This document provides an overview of microbiology laboratory safety and techniques. It discusses different types of growth media including general purpose, enriched, selective, and differential media. It also describes common microbiology lab equipment and microorganism isolation techniques like streak plating. Procedures for transferring microorganisms to slants and broth are outlined. Methods for identifying bacteria cultures through colony morphology and staining techniques such as Gram stain and acid-fast stain are summarized. Biosafety levels and guidelines for safe handling of microorganisms are briefly introduced.
2. 2
Media Types
• General Purpose
Media
• Enriched Media
• Selective Media
• Differential
Media
3. 3
Media Types
• General Purpose Media:
• Supports the growth of many microorganisms
• i.e. Luria Agar
• Enriched Media:
• Has special nutrients to encourage the growth of fastidious
heterotrophs
• i.e. Blood Agar
• Selective Media:
• Favors the growth of one type of microorganisms and inhibits the
growth of others
• Luria + penicillin Agar
• Differential Media:
• Distinguishes between different groups of bacteria on the basis of
biochemical characteristics
• i.e. Eosin Methylene Blue Agar
4. 4
Microbiology Lab Equipment
• Microscope (with accessories)
• inoculation loops
• source of flame (Bunsen burner)
• Microscope slides and Cover slips
• Gram staining kits (can purchase from science supply store)
• Petri dishes and proper growth media
• incubators
• identification kits
• autoclave
• Clorox bleach, like you buy at the supermarket, diluted to 5-10% is the
best cleaning agent for labs.
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Streaking and Flaming
Procedure
• Flame the loop to sterilize it and let cool.
• Position the plate so that the spot of inoculum is nearest the hand not holding
the loop (the opposite hand).
• Lift the plate lid with the opposite hand; just enough to get the loop inside and
touch the loop to the inoculum spot. It is often helpful to treat the inoculating
loop as if it were a pencil - steadying the loop by resting the heel of the hand
against the lab bench.
• Move the loop back and forth across the spot and then gradually continue toward
the center of the plate as you sweep back and forth. Use a very gentle and even
pressure.
• When creating each phase, do not worry about keeping each pass across the
plate separate from previous ones.
• When about 30% of the plate has been covered by the first streaking phase,
remove the loop and flame sterilize it.
• Repeat the above procedure for the second phase, but this time pick up some
inoculum by crossing into the first phase 2-3 times and then not passing into it
again (Figure 1-5).
• Repeat as necessary for the third and fourth phases. After streaking the plate,
flame sterilize the loop before setting it down.
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Procedure for Making a
‘Smear’
• Using aseptic technique remove a colony from a plate or cells from
your slant. Be carefully to gently touch the surface of your culture
with the inoculating loop.
• Make a circular motion in the middle of the circle to spread the cells
equally in this region of the slide
• Add a drop of water in the middle
• Mix again
• Let Air dry
• Run the slide through the flame until the slide is warm ( The frosted
side should be down) This fixes the bacteria to the slide
• Let the slide cool
• Place in the metal tray or in the rack
12. 12
Procedure for Transferring
Microorganisms to a Slant
• 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer
• 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in
your pinkie and do not place it on the counter
• 3. Pass the mouth of the culture tube across the flame
• 4. Direct the inoculating needle into the broth.
• 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack
• 6. Pick up the slant in your non dominant hand
• 7. Twist off the red cap
• 8. Flame the mouth of the slant tube
• 9. Direct the inoculating needle into the tube and “ stab” the agar in the base( butt)
• 10. Withdraw on the entry line and when you reach the surface make a simple streak along
the face.
• 11. Flame the mouth of the tube and replace the cap.
• 12. Flame your inoculating needle and replace in your rack.
16. 16
Procedure for Transferring
Microorganisms to Broth Test Tubes
• Steps for Transfer of Broth to Broth
• Hold loop or needle with dominant hand( right )
• Flame the loop
• Hold culture tube in left hand
• Remove red cap with pinkie of right hand
• Flame mouth of culture tube
• Place loop into broth( water)
• Flame mouth of culture tube and close
• Open culture tube with broth( should be labeled)
• Dip loop into new broth and mix
• Flame mouth of tube and close
• Flame loop
• Place to the side of your rack
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Procedure for Gram Stain
• All staining work is to be done at the sink
• Care should be taken to work directly over the sink
• Place 1 drop of crystal violet stain on the smear ( 1 minute)
• Rock or roll the slide to cover the area
• Use the water bottle to drip water down the slide
• Place 1 drop of iodine on the slide ( 1 minute)
• Place 1 drop of alcohol on the slide 10 seconds ( KEY – do not leave on longer
than 10 seconds or it will decolorize)
• Place 1 drop of saffranin on the slide for 1 minute
• Rinse with water from the bottle
• Let the slide air dry
•
29. 29
Microorganism
Categories
• How are microorganisms categorized?
By genetics to show how they are
related
By tissues they infect to show how they
cause disease
By pathogenicity and communicability
(also known as their BioSafety Level)
30. 30
Guidelines for
Microorganism Use
• Besides federal law and regulations
other guidelines exist for the use and
control of microorganisms:
CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories (BMBL)
WHO (World Health Organization)
Biosafety Manual
USDA (United States Department of
Agriculture) protocols
32. 32
BSL Labs
• Microbiology Laboratories are set
up and maintained to meet a
specific containment level. The
designated level conveys
information about infection
potential and engineering controls
implemented to protect workers.
33. 33
BSL Agents
1 Not known to consistently cause disease in healthy
adults
2 Associated with human disease, hazard =
percutaneous injury, ingestion, mucous
membrane exposure
3 Indigenous or exotic agents with potential for
aerosol transmission; disease may have serious or
lethal consequences
4 Dangerous/exotic agents which pose high risk of life-
threatening disease, aerosol-transmitted lab
infections; or related agents with unknown risk of
transmission
Biosafety Levels for Infectious Agents
34. 34
BSL Practice
1 Standard Microbiological Practices
2 BSL-1 practice plus: Limited access, Biohazard
warning signs, "Sharps" precautions, Biosafety
manual defining any needed waste
decontamination or medical surveillance policies
3 BSL-2 practice plus: Controlled access,
Decontamination of all waste, Decontamination
of lab clothing before laundering,
Baseline serum antibody analysis
4 BSL-3 practices plus: Clothing change before
entering, Shower on exit, All material
decontaminated on exit from facility
Recommended Biosafety Level Practices*
35. 35
BSL
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
1 None required Open bench top & sink required
2 Primary barriers = Class I or II
BioSafety Cabinets; laboratory
coats; gloves; face protection as
needed
BSL-1 plus:
• Autoclave available
3 Primary barriers = Class I or II
BioSafety Cabinets; protective
lab clothing; gloves;
respiratory protection as
needed
BSL-2 plus:
• Self-closing, double-door access
• Exhausted air not recirculated
• Negative airflow into
laboratory
4 Primary barriers = Class III
BioSafety Cabinets or in
combination with full-body,
air-supplied, positive pressure
suit
BSL-3 plus:
• Separate building or zone
• Dedicated supply and exhaust,
vacuum, and decon systems
Engineering Controls by Biosafety Level
37. 37
Biosafety Level 1
Standard Microbiological Practices
• Restrict or limit
access when working
• Prohibit eating,
drinking and smoking
in the laboratory
• Pipetting by mouth
strictly forbidden
2.3
39. 39
Standard practices also
include:
• Keep work areas uncluttered and
clean
• No food in lab refrigerator
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Maintain insect & rodent control
program
42. 42
• Disinfection
The use of a physical or chemical
procedure to virtually eliminate
all recognized pathogenic
microorganisms but not all
microbial forms (bacterial
endospores) on inanimate objects.
Disinfection
Definition
44. 44
• Types
Moist – steam
Dry
Incineration
*The most effective method of
sterilization
Decontamination
Heat
45. 45
• Types
Liquids, i.e.
chlorox, hydrogen
peroxide
Gases, i.e.
ethylene oxide
Decontamination
Chemical
46. 46
• General Lab Use - Hypochlorite
Solutions
Large Spills/Large Organic Load
undiluted from bottle
Small Spills/Virus Inactivation
10% - 1:9
General Surface Disinfection
1% - 1:99
Decontamination
Chemical
47. 47
In case of a spill
• Wear disposable gloves
• Cover large blood spill with paper towels and
soak with 1% (10000 ppm) of household
bleach and allow to stand for at least 5
minutes
• Small spill - wipe with paper towel soaked in
1% bleach
• Discard contaminated towels in infective
waste containers
• Wipe down the area with clean towels soaked
in a same dilution of household bleach
48. 48
Aseptic Technique
• First requirement for study of microbes
pure cultures, free of other microbes
• Maintain a clean environment; work close to
the flame