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Introduction to Microbiology
and Laboratory Safety
Biosafety
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Media Types
• General Purpose
Media
• Enriched Media
• Selective Media
• Differential
Media
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Media Types
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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
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Microbiology Lab Equipment
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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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Microorganism Isolation
Techniques
• Using an Inoculating Loop
• Streaking Methods
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How to hold an Inoculating Loop
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Streaking and Flaming
Procedure
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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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Triple Streak Method
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Streak Plate
http://www.sumanasinc.com/webcontent/anisamples/microbiology/streakplate.html
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Streak plate method of isolation
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Procedure for Making a
‘Smear’
• Using aseptic technique remove a colony from a plate or cells from
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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
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Procedure for Transferring
Microorganisms to a Slant
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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.
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Flaming tubes
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Transferring Microorganisms to Slant
Test Tubes
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Streaking a slant
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Procedure for Transferring
Microorganisms to Broth Test Tubes
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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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Identifying Bacteria
Cultures:
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Colony Morphology
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Colony Morphology
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Colony morphology
Color
Shape
Margin
Elevation
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Stains and Staining
• Bacteria are slightly negatively charged at
pH 7.0
 Basic dye stains bacteria
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Acidic dye stains background
• Simple stain
 Aqueous or alcohol solution of single
basic dye
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Procedure for Simple Stains
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Differential Stains
• Gram stain
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Crystal violet: primary stain
Iodine: mordant
Alcohol or acetone-alcohol:
Safranin decolourizer :
counterstain
Gram positive: purple
Gram negative: pink-red
Staphylococcus aureus
Escherichia coli
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Differential Stains
• Acid-fast stain
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Used to detect Mycobacterium
species
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Procedure for Gram Stain
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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
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Streptococcus
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Staphylococcus aureus
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Gram negative bacilli
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Safety in the
Microbiology Lab
An Introduction to Principles and
Practices at
Biosafety Levels 1, 2, 3, & 4
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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)
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Guidelines for
Microorganism Use
• Besides federal law and regulations
other guidelines exist for the use and
control of microorganisms:
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CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories (BMBL)
WHO (World Health Organization)
Biosafety Manual
USDA (United States Department of
Agriculture) protocols
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Guidelines for
Microorganism Use
The microbes are placed into 4
categories called :
Biosafety Levels (BSL 1-4)
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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.
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Biosafety Levels for Infectious Agents
BSL
Agents
1
Not known to consistently cause disease in healthy
adults
Associated with human disease, hazard =
percutaneous injury, ingestion, mucous
membrane exposure
Indigenous or exotic agents with potential for
aerosol transmission; disease may have serious or
lethal consequences
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Dangerous/exotic agents which pose high risk of lifethreatening disease, aerosol-transmitted lab
infections; or related agents with unknown risk of
transmission
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Recommended Biosafety Level Practices*
BSL
Practice
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Standard Microbiological Practices
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BSL-1 practice plus: Limited access, Biohazard
warning signs, "Sharps" precautions, Biosafety
manual defining any needed waste
decontamination or medical surveillance policies
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BSL-2 practice plus: Controlled access,
Decontamination of all waste, Decontamination
of lab clothing before laundering,
Baseline serum antibody analysis
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BSL-3 practices plus: Clothing change before
entering, Shower on exit, All material
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decontaminated on exit from facility
Engineering Controls by Biosafety Level
BSL
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
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None required
Open bench top & sink required
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Primary barriers = Class I or II
BSL-1 plus:
BioSafety Cabinets; laboratory
• Autoclave available
coats; gloves; face protection as
needed
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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
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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
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Safety Resources
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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
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Biosafety Level 1
Standard Microbiological Practices
38 2.3
Standard practices also
include:
• Keep work areas uncluttered and
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clean
No food in lab refrigerator
Minimize splashes and aerosols
Decontaminate work surfaces daily
Maintain insect & rodent control
program
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Decontamination
• Sterilization
• Disinfection
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Decontamination
Definition
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Sterilization
The use of a physical or chemical
procedure to destroy all microbial
life, including large numbers of
highly resistant bacterial spores.
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Disinfection
Definition
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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.
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Decontamination
Methods
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Heat
Chemical
Radiation
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Decontamination
Heat
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Types
 Moist – steam
 Dry
 Incineration
*The most effective method of
sterilization
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Decontamination
Chemical
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Types
 Liquids, i.e.
chlorox, hydrogen
peroxide
 Gases, i.e.
ethylene oxide
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Decontamination
Chemical
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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
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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
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Aseptic Technique
• First requirement for study of microbes
 pure cultures, free of other microbes
• Maintain a clean environment; work close to
the flame
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Thank you
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