PHA 6114 LAB Sterilty Test Merged

MICROBIOLOGY AND PARASITOLOGY
Lecture or Laboratory \ FIRST SEMESTER
UNIT 07: STERILITY TESTING

OUTLINE • Sterility assurance - this will make sure that our drug
product does not have any microorganisms at all
I. STERILITY
A. Factors Affecting the Sterile Products
i. Environment FACTORS AFFECTING THE STERILE PRODUCTS
ii. Equipment
iii. Product 1. ENVIRONMENT
II. STERILE PRODUCTS
A. Aseptic Processing • Plays an important role in sterility of our drug products
B. Terminally-Sterilized Products • It covers the facility design (how do you design your
III. STERILITY TESTING facility that a manufacture’s that the drug products
A. Membrane Filtration Method needs to be sterile)
B. Direct Inoculation Method • Most of the manufacturing companies prefer or would
IV. GENERAL PROCEDURE
have separate buildings if they are producing
A. Membrane Filtration Method
antibiotics and parenteral preparations because these
i. Growth Promotion Test
are strictly needs to be sterile
ii. Culture Media for Sterility Testing
B. Direct Inoculation Method • Right equipment
i. Preparation of Test Culture Suspension • Personal traffic (how many people can access the area)
V. METHOD SUITABILITY TEST • Personnel hygiene (PPE that they used, trainings
A. Growth Promotion Test provided to them)
B. Suitability Test • Heating, ventilation, and air conditioning or what we so
C. Product Testing called HVAC System
VI. INTEGRATION
STERILITY
• Complete absence of viable microorganisms (bacteria,
yeasts, and molds)
• So, the microbiological safety is achieved through the
implementation of interrelated safety protocols in a
combination with all of these [picture seen below] to
provide the confidence that the items or the drug
products that we will be produced suitable for as it is
label.
2. EQUIPMENT
• The design is important to deliver the need of the
company and the sterility that a drug product needs
• The area on where this equipments are located,
sterilization before and after using them is equally
important as well as the qualification or the training of
the personnel operating this equipments
2. PRODUCT (DRUG ITSELF)

• For the verification of the safety of the product that’s
labelled as sterile, we do sterility assurance
DGU, GBS, JJC, QME | 2F-PH 1


• For us to maintain the sterility of the drug product → (1) 2. Cannot be terminally sterilized must be aseptically
raw material plays a vital role (the source must be prepared 
reputable and we must be sure that they do proper
ASEPTIC PROCESSING
handling of the material prior to giving it to the
manufacturing firm) 
• (2) Sterilization and depyrogenation (removal of
pyrogens) of drug products → this must be done to
assure that our drug product will not introduce other
effects as it is intended or as it is labelled.  
• (3) storage conditions matter → the method validation
and the quality control are also equally important that • Designed to prevent the introduction of viable
the drug products are sterile   microorganism into separately sterilized materials
during their assembly into a sealed sterile package  
• We prepare this from raw materials up to the final
product (it should be sterile and prepared aseptically)
• Each component is individually sterilized, or several
components are combined with the resulting mixture
sterilized. 
• Preparation of solution filtered through sterilizing filter
STERILE PRODUCTS
then filled into sterile containers. 
• Process is designed to prevent the introduction of • All materials should be protected from contamination  
viable microorganisms into/onto separately sterilized
• Point of sterilization up to the closure of the primary
materials during their assemble into a sealed sterile packaging 
package  
TERMINALLY-STERILIZED PRODUCTS
• Each package should be sterilized and most
• Lowest-risk category of sterile pharmaceutical
importantly the primary packaging should be
products 
sterilized as well because it comes in contact with the
• Sterilization process imparts quantifiable safety level
product  
achieved by delivering measurable physical conditions
• Follow aseptic techniques (steps) 
that corresponds to microbial lethality  
• Injections, ophthalmic preparations, irrigation solutions,
• Probability of the terminal sterilization process
hemodialysis solutions (PRODUCTS THAT MUST BE
generating a non sterile unit of ≤ 10-6 
STERILE BEFORE ADMINISTERING IT TO
• The probability of 1 non sterile unit in 1 million units
PATIENT)  
produced; If you have 2 non sterile unit or more, the
• Two categories: 
batch should be rejected  
1. Can be sterilized in final container (terminal
• You sterilize the drug product in its final container  
sterilization) 


• For products that can withstand steam sterilization (unless otherwise justified and
cycle authorized)
o E.g., ampoules and vials (dosage forms that Parenteral preparations
can be terminally-sterilized)
• Assure sterility of finished goods
• Product is sterilized in its final container
• Ionizing radiation Not more than 100 10% or 4 containers, whichever is
• Steam sterilization (e.g., autoclaving) containers the greater
• Dry heat sterilization (e.g., oven sterilization) More than 100 but 10 containers
not more than 500
STERILITY TESTING containers
More than 500 2% or 20 containers, whichever is
• Applied to substances, preparations, or articles which containers less
*For large-volume 2% or 20 containers, whichever is
are required to be sterile 
parenterals less
• Injections, ophthalmic preparations, irrigation solutions, Antibiotic Solids
hemodialysis solutions   Pharmacy bulk 20 containers
packages (<5 g)
• Indicates that no contaminating microorganism has
Pharmacy bulk 6 containers
been found in the sample examined under the packages (>/= 5g)
Bulks and blends See Bulk solid products
conditions of the test 
• Carried out under aseptic conditions 
Ophthalmic and other non-injectable preparations
MEMBRANE FILTRATION METHOD Not more than 200 5% or 2 containers, whichever is

containers greater
• Filters with nominal pore size of n.m.t. 0.45 nm 
More than 200 10 containers
• Cellulose nitrate filter: aqueous, oily, and weakly containers
If the product is presented in the form of single dose
alcoholic solutions  containers apply the scheme shown above for
• Cellulose acetate filters: strongly alcoholic solutions  preparations for parenteral use
Catgut and other 2% or 5 packages, whichever is
surgical sutures for the greater, up to a maximum
DIRECT INOCULATION METHOD veterinary use total of 20 packages
*Not more than 100 10% or 4 articles, whichever is
articles greater
More than 100, but 10 articles
not more than 500
articles
More than 500 2% or 20 articles, whichever is
articles less
Bulk solid products
Up to 4 containers Each container
containers, but not greater
more than 50
containers
containers greater
Table 1. Minimum number of articles to be tested in relation
to the number of articles in the batch
E.g.
Number of items in Minimum numbers of items to
the batch be tested for each medium


1. A batch of 120 pcs Paracetamol ampules were d. If the answer is in decimal form, we need to
produced today, how many ampules should you need round it up into a whole number 
for sterility testing? 
a. first thing that you need to do is to know what
the drug product is as well as the dosage form
and quantity 
b. 120 pcs Paracetamol ampule: parenteral
preparation 
c. Based on the table, on parenteral
preparations, it fits the description of More
Table 2. Minimum quantity to be used for each medium
than 100 but not more than 500 hence, the
answer is 10 ampules.  Quantity per container Minimum quantity to be
used (unless otherwise
2. A lot of 500 Timentin 3.1g powder for reconstitution is
justified and authorized)
on the batch record, how many vials should you need Liquids
Less than 1 mL The whole contents of each
to perform sterility testing?  container
a. Timentin is a combination of ticarcillin and 1-40 mL Half of the contents of each
container but not less than 1
clavulanic acid which is an antibiotic. Also, the mL
dosage form is a powder. Thus, it is a solid Greater than 40 mL, and 20 mL
not greater than 100 mL
dosage form  Greater than 100 mL 10% of the contents of the
container, but not less than
b. Based on the table in under parenteral
20 mL
preparations, antibiotic solids, we need to test Antibiotic liquids 1 mL
Insoluble preparations, Use the contents of each
20 containers for the sterility testing 
creams, and ointments to container to provide not less
3. The last batch of NSS for irrigation produces 10, how be suspended or than 200 mg
emulsified
many should you need for sterility testing?  Solids
a. Based on the table, it is not an opthalmic Less than 50 mg The whole contents of each
container
preparation, but IT IS A NON-INJECTABLE 50 mg or more, but less Half of the contents of each
PREPARATION   than 300 mg container but not less than 50
mg
b. Based on the given, only 10 products are 300mg - 5g 150 mg
Greater than 5g 500 mg
produced, so it fits the description of Not
Catgut and other surgical 3 sections of a strand (each
more than 200 containers hence, the sutures for veterinary use 30-m long)
*Surgical dressing/ 100 mg per package
answer is 5% or 2 containers, whichever is cotton/ gauze (in
greater  packages)
Sutures and other The whole device
c. 2 containers will be utilized because 5% of individually-packaged
10 is 0.5 and 0.5/ 1 is less than 2, thus we use single-use material
Other medical devices The whole device, cut into
2 containers  pieces or dissassembed


E.g.
1. Quinoflox i.v. Infusion 
a. Type: Surgical dressing/ cotton/ gauze (in

a. Dose: 200mg/ 100mL  package) 
b. Dosage form: antibiotic 
b. Hence, the minimum quantity to be used is
c. Therefore, if it’s an antibiotic liquid, we need 100 mg per package 
1mL since it is Greater than 10 mL, and not
GENERAL PROCEDURE
greater than 100 mL 
• Preparation of Test Strains 
2.  Insulin syringe  • Method Suitability Test 
• Product Testing 
• Steps of General Procedure 
o Before a product could be labelled as sterile,

it must undergo sterility testing 
o To assure sterility, one should have done the
formulation using the aseptic conditions  
o Next, products must be sealed in aseptic
conditions 
a. Look at the table and classify the type of
o Afterwards, the final sterilization must be
preparation: Sutures and other individually-
done using the suitable process (as
packed single-use material 
discussed by Sir Renson). 
b. Hence, the minimum quantity to be used is
the whole device  METHOD SUITABILITY TEST (METHOD
VALIDATION)
3.  Cotton  • Performed prior to actual product test, specifically if it
is a new formulation or whenever there is a change in

its experimental conditions in the test. 


• Conducted to demonstrate the applicability of the
method for detection of microbial contamination in the

test product 
• Suitability tests:
o Growth promotion test
§ to demonstrate that the medium
supports growth and has the ability
to detect organisms in the presence
of the test sample.
o Suitability of counting method

• Staphylococcus aureus, Bacillus subtilis, and
§ based on sample characteristics
Pseudomonas aeruginosa will be incubated in an SCD
and required microbial limits.
agar at a temperature of 30°-305°C for about 18 to 24
GROWTH PROMOTION TEST
hours. 
• Seed-lot systems are recommended for long-term
storage, and stock culture of each organism is limited
to not more than five passages removed from the

master seed-stock. 
• As an alternative to preparing and diluting the fresh
suspension of vegetative cells of Aspergillus
brasiliensis for the bacillus subtilis, a stable spore
suspension must be prepared and then appropriate
volume of the spore suspension is used for test

inoculation.  
• Candida albicans (Yeast) and Aspergillus brasiliensis
• The stable spore suspension may be maintained at 2–
(mold) will be incubated in an SDA at 20°C - 25°C for 5
8 °C for a validated period of time. 
-7 days. 
• Standardized cell suspensions should be used within 2
hours or stored at 20- 8°C for not more than 24 hours.

• The suitable culture media for product testing are the
fluid thioglycolate media (FTM) that supports both

• Clostridium sporogenes will be incubated under the
aerobic and anaerobic bacteria. The thioglycolate in
anaerobic reinforced medium at 30°C to 35°C for 24 to
48 hours.  the medium reduces the oxygen to water. This
decreases the amount of oxygen and promotes the

growth of anaerobes. 
• On the other hand, the second medium will be the
Soyabean Casein Digest Medium. It is a versatile
medium that supports the growth of fungi and your

aerobic bacteria. 
MICROBIAL ENUMERATION TESTS (MET)

PREPARATION OF TEST CULTURE SUSPENSION
 
CULTURE MEDIA FOR STERILITY TESTING


1. To prepare a test culture suspension, the turbidity of 3. Incubate Fluid Thioglycollate Medium at 30°-35°C, and
McFarland’s solution must be measured, and it is SCD Medium at 20°- 25°C for 3 days for bacteria, and
equivalent to 1.5 x 10⁸ cfu/mL.  for 5 day for fungi. 
2. Measure 0.1 mL and dilute it with NSS, until the 100mL 4. Clearly visible growth of the microorganisms must be
mark is reached.  observed in order to confirm that the growth promotion
3. From the volumetric flask, you need to collect 0.5 mL test was successful. 
of the diluted suspension.  SUITABILITY TEST METHOD

4. Transfer it into another volumetric flask to further dilute
it with 100mL NSS. 
5. The final concentration will be estimated as 0.1 mL or

75 cfu. 
STERILITY TESTING: METHOD SUITABILITY TEST

(METHOD VALIDATION)
GROWTH PROMOTION TEST
1. Two individual tubes containing Fluid Thioglycollate
Medium or Soybean-Casein Digest Medium, transfer
quantity of product to be examined (such that product

volume is not more than 10% of volume of medium) 
2. Refer to the table of the product volume that you should

use. 
3. Inoculate Fluid Thioglycollate Medium with not more

1. For the growth promotion test, inoculate the medium
than 100 cfu of Clostridium sporogenes, Pseudomonas
with 0.1 mL of the final culture suspension to provide
aeruginosa, and Staphylococcus aureus; 
75 cfu. Clostridium sporogenes, Pseudomonas
4. Inoculate SCD medium with not more than 100 cfu of
aeruginosa, and Staphylococcus aureus for the Fluid
Aspergillus brasiliensis, Bacillus subtilis, and Candida
Thioglycollate Medium. Inoculate Fluid Thioglycollate
albicans. 
Medium with nmt 100 cfu of Clostridium sporogenes,
5. Incubate Fluid Thioglycollate Medium at 30°-35°C, and
Pseudomonas aeruginosa, and Staphylococcus
SCD Medium at 20°-25°C for 14 days. 
aureus.  
6. Clearly visible growth of the microorganisms should
2. Inoculate SCD medium with nmt 100 cfu of Aspergillus
appear. If there is no growth in the medium, it means
brasiliensis, Bacillus subtilis, and Candida albicans. 
that the product contains antimicrobial activity, which
has not been eliminated in the test.  


7. Modification of the conditions is recommended in order
to eliminate the antimicrobial activity and to repeat the

suitability test.  
PRODUCT TESTING
Growth promotion & Suitability of Test (Pseudomonas

aeruginosa)
1. Appropriate volume of the product must be used, such Results of Product Testing:
that the volume is not more than 10% of the volume of • Observations: Clear, yellow solution
• Interpretation: No microbial growth because there is
the medium. To individual tubes containing Fluid
no turbidity. There is no microbial growth.
Thioglycollate Medium or Soybean-Casein Digest • Disposition: Accept, because there is no turbidity, no
bacterial growth, and navalidate yung culture media
Medium, transfer the quantity of product to be
What is the importance of doing method validation prior to
examined.  sterility testing?
2. Incubate Fluid Thioglycollate Medium at 30°C-35°C)
• To test if our media can grow our culture
and SCD Medium at 200-25°C for 14 days.  • If there is no growth, vial ABC is clearly sterile and
does not contain any microbial properties.
3. At intervals during the incubation period and at its • It is important to ensure that the product is sterile to
conclusion, examine the media for macroscopic avoid causing infections.
evidence of microbial growth.  Why is FTM used as the media?
4. There should be no evidence of microbial growth for • FTM can detect aerobic and anaerobic bacteria
the examined product to comply with the sterility test. 
5. If you have observed clear growth, it means that the REFERENCES
product should fail the test.  - Asynchronous Discussion of MicroPara Professors

- Synchronous Integrative Discussion
- Exercise 7 Handout
INTEGRATION
Optional: add memes, songs, QR codes
ABC Vial was subjected to sterility testing using FTM. The LETS GO 2F! <3
following observations are recorded:

Laboratory \ FIRST SEMESTER
EXERCISE 8: BACTERIAL ENDOTOXIN TEST

OUTLINE • Part of the outer portion of the cell wall of gram-negative
bacteria.
I. Endotoxin and Exotoxin • They are liberated when the bacteria die, and the cell wall
A. Endotoxin breaks apart
B. Exotoxin
II. Gram Negative Bacteria Cell Wall Structure • Only found in gram-negative bacteria
A. Review • Called as the lipopolysaccharide
III. Pyrogen
A. Mechanism of Action EXOTOXIN
B. Pyrogen Testing Assays
C. Review
D. Rabbit Pyrogen Test
E. Monocyte Activation Test
IV. Bacterial Endotoxin Test (BET)
A. USP-NF
B. Depyrogenation
C. Three Techniques for BET
D. Mechanism
E. Pyrogens and Endotoxin
F. Bacterial Endotoxin
G. LAL Test
H. Three techniques for BET
I. Theory
V. Theory behind the Gel Clot Technique
A. Rationale Behind the Clotting Mechanism
B. LAL-Clotting Cascade
C. Gel-Clot Technique • Are produced inside mostly gram-positive bacteria as part
i. Procedure of their growth and metabolism. They are then secreted or
ii. Reading of results released following lysis into the surrounding medium
VI. Experiment Procedure (Part 3 of the Lecture)
A. Introduction
B. General Procedure GRAM NEGATIVE BACTERIA CELL WALL STRUCTURE
C. Preparation of Standard Endotoxin Solution
D. Determination of Maximum Valid Dilution
E. Endotoxin Limit per Route of Administration
F. Concentration of Sample Solution
G. The labelled Lysate Sensitivity
i. Determination of Maximum Valid Dilution
VII. Preparatory Testing
A. Test for Confirmation of Labelled Lysate Sensitivity
i. Sample Preparation
ii. Test for Interfering Factors
iii. Limit Test
VIII. Integration
ENDOTOXIN AND EXOTOXIN

ENDOTOXIN
• Not all endotoxins are the same.

• Some are more pyrogenic than others
• Eg. Endotoxin from E. coli is 1000x more pyrogenic
than from H. pylori
• In the gram negative bacteria, the lipopolysaccharide
is composed of
o Lipid A- active component
Causes pyrogenicity of the
endotoxin
o Core polysaccharide
JJC, QME, GBS, DGU | 2F-PH 1


o Repeating oligosaccharides
REVIEW RABBIT PYROGEN TESTING

• Do gram positive bacteria have LPS • The pyrogen test is designed to limit to an acceptable level
o No, only gram-negative bacteria the risks of febrile reaction in the patient to the
• Do all gram-negative bacteria have LPS administration, by injection, of the product
o No, not all gram-negative bacteria (sphingomonas) • The test involves measuring the rise of temperature of
rabbits after the intravenous injection of a test solution
PYROGEN
• Endotoxin, LTA, PG, fungi, synthetic substances, IL-1𝛽, MONOCYTE ACTIVATION TEST
TNF-𝛂, IL-6M
• Employing human blood to stimulate the first stages of the
• Anything can be considered as a pyrogen (fungi, synthetic human immune system, it is used to detect both endotoxins
substances) and non-endotoxin pyrogens in parenteral products, such
• Anything that can cause or propagate fever or other • as pharmaceuticals and medical devices, the MAT gives an
inflammatory responses in vitro alternative to conventional animal testing
• Can be done in vitro
MECHANISM OF ACTION
• Mimic the human immune system and detect endotoxin
• Binding to TLR4-MD2 complex
• Release of IL-1𝛃, TNF𝛂, IL-6 BACTERIAL ENDOTOXIN TEST (BET)
• COX-2 expression in CNS
• The bacterial endotoxins test, also known as Limulus
• Release of Prostaglandin E2 amoebocyte lysate (LAL) test, is an alternative in vitro
• Propagation of prostaglandin/ cytokines endotoxin assay, accepted by main regulatory drug
• Immune response towards the foreign substance agencies like FDA, European Medicines Agency (EMA) or
Pharmaceuticals and Medical Devices (PMDA) among
PYROGEN TESTING ASSAYS others
• Rabbit pyrogen test (RPT) • In vitro test of endotoxin
• Limulus Amoebocyte Lysate Test (LAL) • Other pyrogenic materials will not be detected, only
• Monocyte Activation Test (MAT) endotoxin assays
• The human health effects of acute exposure to endotoxin
include clinical symptoms such as fever, shaking chills, and USP-NF
septic shock • The bacterial endotoxins test (BET) is a test to detect or
o Histamine quantify the endotoxins from Gram-negative bacteria using
o Interleukin-1 and Interleukin-2 amoebocyte lysate from the horseshoe crab (Limulus
o Myocardial depressant factor polyphemus or Tachypleus tridentatus).
o Anaphylatoxins • Get the blood of the horseshoe crab (amoebocyte in the
o Platelet-activating factor blood) and destroy the cells enzyme that will be used in
o Oxygen-derived free radicals the test
o Bradykinin
o Beta-endorphins
o Prostaglandin, thromboxane, leukotriene and Rabbit Pyrogen Test (RPT) Limulus Amoebocyte Lysate (LAL)
prostacyclin release Rabbits have a similar Horseshoe crab blood clots in the
o Activation of coagulation system endotoxin tolerance to presence of endotoxins
o Tumor necrosis factor humans
• Similar body
REVIEW temperature
The sample is injected into Extract from the amoebocyte
• Only endotoxins are pyrogenic the rabbit and changes in (equivalent in function to white blood
o False, anything can be pyrogenic as long as it causes body temperature (fever) are cells in humans) is mixed with the
fever observed sample
• All gram-negative bacteria have endotoxin
o False
• Endotoxin is potentially lethal
o True

Detects all pyrogens Highly sensitive to bacterial • Factor C in the presence of the Endotoxin will become
endotoxin activated
• Only detects gram • Activated Factor C will activate Factor B
negative bacteria
Costly, time consuming, directly Significantly cheaper, rapid (30-60
• Activated Factor B with Activated Factor G in the presence
harms animals, cannot quantify min), does not directly harm of glucan will act upon the pro-clotting enzyme
results animals, can quantify results • Pro-clotting enzyme will turn into the activated clotting
Lysate sourced from Tachypleus is enzyme
referred to as TAL. The method is
equivalent and accepted • The clotting enzyme will turn the substrate Coagulogen into
the coagulin
• The coagulin will develop clots hence, gel formation
DEPYROGENATION
• Remove whatever that causes the fever
• Even if the bacteria is dead, it can still cause problems to
PYROGEN AND ENDOTOXIN
the immune system
• A heterogenous group of chemical entities that share the
• We have to remove them
characteristic of (when injected) being able to cause fever
• Traditional sterilizing methods such as moist heat,
• The main pyrogen encountered in the pharmaceutical
irradiation, ethylene oxide, etc. do not significantly
industry is of Gram-negative bacterial origin
destabilize endotoxin
• Must be heat inactivated or removed using mechanical
• Bacterial endotoxin is the lipopolysaccharide (LPS)
component of the cell wall of Gram-negative bacteria
means such as distillation, reverse osmosis or ultrafiltration
o Lipopolysaccharide (LPS) component is the one
• Moist heat, irradiation, and ethylene oxide can kill the responsible for providing the activation of the
bacteria, but cannot effectively do depyrogenation endotoxins
It is pyrogenic and is a risk to patients who are
THREE TECHNIQUES FOR BET administered with intravenous and intramuscular
• Gel clot technique, based on gel formation (positive result) preparations
• Turbidimetric technique, based on the development of
turbidity after cleavage of an endogenous substrate BACTERIAL ENDOTOXIN
• Chromogenic technique, based on the development of • Ubiquitous in nature
turbidity after cleavage of an endogenous substrate • Has potent toxicity
• In the event of doubt or dispute, the final decision is made o Cause shock, fever, and even death
based upon the gel clot limit test unless otherwise indicated • Stable under extreme conditions
on the product monograph • It can survive even with the high temperature and
whatever environment it is
MECHANISM • Likely to occur in the manufacturing process
o This may present a significant risk to many
pharmaceutical products, specially the parenteral
products
LAL TEST
• Most widely used


• Also known as Limulus Amoebocyte Lysate (LAL) Test • First, endotoxin comes in contact with LAL
• An alternative in-vitro endotoxin assay • Upon the contact of these two substances, this would
• It is used to detect or quantify endotoxins from gram- initiate a series of enzymatic reactions that will result into
negative bacteria using aqueous extract of blood cells activation of a pathway
(amoebocyte) from the horseshoe crab (Limulus • After the activation of pathway, there will be a production of
Polyphemus or Tachypleus tridentatus) at leat three serine protease zymogens namely Factor B,
• The Pharmacopeial monographs for this test with the USP Factor C, and proclotting enzymes
and other Pharmacopeia have already established this long • After producing these zymogens, it will alter amoebocyte
ago for a relatively comprehensive and have been applied coagulogen
to the testing of parenteral products and water systems for o The coagulogen is the invertebrate-like fibrinogen
bacterial endotoxicity ever since 1980s clottable protein
• Lastly, there will be a formation of coagulin gel, thus the
THREE TECHNIQUES FOR BET word gel
• Gel Clot Technique
o Used to get the final decision of a certain batch of RATIONALE BEHIND THE CLOTTING MECHANISM
pharmaceutical products • The clotting mechanism of the blood of the crab is designed
• Turbidimetric Technique to prevent the spread of bacterial contamination throughout
• Chromogenic Technique the horseshoe crab's biochemical system.
• In the event of doubt and dispute, the final decision is made • The defense mechanism is also effective against fungi,
based upon the Gel Clot Limit Test hence a similar reaction occurs in response to a fungal
infection, which triggers the clotting cascade.
THEORY
1) Endotoxin comes in contact with LAL LAL-CLOTTING CASCADE
2) Activation of a pathway (enzymatic reaction)
3) Production of at least 3 serine protease zymogens
(Factor B, Factor C, Pro-clotting enzymes)
4) Alters amoebocyte coagulogen (Invertebrate-like
fibrinogen)
5) Formation of coagulin gel
THEORY BEHIND THE GEL CLOT TECHNIQUE
• The endotoxin will activate Factor C to become Activated

Factor C
• And activated factor C will activate factor B to become
activated factor B
• Activated Factor B and Factor G that is activated by the
lipopolysaccharide presence, this will activate with enough
number Factor G
• The Activated Factor G together with the Activated Factor
B will metabolize the proclotting enzyme which will become
the clotting enzyme
• This clotting enzyme will activate Coagulogen to become
Coagulin
• The Coagulin will form the gel-like structure or the clot itself


• This pathway alters amoebocyte coagulogen into a • Negative for endotoxin: no clot forms or if the clot breaks
fibrinogen-like clottable protein through inversion of the tube
• Final product: coagulin gel
• In the reaction, beta glucans (lipopolysaccharide of the PART 3: EXPERIMENT PROCEDURE
gram-negative bacteria) triggers the protease enzyme INTRODUCTION
Factor G • Endotoxin (a.k.a lypopolysaccharide), is a pyrogenic
• Endotoxin triggers Factor C to produce Factor B substance that is found in the cell wall of Gram-negative
• At least 1,000x more lipopolysaccharide than endotoxin bacteria.
is required to trigger the clotting cascade • Pyrogenic substance (or pyrogen) can induce fever when
• Glucan required to trigger the factor G can be of vary: injected into the blood or cerebrospinal fluid.
o Molecular Weight: 3-100 kDa • It is associated with injectable products.
• Sterile production procedures are needed but does not
ensure the removal of endotoxin (because it is heat-stable).
GEL-CLOT TECHNIQUE • Consequences if there’s an endotoxin unit that will in
• A method that utilizes the endotoxin-mediated clotting injectable products, it may cause fever, headache, chills,
cascade, that naturally occurs within horseshoe crabs, to hypotension, miscarriage, acute lung injury, and even death
produce a gelatinous clot after incubation at 37+/-1°C with • Fatal even in small amounts
endotoxin.
• The gel-clot mimics the clotting of Limulus blood in vivo. GENERAL PROCEDURE
Here a clotting protein is cleaved by an activated clotting
enzyme, at which point the insoluble cleavage products
• Depyrogenate all glasswares and other heat-stable
materials in a hot-air oven at 250 ̊C for not less than 30
coalesce and form a gel.
minutes
• LAL reagent used for the gel-clot is usually supplied with an o If employing plastic apparatus such as microplates and
identified sensitivity or label claim (λ). pipet tips for automatic pipettes, use apparatus that is
• For example, 0.03EU/mL. This means that when mixed with shown to be free of detectable endotoxin and does not
an equal volume of the material under test, a gel or clot will interfere in the test. This instrument might be the one
form if the material contains 0.03EU/mL or greater. to introduce the endotoxin into the injectable substance
o It will only clot if it’s equal
• The units of measurement for the LAL test are EU.
(measure of the activity of endotoxin)
• Pyrogenicity of LAL reactivity of one endotoxin preparation
may be very different from that of another of the same
weight. Conversely, two endotoxin molecules may be
different sizes and different weights but may have the same
reactivity in an LAL test.
PROCEDURE:
• The gel clot method is performed using depyrogenated
glass tubes:
o To prevent false positive reactions, which may happen
with the presence of contaminants outside the working • Reagents:
area. o Amoebocyte lysate - a lyophilized product obtained
• The assay comprises an equal volume of test solution and from the lysate of amebocytes (WBCs) from the
lysate (typically 0.1mL of test solution and 0.1mL of lysate) horseshoe crab (Limulus polyphemus or Tachypleus
that are gently mixed together. tridentatus)
• Due to its sensitivity to vibration this will be incubated in an o Water for Bacterial Endotoxins Test (BET) - water
unstirred water bath or dry block heater at 37°C (to replicate for injections or water produced by other procedures
human body temp.) for 1 hour. that shows no reaction with the lysate employed, at the
• The end point is read by carefully inverting the tube through detection limit of the reagent only. Prepared to have no
180° endotoxins. Acts as diluent
o Lysate TS - Dissolve amebocyte lysate in water BET
READING OF RESULTS or in a buffer recommended by the lysate manufacturer
by gently stirring. Store the reconstituted lysate,
• Positive for endotoxin: after inversion through 180°, a solid
clot (gelation) has formed and remains intact.


refrigerated or frozen, according to the specifications of o Concentration of sample solution = mg/mL or units/mL
the manufacturer. or mL/mL
o 1EU=1IU (EU = endotoxin unit)
ENDOTOXIN LIMIT PER ROUTE OF

ADMINISTRATION
• For parenteral preparations: defined on the basis of dose =
K/M
• Suggested values for K are:
o IV route: K = 5 IU endotoxin/kg body weight
o IV route for radiopharmaceuticals: K = 2.5 IU
endotoxin/kg body weight;
o Intrathecal route: K = 0.2 IU endotoxin/kg body weight
o M = maximum human dose of the product
o K = maximum allowable endotoxin exposure
• Control Standard Endotoxin - usually E. coli which is a
gram-negative bacterium with lipopolysaccharide and is an 5 EU/kg/hour for a 70 kg person
endotoxin • Endotoxin Limit = K/M
• Example:
Endotoxin limit for Enfurvitide is < 1.2 EU/mg and is not stated
PREPARATION OF STANDARD ENDOTOXIN in any reference. Max dose of enfuvirtide is 1.5 mg/kg/h. (Not
SOLUTION stated therefore use K based on guidelines) Therefore:
• After mixing the Standard Endotoxin Stock Solution
Endotoxin Limit = (5 EU/kg/h)/(1.5 mg/kg/h) = 3.33 EU/mg
vigorously, prepare appropriate serial dilutions of Standard
Endotoxin Solution using water BET. Serial dilution is done
by transferring small aliquots into a diluent. We dilute to the If the endotoxin limit is < 1.2 EU/mg, we can accept 3.33 EU/mg
point where the endotoxin limit can still be determined. as computed because it has a three-fold safety margin
• Use dilutions as soon as possible to avoid loss of activity by
adsorption and to have many samples to test on. Do not CONCENTRATION OF SAMPLE SOLUTION
over dilute or else the lysate test cannot detect the • mg/mL in the case of endotoxin limit specified by weight
endotoxin. (IU/mg)
• units/mL in the case of endotoxin limit specified by unit of
biological activity (IU/unit)
• mL/mL when the endotoxin limit is specified by volume
𝜆: THE LABELLED LYSATE SENSITIVITY

• Expressed in IU/ml
DETERMINATION OF MAXIMUM VALID DILUTION

• Example:
Compute for the MVD of azithromycin IV injection 100 mg/mL
with endotoxin limit of 0.17 EU/mg and 𝜆 = 0.03 EU/mL
MVD= (0.17 EU/ mg)(100 mg/mL)/ 0.03 EU/mL= 566 (rounded

off)
DETERMINATION OF MAXIMUM VALID DILUTION
• MVD = maximum allowable dilution of a sample at which THEREFORE: Maximum Valid Dilution is 1:566. If diluted at this
the endotoxin limit can still be determined point, there can still be a positive result for endotoxins. Going
• Formula: (endotoxin) (concentration of sample beyond this will hinder the lysate’s ability to detect endotoxins.
solution)/ 𝜆
• 𝜆= sensitivity requirement for your endotoxin test or labelled PREPARATORY TESTING
sensitivity in the gel-clot technique (EU/mL) • Test for Confirmation of Labelled Lysate Sensitivity
• Where: o The reason why we need to confirm the labelled lysate
o Endotoxin limit is specified in the individual drug sensitivity is to know the exact value of the endotoxins
monograph (EU/mL, EU/mg, EU/unit activity) present.


o The confirmation for lysate sensitivity is usually carried concentrations of standard endotoxin that clots the
out when a new batch of lysate is used or whether there lysate) of the dilution series used
is a change in experimental conditions, which may • f is the number of replicate test tubes
affect the outcome of the test.
G. The labelled sensitivity of the lysate is confirmed if
TEST FOR CONFRIMATION OF LABELLED LYSATE geometric mean endpoint concentration is not less
SENSITIVITY than 0.5λ and not more than 2λ.
A. Prepare four replicates of standard endotoxin solutions
having at least four concentrations (each) to 2λ, λ, 0.5λ,
and 0.25λ by diluting USP Endotoxin RS with water for SAMPLE PREPARATION
BET. • For any given route of administration, the endotoxin limit is
B. Mix 0.1 mL aliquot portion of the Lysate TS (prepared
inversely proportional to the dose. The lower the dose, the
according to manufacturer's instructions) with an equal higher the limit per unit dose. Think about it this way:
volume of the standard endotoxin solutions in individual
test tubes. We should mix it gently because it is • If the dose is 1 mg/kg/hr, the endotoxin limit is (5 EU/kg/hr)
sensitive to vibrations. ÷ (1 mg/kg/hr) = 5 EU/mg
• (*5 EU/mg is the endotoxin limit that we are looking for.)
Replicate Observation at different End Log of

concentrations point Endpoi
nt
2λ (20 λ (10 0.5 0.25λ
EU/m EU/m λ (5 (0.02
L L EU 5 EU
/ / mL
C. Incubate the reaction mixture at 37°C ±10 for 60 ±2 min mL)
(or as directed by lysate manufacturer), avoiding
vibration. I + + - - 10 1
D. To test the integrity of the gel, take each tube in turn EU/mL
directly from the incubator, and invert it through about
180° in one smooth motion. II + + - - 10 1
EU/mL
Lysate is a good/positive test agent because it forms a
positive result. III + + - - 10 1
Positive Result: Firm gel has formed that remains EU/mL
in place upon inversion.
Negative result: No intact gel is formed. IV + + - - 10 1
Therefore, you cannot use this lysate for the EU/mL
bacterial endotoxin test, because if there is a
negative result, there will be no agent that will form Mean log of endpoint / 4 1
the clot, which is an indicator for a positive result.
E. The test is considered valid when the lowest Geometric Mean = Antilog of Mean of Log 10
concentration of the standard solutions shows a (endpoint) / 4 EU/mL
negative result in all replicate tests.
F. Determine the geometric mean endpoint by
The USP requires that the sample is performed at quadruple kit.
calculating the mean of the logarithms of the
Four replicates for each concentration of sensitivity. As
endpoint concentrations of the four-replicate series,
mentioned before, The labelled sensitivity of the lysate is
then taking the antilogarithm of the mean value based
confirmed if geometric mean endpoint concentration is not less
on the following formula:
than 0.5λ and not more than 2λ, which means the sensitivity of
the lysate confirms for the validity in terms of using it for bacterial
Geometric mean endpoint concentration (EU/mL) = antilog
endotoxin test if the three concentrations (2λ, λ, 0.5λ) are the
(Σe/f)
only ones that contain positive results.
Where:
• Σe is the sum of the log endpoint concentrations
(smallest concentration in the series of decreasing


• Procedure:
• Remember: 0.1 mL concentration of the solution (or dose A. Prepare replicate samples of solutions A-D as shown
of solution) is divided from the value of λ to get the value of in the table below:
EU/mL.
Example: 2λ ÷ 0.1 mL = 20 EU/mL. (found in the table above) Endotoxin Diluent Dilution Endotoxin Number of
Solution
• How do we know it’s positive? concentration factor concentration replicates
o If it forms a clot that is so strong that it won’t be affected

None/Sample
by inverting the tube 180° and the clot is stable, it is a A - - - 4
Solution
positive result.
• How do we compute for the endpoint? 1 2λ 4
o We simply copy the value of EU/mL based on where 2λ / Sample Sample 2 1λ 4
B
the last positive is seen on the table. Solution Solution 4 0.5λ 4
• How to calculate the log of the endpoint? 8 0.25λ 4
o *Log of endpoint = log(value of endpoint)
1 2λ 2
o For example, log of 10 EU/mL = log(10) = 1 2λ / Water Water 2 1λ 2
C
BET BET 4 0.5λ 2
We can confirm that the labelled lysate sensitivity is good as a 8 0.25λ 2
null kit, because it complies with the specifications said in the
USP to be used in a bacterial endotoxin test. None/Water
D - - - 2
BET
Conclusion: With respect to the observations above, LAL kit

tested complies with the (USP) specifications, because it is said According to the table shown, sample B requires 4 replicates for
that the labelled sensitivity of the lysate is confirmed if the each concentration, while sample C requires 2 replicates for
geometric mean endpoint concentration is not less than 0.5λ each concentration.
and not more than 2λ.
Water BET = Water for Bacterial Endotoxin Test
TEST FOR INTERFERING FACTORS Solution A = no endotoxin concentration present
Solution B = test for interference
• We need to test for interfering factors, because we should Solution C = control for labelled lysate sensitivity, because the
detect any endotoxin samples that are not really included in water will be used for Bacterial Endotoxin Test;
the testing. Solution D = no endotoxin concentration present; negative
• Also known as Inhibition or Enhancement test control
• This test must be performed on the sample solutions at a
dilution less than the maximum valid dilution, not containing B. Mix 0.1 mL aliquot portion of the lysate TS (white blood
any detectable endotoxins. cell of the horseshoe crab, which will react with the
• Purpose: endotoxin if ever there are any; test solution = drug
o If we want to change the biological nature within the product) with an equal volume of the test solutions in
test (Example: changing the pH of the sample, individual test tubes.
changing the enzymatic activity, etc.), these will C. Incubate the reaction mixture at 37° ±1° C for 60 ±2 min
interfere with the endotoxin test, which is why the Test (or as directed by lysate manufacturer), avoiding
for Interfering Factors is necessary. vibration.
o Perform the inhibition/enhancement test on the sample D. To test the integrity of the gel, take each tube in turn
solutions at a dilution less than the MVD not containing directly from the incubator, and invert it through about
any detectable endotoxins, operating as described 180° in one smooth motion. If a firm gel has formed that
above under Test for confirmation of labelled lysate remains in place upon inversion, record the result as
sensitivity. positive. A result is negative If an intact gel is not formed.
o The geometric mean endpoint concentrations of B and E. The test is considered valid when all replicates of
C solutions are determined by using the formula Solutions A and D show no reaction (it should show no
described above under Test for confirmation of labelled result of clotting), and the result of Solution C confirms
lysate sensitivity. the labelled sensitivity.
o The test for interfering factors must be repeated when F. If the sensitivity of the lysate determined in the presence
any condition changes which is likely to influence the of Solution B is not less than 0.5 λ and not greater than
result of the test. 2 λ, the sample solution does not have factors that
interfere under the experimental conditions used.

Solution Endotoxin Concentration / Number of

Solution to which endotoxin is Replicates
added
A None/diluted sample solution 2
B 2λ / Diluted sample solution 2
C 2λ / Water for BET 2
D None / Water for BET 2
(The positive control solutions B and C contain the standard

endotoxin, at a concentration corresponding to twice the labelled
lysate sensitivity. The negative control solution D consists of
water for bacterial endotoxin test.)
• Note: B. Mix 0.1 mL aliquot portion of the lysate TS (will react

with the endotoxin, if there are any) with an equal
o If the sample under test does not comply with the test volume of the test solutions in individual test tubes.
at a dilution less than MVD (maximum valid dilution),
(This will determine that the product only has a certain
repeat the test using a greater dilution, not exceeding
limit for the endotoxin. If it conforms to that standard,
MVD.
we can proceed with the manufacturing of this batch of
(Of all the dilutions that you have with the tiniest parenterals or injectables.)
endotoxin unit that the sample has, you can C. Incubate the reaction mixture at 37° ±1° C for 60 ±2 min
acquire more samples from it, as long as it does (or as directed by lysate manufacturer), avoiding
not exceed the maximum valid dilution.) vibration.
o The use of a more sensitive lysate permits a greater D. To test the integrity of the gel, take each tube in turn
portion of the sample to be examined, and this may directly from the incubator, and invert it through about
contribute to the elimination of interference. 180° in one smooth motion. If a firm gel has formed that
o Interference may be overcome by suitable treatment remains in place upon inversion, record the result as
such as filtration, neutralization, dialysis, or heating. positive. A result is negative If an intact gel is not
o To establish that the chosen treatment effectively formed.
eliminates interference without loss of endotoxins, E. The test is considered valid when both replicates of
repeat the test. Solutions B and C are positive, and those of solution D
o For the confirmation of labelled lysate, we are testing are negative.
the capabilities of the lysate to interact with the When a negative result is found for both
endotoxin. replicates of Solution A, the preparation under
o We cannot proceed to the Limit testing if the labelled test complies with the test.
lysate does not conform to the standards. When a positive result is found for both of the
replicates of Solution A, the preparation under
test does not comply with the test.
LIMIT TEST
• The “real bacterial endotoxin test”
• Note: When one replicate of Solution A is positive and the
• Procedure other is negative, repeat the test.
A. Prepare replicate samples of solutions A-D as shown
o In the repeat test, both replicates should be negative to
in the table next slide:
comply with the test.
o If the preparation does not comply with the test at a
dilution less than the MVD, the test may be repeated
using a greater dilution, not exceeding the MVD.


Summary: o If it has been diluted and resulted to a
• We are performing the Bacterial Endotoxin Test, to negative, accept the solution
ensure that the intravenous preparations are
conforming to the standard and will not cause any Disposition: ACCEPT
toxicity such as immediate hyperthermia (deadly and
fatal), or even death, which is why we must carefully Why is it still valid even if 0.25λ does not form a clot? Why
check the values. do we perform method validation?
• If it conforms to the standards, it is ready for o We are checking the LAL
manufacturing. o The 0.25λ is acceptable in the body that it will not cause
fever
• Some manufacturing companies in the Philippines are
o MVD- If a concentration is too diluted, we would get
using the 0.03 endotoxin level.
lower chances of detecting endotoxins. Pag sumobra
• In the USP, 5 is the endotoxin limit for the preparation. ang dilution, it can give us a false positive result.
In reality, most companies in the Philippines do not o Method Validation Test indicates if there are any toxins
even follow that limit. They are lowering the value to present in the solution, which would allow us to ensure
0.03 to be more sure that their products conform to the if the pharmaceutical product is ready for
standards and will not induce the endotoxins that may administration.
cause toxicities in the body. o Pag dinilute ng todo, lahat ng endotoxin napunta sa
isang portion. When you distribute it to different vials, a
INTEGRATION vial may contain the one with endotoxins and can be
1. Gram-positive possesses “endotoxins” which can cause lethal to the patient.
fever o Usually, manufacturing firms use the lowest 0.25λ /
o FALSE 0.3λ to be safe
o Gram-negative bacteria can be toxic and lethal in high o Higher concentration can be lethal to the patient
concentrations / doses. This leads to sepsis and fever. o Example:
o Dosage forms needed to test for endotoxins are the
parenterals (or any product that passes through the
bloodstream)
Syrup is not needed because the digestive tract
will destroy the endotoxins
2. ABC Vial was subjected to bacterial endotoxin test using

LAL. The following observations are recorded:
Limit Test
• 4 solutions, 2 replicates each= 8 test tubes
• Solution A = None / diluted sample solution
o No lysate
o Diluted drug sample
Interpretation: Method Validation Test is valid if A and D did not o No clot
clot, meaning there is no endotoxin/ lysate present in the • Solution B = 2 lambda/diluted sample solution (lysate
sample. content)
• If there is a clot formation in test tube A and D, then there is o With lysate content
endotoxin present o With diluted sample
• If there is one that formed a clot and one that didn’t in tube o With clot
A, then we have to retest • Solution C = 2 lambda/water for BET
o Increase the dilution but not exceeding the MVD o With lysate content
because the drug product might have a high o With water for BET
concentration that’s why it resulted to a positive o With clot
• Solution D = None/Water for BET


o No lysate content
o With water for BET
o No clot
• λ = lysate content
• Presence of lysate ensures positive result, because the
lysate contains endotoxins.
• False positives are only possible if Method Validation Test
is not performed.
• 2λ concentration is used to ensure that there is clotting.
(If nag positive and solution D or ung mga replicates, it means

na may endotoxin present sa ABC Vial. So, need mag test ulit
and increase the dilution and should not exceed MVD →
dapat parehong negative result na)
REFERENCES
Recorded Lecture by Dr. Darwin Kiong & Sir Renson

Mendoza

EXERCISE 9: ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Antibiotics is a more specific term used to combat bacterial
infections.
OUTLINE
I. Antimicrobial and Antibiotics DESIRED PROPERTIES OF NEW ANTIMICROBIALS

II. Desired Properties of New Antimicrobials • Currently, there are numerous antimicrobials available in
III. Mechanisms of Action of Antibacterial Agents the market, however, with the advent of antimicrobial
IV. Antimicrobial Susceptibility Testing resistance, the search for newer agents is necessary
A. Broth Dilution Test • Selectivity for microbial rather than mammalian targets
B. Antimicrobial Gradient Method (E-Test)
o the selective toxicity of antimicrobials means that they
C. Agar Well Diffusion Method
must be highly effective against microbial cells that
D. Disk Diffusion Method (Kirby-Bauer Method)
have minimal or no toxicity to human cells.
E. Mueller-Hinton Agar (MHA)
F. Important Considerations (Kirby-Bauer Method) • Cidal activity (antibacterial and antifungal agents)
G. Interpretation o the cidal activity, because of their more aggressive
H. Supplemental Methods for the Detection of antimicrobial action bactericidal or fungicidal agents
Antimicrobial Resistance are often the drugs of choice in seriously ill patients.
V. Antimicrobial Susceptibility Testing • Slow emergence of resistance
A. Importance o Furthermore, we want slow emergence of resistance,
VI. Kirby Bauer Diffusion Method for this purpose, antivirulence drugs are actively
A. Minimum Inhibitory Concentration (MIC) searched for, these are those that are aimed in
B. Minimum Bactericidal Concentration (MBC) destroying structures or interfere with processes that
C. Modified Kirby Bauer Method might contribute to antimicrobial resistance.
D. Always Use
• Narrow spectrum of the activity
E. The Choice of Antimicrobial will Depend on:
o Narrow spectrum of activity is more ideal. Narrow
F. Preparation
spectrum antimicrobials are chemotherapeutic agents
G. Inoculum Turbidity Standard
H. Mueller-Hinton Agar
acting only on a single or a limited group of
I. Additional Notes microorganisms. They are preferred over broad
VII. Antimicrobial Susceptibility (Video) spectrum agents because the administration of broad
VIII. Kirby Bauer Antimicrobial Susceptibility (Teach- spectrum agents can drastically alter the nature of the
Back-Demo) normal bacterial flora in our body
IX. Antibiotic Susceptibility • Non-toxic to the host
A. Broth Dilution Test/Method o minimal to no adverse effects.
B. Epsilomer Test or E-Test • Long plasma half-life (once-a-day dosing)
C. Synergistic o It is also ideal for it to have long plasma half-life to
D. Antibiotic Synergy enable once a day dosing.
X. Preparation of Plate and Bacteria
• Good tissue distribution including CSF
A. Preparing McFarlan Turbidity Standard Number
0.5 • Low-plasma binding
B. Inoculating the Plate • A high degree of protein binding of a drug in the serum
XI. Determining MIC using E-Test restricts its entry into the cerebrospinal fluid or CSF.
XII. Synergy Testing: Cross Approach Therefore, the amount of free or unbound drug in serum
XIII. Synergy Testing: Non-Cross Approach rather than the total amount of drug present is important for
XIV. MIC Determination using Broth Dilution CSF penetration.
XV. Data Analysis and Results: Broth Microdilution • Oral and parenteral dosage forms
XVI. Data Analysis and Results: Synergy Testing o We want oral and parenteral dosage forms available to
XVII. Integration cater different needs or urgencies
• No interaction with other drugs
ANTIMICROBIAL AND ANTIBIOTICS o It shall not have any interaction when co-administered
• Agents used against infectious diseases with other medications.
• Antimicrobials are compounds that kill or inhibit
microorganisms
• Antimicrobials used for different microbial infections - be it
bacterial, fungal, or protozoal.
• Antibiotics are antimicrobials naturally-produced by
microorganisms; they are usually chemically altered to
enhance their modes of action. (semi-synthetics)
JL. SJC, JDL, DCD, GN | 2F-PH 1

MECHANISMS OF ACTION OF ANTIBACTERIAL AGENTS ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Employed in the direct measurement of antimicrobial
activity of the drugs of choice for certain infections and
detection of possible drug resistance in common pathogens.
o Methods:
▪ Broth dilution test
▪ Antimicrobial gradient method (E-test method)
▪ Agar well diffusion method
▪ Disk diffusion test (Kirby-Bauer method)
BROTH DILUTION TEST

• Broth dilution test is the oldest antimicrobial susceptibility
testing method and involves the preparation of two-fold
• Some antibiotics selectively interfere with the synthesis of dilutions of antibiotics in a liquid medium dispensed in test
the bacterial cell wall, a structure that mammalian cells do tubes.
not possess. Thus, they exhibit a high potential for selective • In this technique, the anti8biotic containing tubes are
toxicity. inoculated with the standardized bacterial suspension. After
• Now, it is important to note that to be maximally effective, overnight incubation, the tubes are examined for visible
inhibitors of cell wall synthesis require actively proliferating bacterial growth as evidenced by turbidity.
microorganisms. They have little or no effect on bacteria • The lowest concentration of the antibiotic that prevented
that are not growing and dividing. The most important growth represents the minimal inhibitory concentration or
members of this group of drugs are the beta lactam the MIC.
antibiotics and the glycopeptide, vancomycin. Next,
antibiotics of the polypeptide class such as the polymyxins
and the lipopeptide drugs, daptomycin - are believed to
interfere with the integrity and function of microbial cell
membranes. When the bacterial plasma membrane is
compromised, its cellular components leak out of the cell
that eventually leads to its destruction. Furthermore, a
number of antibiotics exert their antimicrobial effects by
targeting the bacterial ribosome which has components that
differ structurally from those of the mammalian cytoplasmic
ribosome in general the bacterial ribosome is smaller 70 S
than the mammalian ribosome which is 80 S and is
composed of 50 S and 30 S subunits. Antibacterial drugs
that belong to this group may either target the 30 S or the
50 S subunit. Other antibiotics may act on metabolic
pathways, examples of which include sulfonamides,
sulfones, and trimethoprim, which inhibit the synthesis of
bacterial dihydrofolic acid, a compound that is utilized by
bacteria to synthesize their nucleic acids. Another example • Advantages:
is isoniazid or INH that inhibits mycolic acid synthesis. o Generation of quantitative result in the form of MIC
Isoniazid is one of the most potent of the anti tubercular (minimum inhibitory concentration).
drugs we have in the market. Lastly, we have antibacterial • Disadvantages:
agents that exert their effect by inhibiting key enzymes o Tedious, manual task of preparing the antibiotic
involved in the replication of bacterial nucleic acids, thus, solutions for each test.
impairing its cell division or proliferation. So those are the o Possible errors in the preparation of the antibiotic
mechanisms of actions of our commonly used antibacterial solutions
agents. The complete list of their specific mechanisms, their o Relatively large amount of reagents and space are
spectrum activity and representative examples are required for each test
tabulated on your handouts.

• With the advancement of technology, the disadvantages • Advantages:

mentioned in the previous slide have already been o Intrinsic flexibility
addressed. Multiple testing or replicates may now be o Best suited when MIC for only 1-2 drugs is needed or
conveniently tested using 96 well plates and is analyzed fastidious organism is tested
using a UV-Vis spectrophotometer. Since this is a micro o Correlates well with MICs from other methods
assay, lesser agents and materials are needed to carry out • Disadvantages
the test and a computerized report may easily be generated. o Different MIC standard versus broth dilution is used
• Advantages:
o Generation of MICS AGAR WELL DIFFUSION METHOD
o Reproducibility and convenience of having prepared
• This technique involves the incorporation of different
meals
concentrations of the antimicrobial substance into a nutrient
o Economy of reagents and space
Agar medium followed by the application of a standardized
o Computerized report
number of cells to the surface of the Agar plate.
• Disadvantages:
o Inflexibility of drug selections available in standard
commercial panels
ANTIMICROBIAL GRADIENT METHOD (E-TEST)

• We have the antimicrobial gradient method, otherwise
known as the Epsilometer test or E-test. It is an exponential
gradient method that determines the antimicrobial
resistance of the microorganism. It is a cost effective tool
that has been developed to provide a direct quantification
of antimicrobial susceptibility of microorganisms.
Advantages of this method will include intrinsic flexibility for
numerous antimicrobial agents may be tested using this
technique. It is best suited under minimal inhibitory
concentration or MIC for only one to two drugs is needed or
fastidious organisms are to be tested. It also correlates well
with the MIC's from other methods. A disadvantage of this
technique is that a standard has to be prepared using the
Broth dilution method that is to be compared with the results
of these tests.
• Advantages:
o May be used for liquid samples
• Disadvantages
o Non-uniform amount of samples tested


sensitive. But if the inoculum is in excess, there may be
DISK DIFFUSION METHOD (KIRBY-BAUER METHOD) a small zone of inhibition which is falsely resistant
• The Kirby Bauer method is the most widely used antibiotic • Medium
o The medium to be used is MHA or the Mueller-Hinton
susceptibility test in determining what choice of antibiotics
Agar. Careful considerations must be given on its
should be used when treating an infection. This method
formulation or composition including the calcium and
relies on the inhibition of bacterial growth measured under
magnesium content
standard conditions. In Kirby Bauer testing, bacteria are
▪ Formulation/Composition
placed on a plate of solid growth medium and disks of
▪ Calcium/magnesium content
antibiotics are added to the plate. After allowing the bacteria
▪ pH – pH must also be carefully measured to
to grow overnight, areas of clear media surrounding the
ensure optimal antibiotic activity
disks indicate that the antibiotic inhibits bacterial growth.
▪ Agar depth – The Agar depth must be 3 to 5
This represents the zone of inhibition.
millimeters too much moisture on the Agar will
exhibit small zone of inhibition which is falsely
resistant while very dry agar surface will exhibit a
large zone of inhibition which is falsely sensitive
• Incubation
o The atmospheric conditions temperature and duration
must be carefully observed to accurately get the results
▪ Atmosphere
▪ Temperature
▪ Duration
• Antimicrobial agent
o Antibiotic disks to be used must be stored prior to use
to ensure maximum efficacy
▪ Disks
INTERPRETATION
• Advantages
o Simple, practical, and well-standardized
o Categorical results/flexibility in selection of disks
• Disadvantages
o Lack of mechanization or automation of test
o Qualitative result
MUELLER-HINTON AGAR (MHA)

• Best medium for routine susceptibility tests
o Readily available commercially in the form of
dehydrated medium and prepared plates
o Low in sulfonamide, trimethoprim, and tetracycline
inhibitors
o Gives satisfactory growth of most bacterial pathogens
• The interpretation of results, once the zone of inhibition has
• Note: Inoculum for disk diffusion may be prepared using
been obtained. You may now refer to the complete table as
trypticase soy broth and other suitable broth.
provided in the experiment handout or antibiotic disk
product literature for appropriate interpretation. The
IMPORTANT CONSIDERATIONS (KIRBY-BAUER resistant category implies that the microorganisms are not
METHOD) inhibited by the usually achievable concentrations of the
• Inoculum agent with normal dosage schedules and demonstrate zone
o For this method, the amount of inoculum must be 1.5 * diameters that fall in the range or specific microbial
10 raised to 8 cfu per mL, this based on the McFarland resistance mechanisms are likely and the clinical efficacy of
0.5 standard. So if the inoculum is insufficient, a large the agent against the microorganism has not been reliably
zone of inhibition may be observed which is falsely shown in treatment studies. Meanwhile, the susceptible or
sensitive category implies that the microorganisms are


inhibited by the usually achievable concentrations of
antimicrobial agents when the recommended dosage is
used. Lastly, the intermediate category includes
microorganisms with antimicrobial MIC’s that approach
usually attainable blood and tissue levels and for which
response rates may be lower than for susceptible
microorganisms. The intermediate category implies clinical
efficacy in body sites where the drugs are physiologically
concentrated or when a higher than normal dosage of the
drug can be used. This category also includes a buffer zone
IMPORTANCE
separating susceptible from resistant strains. Generally,
reporting of a category result of sensitive, intermediate, or • Guides the clinician in choosing the right antibiotic for a
resistant, provides the clinician with the information particular infection
necessary to select appropriate therapy. • Helps in identifying susceptibility patterns of common
isolates in a particular hospital or community
SUPPLEMENTAL METHODS FOR THE DETECTION • Helps in choosing the right empirical treatment for critically
OF ANTIMICROBIAL RESISTANCE ill patients even before their culture results are obtained
Table 1.0 Supplemental methods for the detection of • This test is performed in a controlled environment under
antimicrobial resistance standard conditions such that results are reproducible
TEST PURPOSE
OXACILIN AGAR SCREEN Detection of staphylococcal KIRBY BAUER DIFFUSION METHOD
resistance to penicillinase • Simple
resistant penicillins (e.g. • Reliable
oxacillin, methicillin, or
nafcillin)
• Widely-used
VANCOMYCIN AGAR Detection of enterococcal
SCREEN resistance to vancomycin MINIMUM INHIBITORY CONCENTRATION (MIC)
AMINOGLYCOSIDE Detection of acquired • Lowest concentration of an antimicrobial agent that visibly
SCREENS enterococcal high-level inhibits the growth of the organism
resistance to
aminoglycosides that would MINIMUM BACTERICIDAL CONCENTRATION (MBC)
compromise synergy with a
• Lowest concentration of the antimicrobial agent that results
cell wall-active agent (e.g.
in the death of the organism
ampicillin or vancomycin)
OXACILIN DISK SCREENS Detection of Streptococcus
pneumoniae resistance to
MODIFIED KIRBY BAUER METHOD
penicillin
D-ZONE TEST Differentiate clindamycin
resistance among S. aureus
resulting from efflux
• Now this table summarizes the different supplemental

methods used for the detection of antimicrobial resistance.
ANTIMICROBIAL SUSCEPTIBILITY TESTING • Antibiotic impregnated filter paper discs are placed on a
Mueller Hinton plate with lawn culture of an organism
• Laboratory test which gives data about the efficacy of • The antibiotic diffuses from the disc into the agar in
antimicrobial agents in treating microbial infections. decreasing amounts as you move further away from the
• In this picture, you have your agar plate and you have 6 disc
different antimicrobials being tested against an organism. • The discs are placed evenly spaced or distance from each
other and this depicts how the antibiotic would diffuse
outside, away from the center. As it diffuses further away,
the amount of antibiotic decreases.

THE CHOICE OF ANTIMICROBIAL WILL DEPEND ON
• If the organism is killed or inhibited by the concentration of

the antibiotic, there will be no growth in the immediate area
around the disc.
• This zone is called the zone of inhibition
• Before putting the agar into the incubator, you place your
antibiotics. Antibiotics A, B, and C. These discs are placed
inside the agar and put into the incubator. After some time,
there will be growth of bacteria, but you will notice a space, • Pathogen
a clear zone, between the disc and the bacteria that is o For example, you know your pathogen is
growing, usually it is circular. The clear zone is measured Mycobacterium tuberculosis. If the pathogen you are
and this is what you call the zone of inhibition going to test is Mycobacterium tuberculosis, of course
• Letter A Antibiotic has no zone of inhibition. Letter C has the you will not use antimicrobial agents such as antifungal.
biggest zone of inhibition and is more effective against the You will not try to use amoxicillin because you already
microorganism. know that those antibiotics will not be effective against
those pathogens. You want to use antibiotics that you
ALWAYS USE know can be effective against the pathogen
• Discs of correct antimicrobial content • Species
o It is important to have the filtered paper disc labelled o For example, the specimen is taken from the spinal
• An inoculum which gives confluent growth fluid meaning there is infection in the brain, so you want
• A reliable Mueller Hinton Agar to use antimicrobials that can penetrate the BBB.
Because using antimicrobials that will not penetrate the
BBB, even though it will show that it is effective against
the pathogen, it will be useless for the patient.
• Range of available antimicrobials
o Even if you’re using novel antimicrobial, it is not
available, the patient would not be able to procure the
antibiotic. It will be useless.
PREPARATION
• Good working environment
• Wear PPE
• After incubation at 35°C for 16-18 hours, zone sizes are • Check materials needed
measured and interpreted • Aseptic technique
• This is the usual setting depending on the microorganism
being tested INOCULUM TURBIDITY STANDARD
• Prepared suspension must be equivalent to 0.5 McFarland
Standard
• Use within 15 minutes of preparation
• Pure inoculum used must be a pure isolate and adjusted to
a count similar or equivalent to the 0.5 McFarland Standard.
Once the bacterial count is adjusted to such, it is advisable
to use it within 15 minutes of preparation


• Before using, dry the plates with their lids slightly raised in
a 35-37C incubator for about 30 minutes.
o If there is moisture, you can affect the diffusion of the
antimicrobial.
ANTIMICROBIAL SUSCEPTIBILITY (VIDEO)

• Using a sterile swab, inoculate a labelled plate of Mueller
Hinton agar by the lawn culture method as described in the
earlier section on culture methods. With a petri dish lid in
place allow three to five minutes but no longer than 15
minutes for the surface of the agar to dry. Using sterile
forceps or a needle mounted in holder or a multi-disc
MUELLER HINTON AGAR dispenser, place the appropriate antimicrobial discs evenly
• Prepare and sterilize the medium as instructed distributed on the inoculated plate.
• Note: The discs should be about 15 millimeters from the
edge of the plate and no closer than about 25 millimeters
from disc to disc. No more than 6 discs should be applied
on a 90 millimeters petri dish. For an inexperienced worker
the back of the plate can be marked with a pen indicating
the position where the discs need to be placed.
• Each disc should be lightly pressed down to ensure its
contact with the agar. It should not be moved once in place.
In 30 minutes of applying the disc, invert the control and test
plates and incubate aerobically at 35 degrees centigrade for
16 to 18 hours. After overnight incubation, examine the
control and test plates to ensure the growth is uniform.
using a ruler on the underside of the plate, measure the
ADDITIONAL NOTES diameter of each zone of inhibition in millimeters. The end
point of inhibition is where the growth starts.
• pH = 7.2 – 7.4
o If <7.2 aminoglycosides, quinolones, macrolides lose
KIRBY BAUER ANTIMICROBIAL SUSCEPTIBILITY
potency
o If > 7.2 tetracyclines can have increased activity (TEACH-BACK DEMO)
• Cation content: Can affect zone of inhibition • Purpose
o Too much cation can result to reduced zone size and o To determine the antimicrobial susceptibility of a given
vice versa in tetracyclines bacterial isolate
o Too much Calcium can increase size of zone in • Mechanism
Pseudomonas vs daptomycin o Disks impregnated with fixed concentrations of
o Too much Zinc can reduce the size zone in antibiotics placed on an agar plate inoculated with a
Pseudomonas vs carbapenems standardized concentration of bacteria give predictable
• Agar depth (4mm) patterns of inhibition that can be correlated to the
o Too shallow may produce false susceptible results and susceptibility of that organism to the antibiotic in
vice versa question
▪ Too shallow may produce false susceptible results: • Components
there is an increase in the zone of inhibition o A known amount of bacteria is spread evenly on a 100
▪ Too deep show false resistant results mm petri dish containing Mueller-Hinton Agar. Disks
• Excessive thymidine or thymine can reverse the inhibitory impregnated with standardized amounts of various
effects of sulfonamides and trimethoprim resulting in antibiotics are placed on the surface of the plate, which
smaller and less distinct zones of inhibition, or no zones at is then incubated overnight.
all • Method
• MH agar should be tested with known strains of organism o Measure the zone (diameter) of inhibition (clear zone =
at least weekly in order to verify that the media and disk are no bacterial growth) around each antibiotic disc in
working as expected. millimeters, using the virtual ruler. This is the zone in
o Make sure Mueller Hinton agar is always reliable. which bacterial growth has been inhibited by the
• Plates cam be stored at 2-8°C for up to 2 weeks antibiotic in the disc. Compare the zone diameters to
the Kirby-Bauer Table of Interpretive Standards.


• Interpretation Those labelled as susceptible, those are what you may
o Record data as R (resistant), I (intermediate), or S want to suggest to give the patient.
(susceptible)
ANTIBIOTIC SUSCEPTIBILITY
• Sensitivity of bacteria to antibiotics
• Can be measured using Broth Dilution Test or
Epsilometer test or E-test
BROTH DILUTION TEST/METHOD

• Standardized number of bacteria is added to a growth
media containing serial antibiotic dilution
• If susceptible, the bacteria cannot grow at the higher
antibiotic concentration but continue to multiply at lower
antibiotic concentrations causing the media to turn turbid.
• AMC
o About 10 mm, so that is less than 13
• CF
o Check for the end of inhibition zone, so that’s about 18
o You need to be very accurate, it’s 1 mm difference to
be labelled as intermediate or as susceptible
• C
o Very big zone of inhibition. About 30 mm
o Make sure when you put the antibiotics, they should be
widely spaced from each other so that you prevent
overlapping
• CIP • Bacterias cannot survive at the lowest antibiotic
o This is about 30 concentration is referred to as a Minimum inhibitory
concentration for a given bacteria.
• CC
o No visible inhibition zone, so that is 0
EPSILOMTER TEST OR E-TEST
• E
o Also 0 • A plastic strip impregnated with Antibiotic gradient is applied
• OX over a freshly spread lawn of bacteria on a Mueller-Hinton
o Also 0 Agar or MH-A-Plate
• P
o Also 0
• S
o It’s 20
• TE
o About 17
• TM
o About 23
• SXT
o It is more about 20
• Given this information, what antibiotics would you not
recommend?
o Based on the culture of E.coli we will not recommend
the use of Amoxicillin, Clindamycin, Erythromycin,
Oxacillin, and Penicillin. All of these antibiotics here
that are interpreted as resistant, are not useful for the
eradication of the infection. • The antibiotic diffuses out to the agar media where it is
• What antibiotics would you want to use? taken up by bacterias
o Ciprofloxacin, Chloramphenicol, Cephalothin,
Streptomycin, Tobramycin, Trimethoprim sulfate.


• If susceptible, the bacterias cannot multiply and will die o The E-strips of antibiotics are placed together in a
forming a clear zone around the e-strip which is called cross formation such that their scales of MIC marks
Growth Inhibition zone. form a 90 degree angle at the intersection.
• When the growth intercepts in the e-strip, the corresponding o Following the incubation of both tests, the MIC value of
value in the scale give the MIC value of the antibiotic. each antibiotic in combination with the antibiotic is red
at the point of the edge Growth inhibition zone
Often the antibiotics are used in combination to prevent the intersects with the edge of the E-strips. Then the FIC
emergence of antibiotic resistance strains of bacteria. This index is calculated.
often results into Synergistic rather than Additive effect.
SYNERGISTIC
• The combined effect of the two antibiotics is greater than
the sum of individual activities. However, the effect is
considered significant only when the MIC value of the
antibiotic combination decreases by at least 2 fold.
• This criterion is evaluated by calculating the Fractional
Inhibitory Concentration Index / FIC Index.
• Formula of FIC Index:
PREPARATION OF PLATES AND BACTERIA

• If it’s equal or lower than 0.5 it indicates synergy • To begin:
o Put on PPE
ANTIBIOTIC SYNERGY o Sterilize the workspace using 70% of ethanol
o Collect 15 milliliters of sterile MHf broth with 50% lysed
• Measured using 2 E-test method, a non-cross test and a
horse blood and 20 milligrams per milliliter Baden
cross test
nicotinamide and 5-8 MHA plates
• In the Non-cross test:
o The 2 different strips with predetermined MIC values
are applied to two separate plates. PREPARING MCFLARLAN TURBIDITY STANDARD
o After the antibiotics have diffused to the medium, the NUMBER 0.5
original E-strips are removed and the E-strips for the 1. Measure out 9.95 milliliters of 1% sulfuric acid solution.
alternate antibiotics are placed such that their MIC 2. Then, add 50 microliters of 1% Barium chloride solution to
scales lay exactly over the MIC scale of the previous the sulfuric acid solution.
strips 3. Vortex the solution well to obtain a turbid suspension. Then,
• In a Cross test which is the faster method of Non-cross test: cover the tube with aluminum foil and set it aside.


4. Next, dispense 1 milliliters of saline solution into a 15 milliliters
tube. Use a sterile loop to scrape out a sample of bacterial SYNERGY TESTING: CROSS APPROACH
growth from your bacterial test plate (Streptococcus grp. g)
• To begin:
5. Then, place the bacterial laden loop to the saline solution, stir
1. Inoculate an MAJ plate with streptococcus group g
gently and vortex the tube well
strain bacteria. Then, label the bottom of the plate with
6. Now place the bacterial suspension and McFarlan turbidity
bacteria’s name, antibiotic to be used and date.
standard side by side and compare them for turbidity
2. Place an e-strips at the center of the plate, then put the
equivalence. Add either additional saline or bacterial colony
second e-strip at 90 degrees above the first e-strips,
until the bacterial suspension turbidity matches that of the
locate its MIC mark, and gently place the second strip
standard.
as where it will intersect with its MIC mark as well. Once
7. Once the desired turbidity is obtained, deep a sterile cut and
placed, do not move them.
tip of the applicator into the bacterial suspension.
3. To ensure accuracy, performing at least three
replicates of the test is recommended. Now, incubate
INOCULATING THE PLATE the plates at 37 degrees celsius for 18-20 hours.
1. Swab the entire surface of the plate gently in zigzag motion
and label the bottom side of the plate with the bacteria’s name SYNERGY TESTING: NON-CROSS APPROACH
and date.
• To begin:
1. After inoculating 2 MAJ plates, place an e-test-strips for
DETERMINING MIC USING E-TEST both plates with different antibiotics separately.
• To begin: 2. Mark the MIC value of each e-strips using an
o Take out a penicillin G-e-test- strips, holding it by the inoculating loop.
edge with forceps and gently place it into the center of 3. Cover the plate, then incubate them at room
a freshly swab MAJ plate then cover with the lid. temperature for 1 hour.
o Gentamicin is also tested in a separate plate 4. After incubation, use forceps to remove the e-strips.
• To determine: Next, collect one of the plates of e-test-strips for the
o Collect the plate with Penicillin, determine the point other antibiotics. Hold the e-strips above the imprint of
where the inhibition zone intersects with the antibiotic the previous e-strips and place it over the MIC value of
o Read the corresponding numerical value in the scale: the previous one to the MIC value of the current e-strip.
Make sure that they align before gently placing the new
e-strip.
5. Repeat number #4 for the second plate.
6. Lastly, incubate the plates at 37 degrees celsius for 18-
20 hours.
MIC DETERMINATION USING BROTH DILUTION

• To begin:
1. Obtain a bacterial suspension with an establish
bacterial concentration and dilute the culture in MAJ
broth to achieve and OD 600 of 0.003
2. Way 16 mg of penicillin G and 128 mg of gentamicin.
o Same with the Gentamicin: Transfer each dry antibiotic into two 15 mL conical
tubes. Add 10 mL of distilled water to each tube.
3. Mix well by vortexing
4. Label the tube with antibiotic name and concentration.
5. In performing the assay in triplicate, add 400 mL of the
working bacterial solution into the first wells of three
row of the 96 wells of microtiter plate.
6. Next, add 200 mL of working bacterial solution in MAJ
broth to the wells of the 3 rows.
7. To generate the 2 fold serial antibiotic dilution. First add
4 microliters of antibiotic stock to the first well.
Generating a 100 fold dilution. Sequentially transfer
200 microliter of bacterial antibiotic solution into each
well. Beginning from the first well through the second
to last well in each row, ensure proper mixing by
pipetting 2-3 times after every transfer.


8. Discard the final 200 microliters of bacteria antibiotic 2. Determine the point where the Growth inhibition zone
solution. intersects with the antibiotic strips. The MIC value
Note: below is Penicillin G in combination with Gentamicin
Bacterial concentration: 10^5 - 10^6 cfu/mL which is 0.064microgram/mL.
Antibiotic stock solution - 1.6 mg/mL of Penicillin G and
12.8 mg/mL of Gentamicin
DATA ANALYSIS AND RESULTS: BROTH

MICRODILUTION
• To determine the result of microdilution test of Penicillin G:
1. Firstly, locate the wells that exhibit no visible bacterial
growth indicated by a lack of turbidity
3. Now, collect the second plate that contains Gentamicin

e-strips and determine the MIC value in combination.
The MIC value for Gentamicin is 4 microgram/mL.
4. Then, to evaluate the effect of combination use the
formula below:
2. From these wells, identify the well with the lowest

antibiotic concentration which represents the MIC
value of Penicillin G for the tested bacteria.
• To determine the result of microdilution of Gentamicin:

1. Firstly, locate the wells that exhibit no visible bacterial
growth indicated by a lack of turbidity
2. From these wells, identify the well with the lowest

antibiotic concentration which represents the MIC
value of Gentamicin for the tested bacteria.
DATA ANALYSIS AND RESULTS: SYNERGY TESTING

• Non-cross test:
1. Collect the first plate containing penicillin G e-strips.

Therefore:
However, there is no synergy between Penicillin G and

Gentamicin against the streptococcus grp G strain. As
the FIC Index is greater than 0.5.
Hence, there is no synergy between Penicillin G and

Gentamicin against the streptococcus grp G strain.
CROSS TEST:
1. Determine the point where the growth inhibition zone

intercepts their respective e-strips.
2. Then, read the numerical MIC value of each e-strips
that corresponds to these interception points.
INTEGRATION
1. The best medium for Kirby Bauer Test

a. MHA
b. Chocolate agar
c. Nutrient agar
d. SSA
2. Factors which influence antimicrobial susceptibility testing
is/are
a. pH
b. Moisture
3. Then, to evaluate the effectiveness of the combination. c. Cations such as Calcium
Use the formula below: d. All of the above
3. An organism is resistant to an antibiotic means
a. Produce large inhibition zones
b. Able to grow well in MHA
c. Cannot grow in the presence on antibiotic disc
d. Can grow around the antibiotic disc
i. Does not inhibit the microorganism
4. An antibiotic that is effectice against a wide variety of
microoganism is called?
a. Synthethic
b. Semisynthetic
c. Narrow spectrum


d. Broad spectrum
5. What measurement unit are used to measure the zone sizes?
a. mm REFERENCES
b. Meter
- PHA 6114 LAB Lec Vids
c. Inch
d. Cm
6. Case: A 19-year-old man presents with a large abscess on
his left thigh. It is painful upon palpation, erythematous, and
has pus draining from several opening. After incision and
drainage, a sample of the pus is sent to the laboratory for
identification and antibiotic susceptibility testing. A gram-
positive, catalase-positive, coagulase-positive coccus is
isolated (S. Aureus). The following results are obtained:
Which of the following is the most effective?
- Tetracycline
LETS GO 2F! <3
7. How is the information from a kirby-bauer disk diffusion test

used for the recommendation of the clinical use of an
antimicrobial drug?
- To know what is the best antibiotic choice for
a specific pathogen
8. If drug A produces a larger zone of inhibition than drug B on
the Kirby-Bauer disk diffusion test, drug A should always be
prescribed.
a. True
b. False
i. Patient can develop drug resistance if
continued tobe prescribed with the same
drug
-END-

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MICROBIOLOGY AND PARASITOLOGY

Lecture or Laboratory \ FIRST SEMESTER

UNIT 07: STERILITY TESTING

2. PRODUCT (DRUG ITSELF)

DGU, GBS, JJC, QME | 2F-PH 1

UNIT 07: STERILITY TESTING

DGU, GBS, JJC, QME | 2F-PH 2

UNIT 07: STERILITY TESTING

MEMBRANE FILTRATION METHOD Not more than 200 5% or 2 containers, whichever is

DGU, GBS, JJC, QME | 2F-PH 3

UNIT 07: STERILITY TESTING

DGU, GBS, JJC, QME | 2F-PH 4

UNIT 07: STERILITY TESTING

a. Type: Surgical dressing/ cotton/ gauze (in

• Steps of General Procedure

o Before a product could be labelled as sterile,

is a new formulation or whenever there is a change in

DGU, GBS, JJC, QME | 2F-PH 5

UNIT 07: STERILITY TESTING

method for detection of microbial contamination in the

o Growth promotion test

§ to demonstrate that the medium

supports growth and has the ability

to detect organisms in the presence

of the test sample.

o Suitability of counting method

storage, and stock culture of each organism is limited

to not more than five passages removed from the

• As an alternative to preparing and diluting the fresh

suspension of vegetative cells of Aspergillus

brasiliensis for the bacillus subtilis, a stable spore

suspension must be prepared and then appropriate

volume of the spore suspension is used for test

DGU, GBS, JJC, QME | 2F-PH 6

UNIT 07: STERILITY TESTING

• The suitable culture media for product testing are the

fluid thioglycolate media (FTM) that supports both

decreases the amount of oxygen and promotes the

• On the other hand, the second medium will be the

Soyabean Casein Digest Medium. It is a versatile

medium that supports the growth of fungi and your

MICROBIAL ENUMERATION TESTS (MET)

CULTURE MEDIA FOR STERILITY TESTING

DGU, GBS, JJC, QME | 2F-PH 7

UNIT 07: STERILITY TESTING

of the diluted suspension. SUITABILITY TEST METHOD

5. The final concentration will be estimated as 0.1 mL or

STERILITY TESTING: METHOD SUITABILITY TEST

1. Two individual tubes containing Fluid Thioglycollate

Medium or Soybean-Casein Digest Medium, transfer

quantity of product to be examined (such that product

2. Refer to the table of the product volume that you should

3. Inoculate Fluid Thioglycollate Medium with not more

DGU, GBS, JJC, QME | 2F-PH 8

UNIT 07: STERILITY TESTING

to eliminate the antimicrobial activity and to repeat the

Growth promotion & Suitability of Test (Pseudomonas

evidence of microbial growth. Why is FTM used as the media?

the examined product to comply with the sterility test.

5. If you have observed clear growth, it means that the REFERENCES

product should fail the test. - Asynchronous Discussion of MicroPara Professors

DGU, GBS, JJC, QME | 2F-PH 9

EXERCISE 8: BACTERIAL ENDOTOXIN TEST

ENDOTOXIN AND EXOTOXIN

• Steps of General Procedure 

of the diluted suspension.  SUITABILITY TEST METHOD

evidence of microbial growth.  Why is FTM used as the media?

the examined product to comply with the sterility test. 

product should fail the test.  - Asynchronous Discussion of MicroPara Professors