intended use
- Calibration and maintenance programs for analytical instruments and equipment
- Validation and security of computerized or automated processes
- Reference standards; source, purity and assay, and tests to establish equivalency to current
official reference standards as appropriate
- System suitability checks on USP compendial tests and/or Microbial Id. system
- Specifications, standards, and representative sampling plans
- Adherence to the written methods of analysis
- Validation/verification of analytical methods
- Control system for implementing changes in laboratory operations
- Required testing is performed on the correct samples
- Complete analytical records from all tests and summaries of results
- Quality and retention of raw data (e.g., microbial identification printout)
- Correlation of result summaries to raw data; presence of unused data
- Adherence to an adequate Out of Specification (OOS) procedure which includes timely

completion of the investigation
- Adequate reserve samples; documentation of reserve sample examination
- Stability testing program, including demonstration of stability indicating capability of the test
methods
Page 73 of 81
DATE: 04-25-2014
FDA Compliance program Guidance Manual for FDA Staff: Sterile

Drug Process Inspections 7356.002A
http://www.fda.gov/downloads/ICECI/ComplianceManuals/ComplianceProgramManual/UCM125
409.pdf
Read sections entitled “Inspectional” and “Analytical” and “Attachment A” Extremely helpful.
III. Compliance Policy Guide

Sec. 100.550- Status and Responsibilities of Contract Sterilizers Engaged in the Sterilization of
Drugs and Devices (CPG 7150.16)(Oct 2006)
(http://www.fda.gov/ora/compliance_ref/cpg/cpggenl/cpg100-550.html)
Sec. 490.100 Process Validation Requirements for Drug Products and Active Pharmaceutical
Ingredients Subject to Pre-Market Approval (CPG 7132c.08) (3/2004)
(http://www.fda.gov/ora/compliance_ref/cpg/cpgdrg/cpg490-100.html)
Manual of Policies and Procedures, CDER, MAPP 5040.1
Product Quality Microbiology Information in the Common Technical Document - Quality (CTD-
Q)
(http://www.fda.gov/cder/mapp/5040.1.pdf)
Compliance Policy Guidance for FDA Staff- Sec. 280.110 Microbiological Control Requirements
in Licensed Anti-Human Globulin and Blood Grouping Reagents
(http://www.fda.gov/ora/compliance_ref/cpg/cpgbio/cpg280-110.html)
IV. US Pharmacopeia (USP) Compendium

Review all relevant product monographs (not all have microbiological requirements); the
following are regulatory chapter that contain enforceable microbiology requirements -
(http://inside.fda.gov/Library/ElectronicResourcesWebLERN/Alphabeticallist/index.htm) FDA
access to the USP is available through this link
General Notices and Requirements (page 1-13), Chart 10- Microbiology
<1> Injections
<51> Antimicrobial Effectiveness test
<55> Biological Indicators-Resistance Performance tests
<61> Microbiological Examination of Nonsterile products: Microbial enumeration tests
<62> Microbiological Examination of Nonsterile products: Tests for Specified Microorganisms

Page 74 of 81
DATE: 04-25-2014
<63> Mycoplasma
<71> Sterility Tests
<81> Antibiotics-Microbial Assays
<85> Bacterial Endotoxins Test
<151> Pyrogen Test
<161> Transfusion and Infusion Assemblies and Similar Medical Devices
<171> Vitamin B12 Activities Assay
<797> Pharmaceutical Compounding-Sterile Preparations
Dietary Supplements General Chapters Information-
<2021> Microbial Enumeration Test-Nutritional and Dietary Supplements
<2022> Microbiological Procedures for Absence of Specified Microorganisms- Nutritional and

Dietary Supplements
<2023> Microbiological Attributes of Non-sterile Nutritional and Dietary Supplements
USP Informational chapters (<1000>through <1999> are not generally enforced by FDA but
found to be extremely informative. These Informational chapters will help explain or expand on
scientific principles established in the regulatory chapter.
<1035> Biological Indicators for Sterilization
<1072> Disinfectants and antiseptics
<1111> Microbiological Examination of Nonsterile Products: Acceptable Criteria for

Pharmaceutical Preparations and Substances for Pharmaceutical Use
<1112> Application of water activity Determination to Non-sterile pharmaceutical products.
<1113> Microbial Characterization, Identification, and Strain Typing
<1116> Microbiological evaluation of clean rooms and other controlled environments
<1117> Microbiological Best Laboratory Practices
<1207> Sterile Product Packaging—Integrity Evaluation
<1208> Sterility Testing –Validation of Isolator Systems

Page 75 of 81
DATE: 04-25-2014
<1209> Sterilization—Chemical and Physicochemical Indicators and Integrators
<1211> Sterilization and Sterility Assurance of Compendial Articles
<1223> Validation of alternative microbiological methods
<1227>Validation of Microbial Recovery from Pharmacopeial Articles
<1237> Virology Test Methods
V. AOAC international-
Includes chapters on disinfectants evaluation (i.e., Phenol coefficient Methods; Hard surface
carrier test methods; Use-Dilution Method) Online access is available through
(http://inside.fda.gov/Library/ElectronicResourcesWebLERN/Alphabeticallist/index.htm)
VI. Association for the Advancement of Medical Instrumentation

(AAMI)/ International Organization for Standardization (ISO).
(http://inside.fda.gov/scripts/first/stdsactivity/csam_committees.cfm?center_id=3&page=1)
There are over fifty (50+) documents available through AAMI/ISO on the topic of “Sterilization
Processes and Validation”. These are internationally recognized standards and procedures
recognized by FDA and Industry.
AAMI/ISO Guidance documents- are available at the FDA intranet weblink given below.
VII. FDA Inspection Guidance documents-

(http://www.fda.gov/cder/guidance/index.htm)
Listed below are all the FDA guidance documents that contain only microbiological information
relevant to inspection. Not all the citations have direct web links to the specific documents. In
most cases these will be listed in the general website for CBER or CBER guidelines
(http://www.fda.gov/cber/guidelines.htm)
Submission of Documentation in Applications for Parametric Release of Human and Veterinary

Drug Products Terminally Sterilized by Moist Heat Processes (draft 8/2008)
(http://www.fda.gov/cber/gdlns/moistheat.pdf)
Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and
Gene Therapy Products (draft guidance, 2/2008) http://www.fda.gov/cber/guidelines.htm.
Guidance for Industry- Container and Closure system Integrity Testing in Lieu of Sterility
Testing as a Component of the Stability Protocol for Sterile Products (2/2008)
(http://www.fda.gov/cder/guidance/index.htm)
Page 76 of 81
DATE: 04-25-2014
Guidance for Industry- Quality Systems Approach to Pharmaceutical cGMP Regulations

(9/2006) (http://www.fda.gov/cder/guidance/index.htm)
Draft Guidance for Industry and FDA Staff- Nucleic Acid Based In Vitro Diagnostic Devices for
Detection of Microbial Pathogens (12/2005) Docket number 2005D-0434
(http://www.fda.gov/dockets/ecomments)
Guidance for Industry- Manufacturing Biological Drug Substances, Intermediates, or Products

using Spore-forming Microorganisms (2/2005) (http://www.fda.gov/cder/guidance/index.htm
Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing

Practice, 9/2004 (http://www.fda.gov/cder/guidance/5882fnl.htm)
Comparability Protocols - Chemistry, Manufacturing, and Controls Information

(http://www.fda.gov/cber/gdlns/cmprprot.htm), Required for industry interested in substituting an
automated/Rapid Microbiological method in place of the USP compendial method cited in their
original application (2/2003)
Guidance for Industry- Sterility Requirement for Aqueous- Based Drug Products for Oral
Inhalation—Small Entity Compliance Guide (11/2001)
(http://www.fda.gov/cder/guidance/4774fnl.pdf)
Guide to Inspections of Quality Systems-Medical Device (8/1999)
Guidance for Industry-Content and Format of Chemistry, Manufacturing and Controls

Information and Establishment Description Information for a Vaccine or Related Product
(1/1999) (http://www.fda.gov/cber/guidelines.htm)
Guide to Inspections of Lyophilization of Parenterals (10/18/97)
(http://www.fda.gov/ora/inspect_ref/igs/lyophi.html)
Guide to Inspections of Cosmetic Product manufacturers (2/1995)

(http://www.fda.gov/ora/inspect_ref/igs/cosmet.html)
Guide to Inspections of Sterile Drug Substance Manufacturers, (7/1994)

(http://www.fda.gov/ora/inspect_ref/igs/subst.html)
Guide to Inspections of Topical Drug Products (7/1994)

(http://www.fda.gov/ora/inspect_ref/igs/topic.html)
Guidance for Industry- Submission Documentation for Sterilization Process Validation in

Applications for Human and Veterinary Drug Products (11/1994)
(http://www.fda.gov/cder/guidance/cmc2.pdf)
Guideline for the manufacture of In Vitro Diagnostic Products (1/1994)

(http://www.fda.gov/cdrh/comp/918.pdf)
Page 77 of 81
DATE: 04-25-2014
Guide to Inspections of Microbiological Pharmaceutical Quality Control Laboratories (7/1993)

(http://www.fda.gov/ora/inspect_ref/igs/micro.html)
Guide to Inspections of High Purity Water Systems, (7/1993)

(http://www.fda.gov/ora/inspect_ref/igs/high.html)
Guide to Inspections of Validation of Cleaning Processes (7/1993)

(http://www.fda.gov/ora/inspect_ref/igs/valid.html)
FDA Biotechnology Inspection Guide, Reference materials and training aids (11/1991)
(http://www.fda.gov/ora/inspect_ref/igs/biotech.html)
Guidance for Industry, Pyrogen and Endotoxins Testing: Questions and Answers (June 2012)
VIII. Inspectors technical guidance (ITG)-

There were a few ITGs that were written regarding microbiological issues. I listed all of them
below with direct link for your perusal. Although they are a little dated the information is still
relevant.
PYROGENS, STILL A DANGER (1/12/79 Number: 32)

(http://www.fda.gov/ora/inspect_ref/itg/itg32.html)
HEAT EXCHANGERS TO AVOID CONTAMINATION (7/31/79 Number: 34)

REVERSE OSMOSIS (10-21-80 Number: 36) (http://www.fda.gov/ora/inspect_ref/itg/itg36.html)
BACTERIAL ENDOTOXINS/PYROGENS (3/20/85 Number: 40)

LYOPHILIZATION OF PARENTERALS (4/18/86 Number: 43)

WATER FOR PHARMACEUTICAL USE (12/31/86 Number: 46)

MICROBIOLOGICAL CONTAMINATION OF EQUIPMENT GASKETS WITH PRODUCT

CONTACT (12/31/86 Number: 48) (http://www.fda.gov/ora/inspect_ref/itg/itg48.html)
Report No. Title- PDA Technical Reports related ONLY to Microbiological Issues Date
1 Validation of Moist Heat Sterilization Processes: Cycle Design, July

Development, Qualification and Ongoing Control 2007
3 Validation of Dry Heat Processes Used for Sterilization and 1981

Depyrogenation
Page 78 of 81
DATE: 04-25-2014
4 Design Concepts for the Validation of Water-for-Injection Systems 1983
5 Sterile Pharmaceutical Packaging: Compatibility and Stability 1984
7 Depyrogenation 1985
11 Sterilization of Parenterals by Gamma Radiation 1988
13 Fundamentals of an Environmental Monitoring Program 1990

(Revised
2001)
15 Industrial Perspective on Validation of Tangential Flow Filtration in 1992

Bio-pharmaceutical Application
20 Report on Survey of Current Industry Gowning Practices 1990
21 Bioburden Recovery Validation 1990
22 Process Simulation Testing for Aseptically Filled Products 2011
23 Industry Survey on Current Sterile Filtration Practices 1996
26 Sterilizing Filtration of Liquids 2008
28 Process Simulation Testing for Sterile Bulk Pharmaceutical 2006

Chemicals (revised)
29 Points to Consider for Cleaning Validation 2012
30 Parametric Release of Pharmaceuticals Terminally Sterilized by 1999

Moist Heat
33 Evaluation, Validation and Implementation of New Microbiological 2000

Testing Methods
34 Design and Validation of Isolate Systems for the Manufacturing and 2001
Testing of Health Care Products
35 A Proposed Training Model for the Microbiological Function in the 2001

Pharmaceutical Industry
36 Current Practices in the Validation of Aseptic Processing -- 2001 2002
40 Sterilization Filtration of Gases 2005
41 Virus Filtration 2005
Page 79 of 81
DATE: 04-25-2014
45 Filtration of Liquids using Cellulose-based depth filter 2008
57 Analytical Method Validation and Transfer for Biotechnology products 2012
61 Steam in place 2013
IX. Miscellaneous FDA Documents and References

This list of references may not be entirely microbiology but very important none the less if you
want to be a serious FDA field investigator
FDA Inspectional Operational Manual (http://www.fda.gov/ora/inspect_ref/iom/default.htm)
FDA Warning Letters and Responses http://www.fda.gov/foi/warning.htm
FDA Bacteriological Analytical Manual (BAM) (1/2001) (http://www.cfsan.fda.gov/~ebam/bam-

toc.html)
FDA Sterility Analytical Manual (2001, contact FDA Division of Field Science for a copy)
X. Important Government and International organizations:

National Institute of Health (www.nih.gov)
Center for Disease Control and Prevention (www.cdc.gov)
CDC report on environmental monitoring

(http://www.cdc.gov/ncidod/dhqp/gl_environinfection.html)
World Health Organization (www.who.org) Those of you interested in world travel and global
inspections with the FDA may want to become familiar with this website. International
pharmaceutical regulations along with monitoring of disease outbreaks around the globe may be
important to you if you have been assigned to work in a high risk area.
XI. Industry Technical references-

Parenteral Drug Association (PDA) Technical Reports- Although the scientific
recommendations in these technical reports are not enforceable by FDA they do contain
industry current manufacturing practices and scientifically sound principles that support
regulatory concerns. This is prime reading material for novice and experts interested in
understand microbiological principles and applications.
(http://eroom.fda.gov/eRoom/CDER7/CDERPDATechnicalReports/0_227d)
XII. Books and Commercial Trade reports:
(This is not an FDA endorsement, just a potential starter list

Page 80 of 81
DATE: 04-25-2014
ASM, Manual of Clinical Microbiology;
Disinfection, Sterilization, and Preservation, by S Block;
Bergey’s manual systematic Bacteriology
Remington’s Pharmaceutical Sciences
F-D-C Monthly Reports
Excellent summary of conferences, FDA regulation changes, Key Industry personnel and often
a list of the most recent Product Recalls and regulatory actions by FDA. Need to sign up for
email membership. Instructions for membership enrollment are available at FDA website below.
(So easy even a Microbiologist can do it)
(http://inside.fda.gov/Library/ElectronicResourcesWebLERN/Alphabeticallist/index.htm)
The “Gold Sheet”- Pharmaceutical & Biotechnology Quality Control
The “Pink Sheet”- Prescription Pharmaceuticals and Biotechnology
The “Gray Sheet”- Medical Devices Diagnostics & Instrumentation
The “Silver Sheet”- Medical Device Quality Control reports
XIII. Free Trade publications available on line-

(Not an extensive list but a fair start)
Pharmaceutical Technology (www.pharmtech.com)
Controlled Environments (www.cemag.us)
American Pharmaceutical Review (www.americanpharmaceuticalreview.com)
International BioPharm (www.biopharminternational.com)
XIV. Professional memberships

These organizations have available searchable references. There are many other professional
societies chose one you can afford and enjoy.
International Society of Pharmaceutical Engineers (www.ispe.org)
American Society for Microbiology (www.asm.org)
Parenteral Drug Association (www.pda.org)
Page 81 of 81

Micro Test Final PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Micro Test Final PDF

Uploaded by

Copyright:

Available Formats

PROTECTION MEASURES TO PREVENT AND CONTROL

Equipment • Safe and proper use of medications

Background (cont.) Background (cont.)

Background (cont.) Background (cont.)

Scope of USP <797> Multidisciplinary • Personnel cleansing and gowning

Personnel Cleansing and Gowning Personnel Cleansing and Gowning (cont.)

• Hand Washing • After washing hands, put on

• Face mask must be worn • Personnel cleansing and gowning

Responsibilities of Compounding Personnel Responsibilities of Compounding Personnel (cont.)

Low-Risk Level Characteristics Low-Risk Level Characteristics (cont.)

• Low-Risk Conditions • Examples of Low-Risk Compounding

Medium-Risk Level Characteristics (cont.) Medium-Risk Level Characteristics (cont.)

High-Risk Level Characteristics High-Risk Level Characteristics (cont.)

• Personnel cleansing and gowning • Physical Inspection

• Sterilization and Bacterial Endotoxin Testing

Selected Sections of Chapter <797> Personnel Training and Assessment

• Personnel cleansing and gowning

Personnel Training and Assessment (cont.) Selected Sections of Chapter <797>

• Critical Site • Cleaning and sanitizing workspaces

• Personnel garb and hand washing (policy PH06-04)

Selected Sections of Chapter <797> Equipment

Selected Sections of Chapter <797> Storage and Beyond-Use Dating

CONTROL OF • In 1847, Dr. Ignaz Semmelweis's close

• Kolletschka soon died of symptoms

Joseph Lister Lister’s Carbolic

– Carbolic acid (phenol)

Control of Microbial Control of Microbial

BACTERIOSTASIS ASEPSIS (A = without)

– Actively growing organisms more

Bacterial Death Rate 1,000,000

PHYSICAL METHODS Physical Methods

MOIST HEAT Autoclave

LIQUID FILTRATION GAS

Used in cell culture

Nitrates/Nitrites Nitrates in food

Disk Diffusion Method Disinfectants

Phenols Chemical Methods -Disinfectants

• In most situations neither condition is met to make

SOAPS Chemical Methods -Disinfectants

Types Of Disinfectants (cont.) Chemical Methods -Disinfectants

Chemical Methods -Disinfectants

Chemical Methods -Disinfectants GASEOUS

Maternal mortality, 1842

The Intervention: Hand Hygiene: Not a New Concept

~ Hand antisepsis reduces the frequency of patient infections ~

Colonized or Infected: The Iceberg Effect

Which method do you use to

Which of the following hand

1. Plain soap and water 1. Plain soap and water

It is acceptable for healthcare How much time would an ICU

How much time would an ICU Healthcare-associated

* Based on 12 opportunities/hour, handwashing time=60 seconds,

When a healthcare worker touches a A co-worker who examines a patient

Can a Fashion Statement

risk to patients. Artificial (n=27)

5. Strongly disagree Avoid wearing artificial nails, keep natural nails

Hand Hygiene Options at

What is the Story on

~ Do not refill lotion bottles ~

Aseptic Processing - Overview

• Two categories of sterile products

1 Manufacture of sterile medicines – Advanced workshop for SFDA GMP 2

Aseptic Processing - Overview Aseptic Processing - Overview