INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY

MED-21A- Mr. Ariel Salvacion

INTRODUCTION TO MICROBIOLOGY

• It is a study of living organisms of microscopic size.

• Study of microbes that can only be observed with various type of microscopes.
• Microbiology is the subdivision concerned with the causative agents of infectious disease of man, the response of the host to
infection and various methods of diagnosis, treatment, and prevention.
• CATEGORIES OF MICROBES
- Virus - Bacteriae
- Fungi - Protozoa
- Archaea - Algae
• Pathogens – can cause disease type of microbes
— can spread easily
—Known as infectious disease
— In distinct shapes and live everywhere.
—3% known microbes
• Non-pathogenic – harmless, and composed 70% of all bacteria.
• —does not cause disease
• —can be beneficial (ex. Saprophytes – that live in the dead matter)
• BACTERIAL SHAPES
▪ Round/ spherical
▪ Rod (bacilli)
▪ Spiral
CATEGORIES OF MICROBES
Acellular microbes • Infectious particles
• Viruses and prions
Cellular microbes • Aka MICROORGANISMS
• Bacteria, protozoa, some algae,archae, some fungi.
• Prokaryotes – Archaea and Bacteria
• Eucaryotes- Algae, Fungi, Protozoa

CATEGORIES OF DISEASE CAUSED BY PATHOGENS

INFECTIOUS DISEASE • Types of disease that colonizes a persons body
• Ex. Neiserria, Mycobacteria, Streptococcus, Staphylococc
MICROBIAL INTOXICATION • Types of diseases in which a pathogen releases toxin that once ingested
by the host can cause intoxication.
• Toxin- a poisonous molecules produced by microorganisms that is
capable of causing harm to the host.

ROUTINELY DETECTED
ORGANISM TOXIN NAME TOXIN TYPE CLINICAL SIGNIFICANCE FOR DEFINITIVE
DIAGNOSIS
Bacillus anthracis Edema toxin (ET) Adenylate cyclase Edema and skin necrosis Yes
plus Protective
antigen (PA)
Lethal Toxin (LT) Metalloprotease YES
plus PA
Bacteroides fragilis Bacteroides fragilis Metalloprotease YES
(strict anaerobes) Enterotoxin
Bordetella pertussis Pertosis Toxin (PT) ADP- riboxylation Tracheobronchitis YES
(whooping cough) ▫️Adenylase cyclase
toxin (ACT) ▫️Adenylase cyclase ▫️NO
Clostridium botulinum Botoinum Metalloprotease Muscle paralysis, botulism YES
(anaerobe) Neurotoxin (BoNT)
Clostridium difficile Toxins A & B Glucosylating toxins Diarrhea Yes
(Post-antibiotic diarrhea)

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
Clostridium perfringens Perfringens Adenylate cyclase Diarrhea NO
enterotoxin
▫️Pore-forming toxin
▫️Perfringiolysin O ▫️Unknown
Clostridium family- strict/ obligate anaerobes.

HISTORY OF MICROBIOLOGY

EARLIEST KNOWN INFECTIOUS DISEASES

• Earliest “pestilence” in EGYPT around 3180 BC.

- First recorded pandemic
- Words like ‘pestilence’ or ‘plague’ were used without definition.
• Near the end of Trojan War around 1900 BC
- Greek Army was decimated by an epidemic of what is thought to have been bubonic plague.
• The Ebers papyrus written around 1500 BC
- describing epidemic fevers, was discovered in a tomb in Thebes, Egypt.
• occurred in China around 1122 BC
- A disease thought to be smallpox
• Epidemics of plague occurred in Rome in 790, 710, and 640 BC and in Greece around 430 BC.
❖ Aside from already mentioned:
- there are early accounts of rabies, anthrax, dysentery, smallpox, ergotism, botulism, measles, typhoid fever, typhus fever,
diphtheria, and syphilis.
❖ Syphilis -It made its first appearance in Europe in 1493. Many people believe that syphilis was carried to Europe by Native
Americans who were brought to Portugal by Christopher Columbus.
o They suggested that disease were caused by "invisible
LUCRETIUS (98-55 B.C) and GIROLAMO FRACASTORO
creatures"
(1478-1553)
o Italian, wrote poems about STDs (Syphilis) “infections
GIROLAMO FRACASTORO
result from tiny self-multiplying bodies that can be spread
by direct/indirect contact "
o He is considered as the "first true microbiologist"
o He is the first person to observe and accurately describe
living microorganisms (rain water, pond, lake, etc.)
o Regarded as the "FATHER OF BACTERIOLOGY AND
ANTONIE VAN LEEUWENHOEK (1632-1723)
PROTOZOOLOGY"
o He used the term "ANIMALCULES" or the tiny living and
moving cells seen under the microscope

o It states that life arises from non-living material if it

contain "pneuma" (vital heat).
o The theory suggests that organisms do not descend from
ARISTOTLE other organism or from a parent and only require that
• THEORY OF SPONTANEOUS GENERATION certain condition in their environment be fulfilled in order
(ABIOGENESIS) for creation to ocCur.
o Living organisms arise from non-living organisms in
combination with energy (earth, water, air, vital heat etc.)

o demonstrated that maggots were the offspring of flies,

not products of spontaneous generation

Francesco Redi

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
John Needham o microbes arose spontaneously in broth from a "life force”.

o Heated but sealed flasks remained clear, without any

Lazzaro Spallanzani signs of spontaneous growth, unless the flasks were
subsequently opened to the air.

o suggested that the

exposure of a broth to air
o was not introducing a "life
force" to the broth
Louis Pasteur o but rather airborne
microorganisms.
o ("Life only comes from
life")
o ...life is a germ and a germ is life".
Rudolf Virchow (1821 - o Proposed the theory of biogenesis, which states that
1902) living cells can arise only from pre-existing living cells.
Ellie Metchnikoff (1845 - 1916) o First to describe the immune system cells and the
process of phagocytosis.
John Tyndall (1820 - 1893) o showed that dusts carry agents that could contaminate a
sterile broth.
o He discovered that there are bacteria that could
Ferdinand Cohn (1828- 1898)
withstand a series of heating and boiling because of
heat-resistant structures known as endospores.
Fanny Hesse (1850 - 1934) o She suggested the use of Agar, a solidifying agent, in
the preparation of culture media
Julius Richard Petri (1852- 1921) o He developed the petri dish.

LOUIS PASTEUR (1822-1895)

▪ BIOGENESIS (making new living things)
o He resolved the issue on Spontaneous Generation with a series of ingenious and persuasive experiment.
o He demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but air itself does not create
microbe.
o He proposed the use of heat in killing microorganisms (ASEPTIC TECHNIQUE) or a method used in preventing contamination
by unwanted organisms.
▪ PASTEUR'S CONTRIBUTION TO MICROBIAL SCIENCE
o He disproved the theory of spontaneous generation.
o He developed the vaccine against anthrax (1881) and rabies (1885)
o He improved the wine-making process (fermentation and pasteurization)

ROBERT KOCH (1843-1910)

o He was the first to show irrefutable proof that bacteria cause diseases
o He discovered: Bacillus anthracis (1876),
Mycobacterium tuberculosis (1882)
o He was the first to cultivate bacteria on boiled potatoes, gelatin, meat extract and protein (ingredients of culture medium)
o He developed a culture media for observing bacterial growth isolated from the human body
▪ Koch's Postulates
✓ Four (4) criteria to assess if a microorganism can cause a disease (Segre, 2013):
1.) The microorganism must be found in diseased but not healthy individuals
2.) The microorganism must be cultured from the diseased individual
3.) Inoculation of a healthy individual with the cultured microorganism
4.) The microorganism must be re-isolated from the inoculated, diseased individua! and matched to the original
microorganism

IMMUNOLOGY (ADVENT OF VACCINATION)

Edward Jenner (1749 - 1823)
LOUIS PASTEUR (1882-1895),
PIERRE PAUL EMILE ROUX (1853-1933)
• He introduced the smallpox vaccination through cowpox
inoculation.
• Pasteur used the term "vaccine" for an attenuated culture
(reduced virulence) They made a series of experiments to
produce an attenuated strains of bacteria.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
MODERN THERAPY: "MAGIC BULLET"

SELMAN WAKSMAN • He discovered the streptomycin and neomycin

(1888-1973) antibiotics.

ALEXANDER FLEMING (1881-1955) • He accidentally discovered the antibiotic Penicillin

(Penicillium notatum) (fungi).
• He Discovered the lysozyme
PAUL EHRLICH (1854-1915) • He discovered salvarsan (arsphenamine for the
treatment of syphilis)

ROBERT HOOKE, ENGLISH-MAN (1665)

▪ After observing a thin slice of cork, reported to the world that life's smallest structural units were "little boxes” or "cells", as
he called them.
▪ Using his improved version of compound microscope, he was able to see individual cells and marked the beginning of "CELL
THEORY". This means
▪ "CELL THEORY"-the theory that all living things are composed of cells.

ANTON VAN LEEUWENHOEK, DUTCH MERCHANT and AMATEUR

▪ Was probably the first to actually observe live microorganisms through the magnitying lenses of more than 400
microscope lenses he constructed.
▪ He made his own microscope magnified to about 270 times and described the different shapes of bacteria.
▪ He made detailed drawings of "animalcules" in rainwater, in his own feces, and in some material scraped from his teeth.
▪ Of his significant discoveries he was known as the "Father of Ancient Bacteriology and Scientific Microscopy".
▪ Thus, was born the Science of Bacteriology Another factor that brought great impact on the development of Microbiology was
the issueSpontaneous Generation Theory.

MICROBIAL TAXONOMY

→ Orderly classification and grouping of organisms into “taxa” or categories.

→ Taxon means “arrangement”
→ Nomos means “law”
Note: Taxon + Nomos = Law of arrangement of microorganisms.
TAXONOMY
▪ is an area of biologic science that comprises three distinct but highly interrelated disciplines:

3 CATEGORIES OF TAXONOMY
→ Classification
→ Nomenclature (naming)
→ Identification
▪ Provides a consistent means to classify, name, and identify organisms. This consistency allows biologists worldwide to use a
common label for every organism studied within the multitude of biologic disciplines (Bailey & Scott.

I. Classification
- It is the organization of microorganisms that have similar morphologic, physiologic, and genetic traits into specific groups or
taxa.
- The formal levels of bacterial hierarchical classification in successively smaller taxa or subsets are as follows:
FORMAL RANK EXAMPLE
Domain Bacteria, Archaea, Eukarya
Kingdom Similar divisions/phyla
Division or Phylum Similar classes
Class Similar orders
Order Similar families
Family Similar genera
Genera Similar species
Species Specific epithelia

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
I.I EUKARYOTES
❖ Distinguishing characteristic:
▪ The "true nucleus" of eukaryotes (from Gr karyon "nucleus") is only one of their distinguishing features.
▪ The membrane-bound organelles, the microtubules, and the microfilaments form a complex intracellular structure.
▪ Flagella and cilia - complex multistranded structures
▪ In general, the genetic transfer among eukaryotes depends upon fusion of HAPLOID (23) gametes to form a DIPLOID cell
containing a full set of genes derived from each gamete (egg and sperm cell)
▪ The life cycle of many eukaryotes is almost entirely in the diploid state (not encountered in prokaryotes)

I.II PROKARYOTES
❖ Organisms in which DNA is not physically separated from cytoplasm no membrane bound organelles)
Distinguishing Characteristics:
▪ Relatively small in size (1 um in diameter)
▪ Absence of nuclear membrane
▪ The DNA of almost all bacteria is a circle with a length of 1mm
▪ Most prokaryotes have only single chromosome
▪ The specialized region of cell containing DNA is termed as NUCLEOID and can be visualized by EM (electron microscope)
▪ Have the capacity to exchange small packets of genetic information this information is carried on PLASMIDS.

I.II FAMILY
- encompasses a group of organisms that may contain multiple genera and
consist of organisms with a common attribute.
- The name of a family is formed by adding the suffix -aceae to the root
name of one of the group's genera, called the type genus
Ex. Family - Streptococcaceae
Genus - Streptococcus
Except: Enterobacteriaceae
- It is named after the "enteric" group of bacteria rather than the type species
Escherichia coli.

II. Nomenclature

- Naming of microorganisms according to established rules and guidelines set forth in the International Code of Nomenclature of
Bacteria (ICNB) or the Bacteriological Code (BC)
▪ Genus and species
▪ RULES:
1. Genus designation -first letter is always capitalized,
2. Species designation -first letter is always lower case
3. Printed in italics
Ex. Staphylococcus aureus
Staphylococcus agalactiae
4. Underlined in script
Ex. Staphylococcus aureus
Staphylococcus agalactiae

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
III. IDENTIFICATION

▪ Process by which a microorganism's key features are delineated.

I IDENTIFICATION METHODS
1. GENOTYPIC CHARACTERISTICS • organism's genetic make-up, including the nature
organism's genes and constituent nucleic acids
a. DNA base composition ratio

2. PHENOTYPIC CHARACTERISTICS • based on features beyond the genetic level and include
both readily observable characteristics and those that
may require extensive analytic procedures to be detected.
1. Macroscopic and microscopic morphology
(inoculated)
2. Staining characteristics (gram staining, any staining
technique)
3. Nutritional requirements (an/aerobic organism, needs
oxygen, doesn't need additional vitamins, etc. for growth)
4. Biochemical Testing (results)

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

INFECTION CONTROL AND LABORATORY

SAFETY

• According to WHO:
- The risk associated with biological materials in the laboratory has a safety and a security componen
1. LABORATORY BIOSAFETY
➢ Containment principles, technologies, and practices implemented to prevent unintentional exposure to pathogens and toxins,
or their unintentional release.
"PROTECTING PEOPLE FROM DANGEROUS PATHOGENS"
➢ Protect the users.
➢ Protect those outside the labs
➢ Protect the environment
2. LABORATORY BIOSECURITY
➢ Institutional and personal security measures designed to prevent the loss, theft, misuse, diversion, or intentional release of
pathogens and toxins
"PROTECTING PATHOGENS FROM DANGEROUS PEOPLE"
➢ PRINCIPLES OF BIOSAFETY
1. PRACTICE AND PROCEDURES a. Standard practices
✓ Most important concept/strict adherence
✓ Aware of potential hazard
✓ Trained and proficient in techniques
✓ Supervisors responsible for: appropriate laboratory
facilities, personnel and training
b. Special practices and considerations -WHO programs
2. SAFETY EQUIPMENT ✓ Primary containment barrier
✓ Minimize exposure to hazard -prevent contact/contain
aerosols
✓ Engineering controls/equipment
✓ PPE -gown, gloves, respirator, face shield booties
✓ BSC
✓ Covered or ventilated animal cage systems

3. FACILITY DESIGN AND CONSTRUCTION ✓ Secondary barrier/engineering controls

✓ Contributes to worker protection
✓ Protects outside the laboratory
Example:
bldg. and lab design, ventilation, autoclaves, cage, wash facilities
4.INCREASING LEVELS OF PROTECTION -BIOSAFETY LEVELS 1-4

BIOHAZARD SYMBOL
There are four circles within the symbol, signifying the chain of
infection.
1. AGENT: The type of microorganism, that causes infection
or hazardous condition.
2. HOST: The organism in which the microorganism Infect.
The new host must be susceptible.
3. SOURCE: The host from which the microorganism
originate. The carrier host might not show symptoms.
4. TRANSMISSION: The means of transmission, mostly
direct or indirect. Some routes of transmission include air,
insect, direct contact and contaminated surfaces.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

BIOSAFETY LEVELS
o Level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility.
o Combination of laboratory practices and procedures, safety equipment (primary barriers and laboratory facilities (secondary
barriers) (based on the risk groups of microorganism)
o Each biosafety level has its own specific containment controls that are required for the following (CDC):
✓ Laboratory practices
✓ Safety equipment
✓ Facility construction

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
CDC BIOSAFETY LEVELS

BSL Dangerous and exotic, posing EBOLA

4 a high risk of aerosol- SMALL POX
transmitted infections. ( no BSL 4 in the
Infections caused by these Philippines)
microbes are frequently fatal
and without treatment or
vaccines

BSL Microbes there can be either HIV, H1N1 Flu,

3 indigenous or exotic, and they Yersinia pestis
can cause serious or (The Plague),
potentially lethal disease Tuberculosis,
through respiratory SARS,
transmission. Rabies, West Nile
Virus, Rickets

BSL Microbes potential hazard to Most Chlamydiae,

2 personnel and the Hepatitis A-C,
environment. Includes Influenza
bacteria and viruses that A, Lyme Disease,
cause only mild disease to Salmonella,
humans, or are difficult to Mumps, Measles
contract via aerosol in a lab
setting. (most laboratories)

BSL Not known to consistently Canine hepatitis,

1 cause disease in healthy non-pathogenic
adult humans, and of minimal Escherichia coli,
potential hazard to laboratory and non-infectious
personnel and environment. bacteria

BIOSAFETY LEVELS
BIOSAFETY LEVEL 1
- For undergraduate and secondary educational training and teaching laboratories.
- Microbes not known consistently to cause disease in healthy adults and present minimal potential hazard to lab and
environment
- Open bench - No containment
• Nonpathogenic strain of Escherichia coli, Bacillus subtilis
BSL 1 PRACTICES SAFETY EQUIPMENT
• Standard microbiological practices are followed • Special containment devices or equipment, such as BSCs
• Work can be performed on an open table or bench - not generally required.
• PPE (Personal Protective Equipment) needed. • Protective laboratory coats or uniforms are
recommended.
• Wear gloves to protect hands from exposure to
hazardous materials.

STANDARD MICROBIOLOGICAL PRACTICES

• Hand washing practices
• Eating, drinking, smoking, handling contact lenses applying cosmetics, and storing food - not permitted in laboratory.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
• Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
• Mouth pipetting is prohibited
• Policies for safe handling of sharps must be implemented.

BIOSAFETY LEVEL 2
- Designed for laboratories that deal with indigenous moderate-risk agents present in the community.
- Practices, equipment and facility design applicable to clinical, diagnostic, teaching laboratories.
- Microbes that possess moderate hazards to laboratorians
- Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures,
or ingestion of infectious materials.
• Hepatitis virus, HIV, Salmonella enterica spp.
• Standard Microbiological Practices (Same as BSL-1)
BSL- 2 PRACTICES • Access to laboratory is restricted when working is being conducted

• Properly maintained BSCs, personal protective equipment (gloves, goggles, mask, face shield)
and/or physical containment devices must be used especially during:
- Procedures with potential for creating infectious aerosols or splashes are conducted.
- Example: Pipetting. Centrifuging
- High concentrations or large volumes of infectious agents are used.
• Autoclave/Decontamination proper
SAFETY
• Self-Closing doors
EQUIPMENT
• Sink with eyewash apparatus readily available
• A sign incorporating:
- Universal biohazard symbol
- Laboratory's biosafety level
- Supervisor's name, Telephone number
- Required procedures for entering

BIOSAFETY LEVEL 3
- Applicable to clinical, diagnostic, teaching, research
- Serious/potentially lethal disease through respiratory transmission
- Primary hazards - relate to autoinoculation, ingestion and exposure to infectious aerosols.
• Mycobacterium tuberculosis, Coxiella burnetiid, St. Louis encephalitis

BSL 3 PRACTICES SAFETY EQUIPMENT STANDARD MICROBIOLOGICAL PRACTICES

• Laboratorians • All procedures must be conducted within a • Same as BSL-1 8 2

- under BSC (preferably Class II or Class III), or • Method for decontaminating all
medical other physical containment devices. laboratory wastes should be
surveillance • Use protective laboratory clothing with a available in the facility, preferably
and receive solid-front, such as tie-back or wrap-around within the laboratory
immunization gowns, scrub suits. • Example: Autoclave, Chemical
• Access to • Eye and face protection (goggles, mask, disinfection, incineration)
laboratory is face shield or other splash guard)
restricted and • Gloves must be worn.
controlled • PPE with respirators
• Biosafety Cabinets
• Sink with eyevgah
• Exhaust air - Not recirculated
• Self-closing doors with automatic locking

BIOSAFETY LEVEL 4
- For working with dangerous and exotic agents that pose a high individual risk of life-threatening disease that may be
transmitted via the aerosol route, for which there are no available treatment or vaccines.
- Highest level of biological safety
- Dangerous and exotic microbes
Montalban, Kimberly N. & Barredo, Althea Kristine
BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
• Example: EBOLA, Marburg Viruses, Crimean-Congo hemorrhagic fever

BSL-4 PRACTICES
• Same with BSL-3
• Change clothes before entering
• Shower upon exiting
• Decontaminate all materials before exiting
• Class III BSC
• Separate building for laboratory
• Vacuum lines and decontamination systems

A Biological Safety Cabinet (BSC)

o mainly used for handling pathogenic biological samples or for applications that require a sterile work zone.
o A biological safety cabinet creates inflow and downflow of air that provides operator protection.
o Air that contains the infectious material is sterilized, either by heat, ultraviolet light, or, most commonly, by passage through a
HEPA filter that removes most particles larger than 0.3 mm in diameter.
• The downflow air passes through an ULPA/HEPA filter and creates an ISO Class 3 work zone to protect samples from the risk
of cross-contamination
Class I Biosafety cabinet

o protects the operator and the environment from exposure to biohazards.

o It does not prevent samples being handled in the cabinet from being
exposed to contaminants that may be present in room air.
o allow room (unsterilized) air to pass into the cabinet and around the area
and material within, sterilizing only the air to be exhausted
o Naturally, there is a possibility of cross-contamination that may affect
experimental consistency. Consequently, the scope and application of
Class I cabinets is limited, and it is largely considered obsolete.

Class II Type A2
o is the most common Class II cabinet.
o It has a plenum from which 30% of air is exhausted, and 70%
re-circulated to the work area as the downflow.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
o both the Class II Type A1 and Type A2 must have the positively-pressurized contaminated plenum to be surrounded by
negative pressure. In case there is a leakage on the positive plenum, the leaking aerosol will be pulled by the negativpressure
back to the positive plenum, and it will not leak out.
o In the A2 cabinet, about 70% of air from the positive plenum is recirculated as downflow, and the remaining 30% is discharged
to the lab through the exhaust filter.

Class III biological safety

• Suitable for work with microbiological agents assigned to biosafety levels 1, 2, 3
and 4.
• They are frequently specified for work involving the most lethal biological
hazards.
• Work is performed through glove ports in the front of the cabinet.
• During routine operation, negative pressure relative to the ambient environment
is maintained within the biosafety cabinet. This provides an additional fail-safe
mechanism in case physical containment is compromised.
• On all Class III BSCs, a supply of HEPA filtered air provides product protection
and prevents cross contamination of samples.
• Exhaust air is usually HEPA filtered and incinerated. Alternatively, double HEPA
filtration with two filters in series may be

LABORATORY FACILITIES IN BSL-2

o Laboratory should be
designed in such a way that
it can be easily cleaned.
o Laboratory contains a sink
for washing.
o Laboratory tops are
impervious to water but
resistant to acids, alkalis
and organic solvents.
o An autoclave to
decontaminate infectious
material is available.
o Illumination is adequate for all laboratory activities.
o Storage space is adequate.
• Issues to Consider
o After reading the patient's case history, consider.
o The role of the laboratory worker and microbiology laboratory in an infection prevention and control program
o The surveillance of health care-associated infections (HAls) and the required laboratory support
o The information needed in an outbreak investigation
o The role of the laboratorian as an educator in infection prevention and control
o Bioterrorism and emerging pathogens

INFECTION PREVENTION AND CONTROL

WHAT IS INFECTION PREVENTION AND CONTROL (IPC)?

- Variety of measures practiced by health care personnel to prevent spread, transmission and acquisition and spread of
infection.
• Patient to Patient
• Patient to Staff Member I v Staff Member to Patient
• Staff Member to Staff Member
GOALS OF INFECTION PREVENTION AND CONTROL
• IPC is a universal discipline with relevance to all aspects of healthcare.
✓ Assures that HCWs understand how pathogens can be transmitted in their work environment.
✓ Apply current scientifically accepted infection control principles appropriately.
✓ Minimize opportunity for transmission of pathogens to patients and healthcare workers.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
HIERARCHY OF SAFETY AND HEALTH CONTROLS
• The National Institute for Occupational Safety and Health (NIOSH)
defines five rungs of the Hierarchy of Controls:
1. Elimination
2. Substitution
3. Engineering controls
4. Administrative controls
5. Personal protective equipment.
❖ The hierarchy is arranged beginning with the most effective
controls and proceeds to the least effective.

➢ GENERAL STRATEGIES OF IPC

STANDARD PRECAUTIONS

HAND HYGIENE PERSONAL PROTECTIVE EQUIPMENT SUITABLY CLEAN ENVIRONMENT

ENVIRONMENT
RESPIRATORY HYGIENE SAFE INJECTION PRACTICES SAFE MANAGEMENT

▪ HAND HYGIENE
- A general term referring to any action of hand cleansing
(WHO)
HAND RUB
- is used when the hands are not visibly soiled using an
alcohol-based antiseptic gel or sanitizer.
HAND WASH
- is used when the hands are visibly soiled (after using gloves
or going to the bathroom).
FIVE MOMENTS FOR HAND HYGIENE
1. BEFORE TOUCHING A PATIENT
2. BEFORE CLEAN / ASEPTIC PROCEDURE
3. AFTER BODY FLUID EXPOSURE RISK
4. AFTER TOUCHING A PATIENT
5. AFTER TOUCHING PATIENT SURROUNDINGS

➢ STEPS OF AN OUTBREAK INVESTIGATION

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BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
TABLE 3.3
HEALTH CARE SETTINGS AND COMMON MICROBES OF
INFECTIN CONTROL SIGNIFICANCE
Health Care Setting Microorganisms and Infectious
Diseases
Salmonella spp., Shigella spp.,
Campylobacter spp., Giardia spp.,
Public health Cryptosporidium spp., syphilis,
gonorrhea, Chlamydia spp., HIV,
Neisseria meningitidis, encephalitis
viruses, hepatitis B, hepatitis C
Staphylococcus aureus, MRSA, VRE,
Acute care
Escherichia coli, Pseudomonas
aeruginosa, Clostridium difficile
Hepatitis B, hepatitis C, HIV, 5.
Ambulatory care aureus, MRSA,
P. aeruginosa, VRE
Extended care Opportunistic pathogens, P.
facilities, home aeruginosa,
care Candida albicans, S. aureus, MRSA,
Acinetobacter, C. difficile, VRE
S. aureus, MRSA, hepatitis C, lice
Communal living

HIV, Human immunodeficiency virus; MRSA, methicillin-

resistant S. aureus;
VRE, vancomycin-resistent enterococci.

❖ PERSONAL PROTECTIVE EQUIPMENT

• EYE PROTECTION
- Goggles - used as eye protectors from flying particles, dust, wind, chemical fumes or other external hazards
• RESPIRATORY PROTECTION
- Mask - a covering for the face that prevents droplets from the mouth and nose from spreading in the air.
- Respirator masks are a form of respiratory protection PPE that protects the wearer from inhaling harmful airborne particles,
gases or vapors that are present in the environment.
• GLOVES - Gloves protect and comfort hands against cold or heat, damage by friction, abrasion or chemicals, and disease
• ISOLATION GOWN / COVERALLS
- Hospital gowns worn by medical professionals as personal protective equipment (PPE) in order to provide a barrier between
patient and professional.
• SHOE COVERS
- Shoe covers are important as they help maintain a sanitary environment by eliminating tracked-in dirt and microbes and they
protect the wearer fromaccidental spills and body fluids.
• HAIR COVERING
- Surgical caps are a part of medical protective clothing and should prevent germs from the hair or scalp of surgical personnel
from contaminating the operating area.
• FACE PROTECTION
- Face shield that protects the mucous membranes of the eyes, nose and mouth during patient-care procedures.
▪ DONNING AND DOFFING OF PPE

SEQUENCE OF DONNING OF PPE

1. Gown
2. Mask or respirator
3. Goggles or face shield
4. Gloves
SEQUENCE OF DOFFING OF PPE
1. Gloves
2. Face shield or goggles
3. Gown
4. Mask or respirator

❖ SAFE INJECTION PRACTICES

- Safe injection practices are intended to prevent transmission of infectious diseases between one patient and another, or
between a patient and health care personnel during phlebotomy.
• Sharps
- are medical devices like needles, scalpels, and other tools that cut or go into the skin.
✓ Do not uncover on unwrap the sharp object until it is time to use it.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
✓ Keep the object pointed away from yourself and other people at all times.
✓ Never recap or bend a sharp object.
✓ Keep your fingers away from the tip of the object.
✓ If the object is reusable, put it in a secure, closed container after you use it.
✓ Never hand a sharp object to someone else or put it on a tray for another person to pick it up.
• DO NOT RECAP!!!
- If it is essential that a needle be recapped due to the nature of your work, the use of a mechanical device (e.g., tongs, forceps,
or the one-handed scoop method) must be used.
PROPER DISPOSAL
✓ Please remember the following:
1. Approved sharps containers must be placed in areas where sharps may be utilized
2. Containers must be labeled with the date that waste accumulation begins
3. Sharps containers must be properly closed and sealed prior to disposal.
➢ DISEASES THAT CAN BE TRANSMITTED THROUGH NEEDLE STICK INJURY
• Human Immunodeficiency Virus (HIV)
• Hepatitis B Virus (HBV)
• Hepatitis C Virus (HCV)
• HBV - 6-30%
• HCV - 1.8%
• HIV - 0.3%
➢ TRANSMISSION-BASED PRECAUTIONS
• Intended to supplement Standard Precautions in patients with known or suspected colonization of infection of highly
transmissible or epidemiologically important pathogens.
AIRBORNE DROPLET CONTACT
Tuberculosis, Measles, Varicella Pneumonia, Influenza, Diphtheria, Scabies, Poliomyelitis, Congenital Rubella
COVID-19*
Use gown / apron and gloves for any
Fitted Respirator (N95 Mask)
Regular Medical Mask procedure involving contact with patient or
their immediate environment
Distance at least 3 feet apart.
May be placed in a private room or
Patient must be placed in a negative Educate about cough etiquette.
cohorting.
pressure room (ideally). May be placed in a private room or
cohorting.

❖ WASTE MANAGEMENT
- waste management is the collection, transport, processing or disposal, managing and monitoring of
Purposes:
✓ To reduce hazardous nature waste.
✓ To reduce volume of waste.
✓ To prevent misuse or abuse of waste.
✓ To ensure occupational safety and health.
▪ All wastes generated by the hospital shall be segregated and disposed property as described in the joint DENR-DOR
Administrative Order No. 02.
▪ Biodegradable, non-biodegradable and infectious wastes shall be placed in green, black and yellow trash bags respectively.
✓ Paper & Paper Products
BLACK ✓ Bottles
NON-INFECTIOUS DRY WASTES ✓ Packaging materials

✓ Leftover food
✓ Used cooking oil
GREEN
✓ Fish entrails, scale & fins
NON-INFECTIOUS WET WASTES
✓ Fruits & vegetable peelings
✓ Rotten fruits & vegetables
✓ Foreign bodies removed from any body parts
✓ Used gloves
YELLOW ✓ Used tubing - IV, nebulizer
INFECTIOUS & PATHOLOGICAL WASTES ✓ Used test strips
✓ Used urine bags
✓ Used mask / face mask

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
✓ lodine 125 (I-125)
✓ lodine 131 (1-1311
✓ H3 - Thymidine
ORANGE
✓ Cesium 137
RADIOACTIVE / NUCLEAR WASTES
✓ Chromium 51
✓ Technetium 99m
✓ Used x-ray films, developers & fixers
✓ Needles & syringes
✓ Scalpel blades
✓ Glass vials - tuberculin / insulin
✓ Stylet
✓ Capillary tubes
RED ✓ Ampules
SHARPS & PRESSURIZED ✓ Blood evacuation tubes
CONTAINERS ✓ Pipette slides / cover slips
✓ Aluminum cover
✓ Blood lancets
✓ Empty aerosol cans
✓ Rusty pins, nails, clips & screws
✓ Broken glasses

✓ MICROBIAL GROWTH CONTROL

Sterilization and Disinfection
• May be accomplished by Physical or chemical means
Sterilization
- is a process whereby all forms of microbial life, including
bacterial spores are killed.
• Target: endospores/bacterial spores
— most important structure present in bacteria being
targeted by sterilization.
• Endospores - highly resistant structures that can withstand
heat, dehydration, freezing, toxic chemicals, UV radiation,
enzymes; which makes the microorganism more pathogenic.
Disinfection
- is a process whereby pathogenic organism, but not
necessarily all microorganisms or spores are destroyed.
• Target: vegetative state (capable of growing/reproducing
without spores)
• Antisepsis- destruction of vegetative state on living tissues.
- Target: vegetative state
• Disinfectant - chemical agent applied to inanimate objects
(table tops, floors, linen)
- Ex. Sodium hypochlorite, Lysol
• Decontamination- is the removal of pathogenic
microorganisms so items are safe to handle or dispose of.

➢ FACTORS THAT LIMIT THE SUCCESS OR DEGREE OF STERILIZATION , DISINFECTION , OR DECONTAMINATION IN A HEALTH CARE
SETTING :
• organic load
o organisms and other contaminating materials such as
blood or body fluids
o Total number of organisms present (microbial
load/bioburden)
• type of organisms present
• concentration and exposure time to the germicide
• physical and chemical nature of the surface (hinges, cracks, rough
or smooth surfaces)
• temperature, pH, humidity
• presence of a biofilm (microorganisms living together in
communities)

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
❖ METHODS OF STERILIZATION
Incineration
o is a method of treating infectious waste.
Hazardous material is literally burned to ashes at temperatures of 870° to 980°C.
o is the safest method to ensure that no infective materials remain in samples or containers when disposed.
Ex. Prions, infective proteins are not eliminated using conventional methods. Therefore incineration is recommended.
Dry heat
o requires longer exposure times (1.5 to 3 hours) and higher temperatures than moist heat (160'
Dry heat ovens are used to sterilize items such as glassware, oil, petrolatum, or powders
TABLE 4-2
Control of Microorganisms Using Heat Methods
Method Temperature (°C) Time Required Applications
Boiling water (steam) 100 15 min Kills microbial vegetative
forms;
endospores survive
Autoclave 121.6 15 min at psi Sterilizes and kills endospores
(steam under pressure)
Pasteurization 63 30 min Disinfects and kills milk-borne
Batch method pathogens and vegetable
forms; endospores survive
Flash method 72 15 secs Same, but shorter time at
higher temperature
Over (dry heat) 160-180 1.5-3 hr Sterilizes; keeps materials dry

• Moist heat (steam under pressure)

- is used to sterilize biohazardous trash and heat-stable objects; an autoclave is used for
this purpose.
- The most commonly used steam sterilizer in the microbiology laboratory is the gravity
displacement autoclave.
✓ The two common sterilization temperatures are 121°C and 132°C.
✓ Moist heat is the fastest and simplest physical method of sterilization.
- Prions require a much more extensive sterilization process.
✓ Several options are recommended for the removal of prions from surgical instruments
or other laboratory materials contaminated with high risk tissue such as brain, spinal
cord, and eye tissue.
✓ 30 minutes at 121°C in a displacement sterilizer
Biologic waste (broth or solid media) ✓ 4 minutes at 132°C in a prevacuum sterilizer.

Infectious medical waste containing body fluids ✓ 132°C for 30 to 60 minutes

According to the eighth edition of Principles and Practices of Infectious Diseases (Elsevier,
2015), there are four options for sterilization:
1. autoclave at 134°C for 18 minutes in a prevacuum sterilizer;
2. autoclave at 132°C for 1 hour in a gravity displacement sterilizer;
3. immerse in 1 N sodium hydroxide for 1 hour, remove and rinse with water, then autoclave at 121°C in a gravity displacement
or 134°C in a prevacuum sterilizer for 1 hour; or
4. immerse in 1 N sodium hydroxide for 1 hour and heat in a gravity displacement at 121°C for 30 minutes, then clean and
subject to routine equipment
✓ Filtration
- is the method of choice for antibiotic solutions, toxic chemicals, radioisotopes, vaccines, and carbohydrates, which are all heat
sensitive
Liquid - pulling the solution through a cellulose acetate or
cellulose nitrate membrane with a vacuum.
- using high efficiency particulate air (HEPA) filters
Air designed to remove organisms larger than 0.3 mm
from isolation rooms, operating rooms, and biologic
safety cabinets (BSCs).
• lonizing radiation
- is used for sterilizing disposables such as plastic syringes, catheters, or gloves before uses
• Chemical sterilant
- The most common chemical sterilant is ethylene oxide (EtO) which is used in gaseous form for sterilizing heat-sensitive
objects.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

End of week 1.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A