Laboratory Manual
Microbiology (BT102)
VIRTUAL UNIVERSITY OF
PAKISTAN
, Contents
Sr. No. Practical P. No.
1 Biosafety levels 2
2 Equipment and Laboratory Materials used in Microbiology 11
3 Microscope and Microscopy 14
4 Sterilization techniques (Physical/Heat method of sterilization) 17
5 Sterilization techniques (Chemical method of sterilization) 22
6 Sterilization techniques (Mechanical/Filtration methods) 24
7 Types of media and culturing 27
8 Culturing of bacteria in liquid medium 33
9 Culturing of bacteria on solid medium 35
10 Colony and cell morphology 40
11 Bacterial cell count 44
12 Gram staining of bacteria, 46
13 Testing sensitivity to antimicrobial substances 49
14 Biochemical tests 51
15 Growth Kinetics 64
1
, Practical No. 1
Biosafety levels
Biosafety
Safety from exposure to Infectious Agents
Biosafety Level:
A biosafety level is a set of biocontainment precautions required to isolate dangerous biological
agents in an enclosed laboratory facility.
Importance:
• Laboratorians recognize hazards of processing infectious agents
• Guidelines developed to protect workers in microbiological and medical labs through
engineering controls, management policies, work practices
• Precautions so people researching or trying to identify organisms do not become infected
• While handling or testing clinical specimens, workers could accidentally infect themselves or
coworkers
• Labs must adhere to very specific safety regulations to work with organisms that pose a threat
to human health
Biosafety levels (1-4):
There are four biosafety levels; protective practices increase with each
Biosafety Level 1 labs - work with least dangerous agents, require fewest precautions.
Biosafety Level 4 labs - have strictest methods because dealing with agents that are most dangerous
to human health
Primary barriers: Physical barriers or personal protective equipment between lab worker and
pathogen. For example: Gloves, masks, lab coat, goggle, shoes cover
Secondary barriers: Structural aspects of the laboratory that make working environment safer
against infection. For example: Sinks for hand washing, special containment areas, special air
ventilation patterns
BIOSAFETY LEVEL 1
• Work is generally conducted on open bench tops using standard microbiological practices.
• Special containment equipment or facility designs is not required nor generally used.
2
, • Laboratory personnel have specific training in the procedures conducted in the laboratory and
are supervised by a scientist with general training in microbiology.
Standard Microbiological Practices:
• Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted
in the work areas where there is reasonable likelihood of exposure to potentially infectious
materials.
• Food is stored outside the work area in cabinets or refrigerators designated and used for this
purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an
approved decontamination method.
• Materials to be decontaminated outside of the immediate laboratory are to be placed in a
durable, leak-proof container and closed for transport from the laboratory.
• Materials to be decontaminated at off-site from the laboratory are packaged in accordance with
applicable local, state, and federal regulations, before removal from the facility.
• Special containment devices or equipment such as a biological safety cabinet is generally not
required for manipulations of agents assigned to Biosafety Level 1.
• It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination.
• Gloves should be worn if the skin on the hands is broken or if a rash exists.
Laboratory Facilities:
• Each laboratory contains a sink for hand washing.
• The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not
appropriate, and should not be used because proper decontamination following a spill
extremely difficult to achieve.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and
moderate heat.
• Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible
for cleaning.
3
Microbiology (BT102)
VIRTUAL UNIVERSITY OF
PAKISTAN
, Contents
Sr. No. Practical P. No.
1 Biosafety levels 2
2 Equipment and Laboratory Materials used in Microbiology 11
3 Microscope and Microscopy 14
4 Sterilization techniques (Physical/Heat method of sterilization) 17
5 Sterilization techniques (Chemical method of sterilization) 22
6 Sterilization techniques (Mechanical/Filtration methods) 24
7 Types of media and culturing 27
8 Culturing of bacteria in liquid medium 33
9 Culturing of bacteria on solid medium 35
10 Colony and cell morphology 40
11 Bacterial cell count 44
12 Gram staining of bacteria, 46
13 Testing sensitivity to antimicrobial substances 49
14 Biochemical tests 51
15 Growth Kinetics 64
1
, Practical No. 1
Biosafety levels
Biosafety
Safety from exposure to Infectious Agents
Biosafety Level:
A biosafety level is a set of biocontainment precautions required to isolate dangerous biological
agents in an enclosed laboratory facility.
Importance:
• Laboratorians recognize hazards of processing infectious agents
• Guidelines developed to protect workers in microbiological and medical labs through
engineering controls, management policies, work practices
• Precautions so people researching or trying to identify organisms do not become infected
• While handling or testing clinical specimens, workers could accidentally infect themselves or
coworkers
• Labs must adhere to very specific safety regulations to work with organisms that pose a threat
to human health
Biosafety levels (1-4):
There are four biosafety levels; protective practices increase with each
Biosafety Level 1 labs - work with least dangerous agents, require fewest precautions.
Biosafety Level 4 labs - have strictest methods because dealing with agents that are most dangerous
to human health
Primary barriers: Physical barriers or personal protective equipment between lab worker and
pathogen. For example: Gloves, masks, lab coat, goggle, shoes cover
Secondary barriers: Structural aspects of the laboratory that make working environment safer
against infection. For example: Sinks for hand washing, special containment areas, special air
ventilation patterns
BIOSAFETY LEVEL 1
• Work is generally conducted on open bench tops using standard microbiological practices.
• Special containment equipment or facility designs is not required nor generally used.
2
, • Laboratory personnel have specific training in the procedures conducted in the laboratory and
are supervised by a scientist with general training in microbiology.
Standard Microbiological Practices:
• Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted
in the work areas where there is reasonable likelihood of exposure to potentially infectious
materials.
• Food is stored outside the work area in cabinets or refrigerators designated and used for this
purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an
approved decontamination method.
• Materials to be decontaminated outside of the immediate laboratory are to be placed in a
durable, leak-proof container and closed for transport from the laboratory.
• Materials to be decontaminated at off-site from the laboratory are packaged in accordance with
applicable local, state, and federal regulations, before removal from the facility.
• Special containment devices or equipment such as a biological safety cabinet is generally not
required for manipulations of agents assigned to Biosafety Level 1.
• It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination.
• Gloves should be worn if the skin on the hands is broken or if a rash exists.
Laboratory Facilities:
• Each laboratory contains a sink for hand washing.
• The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not
appropriate, and should not be used because proper decontamination following a spill
extremely difficult to achieve.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and
moderate heat.
• Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible
for cleaning.
3
