Download Aseptic Technique

Introduction to Microbiology and Laboratory Safety
Biosafety
Microbiology Techniques
SAFETY
NO FOOD OR DRINKS!
Wash hands thoroughly
Disinfect counters and work area
Tie hair back
Smock, apron, or lab coat optional
Gloves and goggles optional
Closed toed shoes required
Eyewash in back sink. Safety shower/fire
extinguishers in back.
 Fire blanket in back above eyewash station.
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STANDARD MICROBIOLOGICAL PRACTICES
STANDARD MICROBIOLOGICAL PRACTICES
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Always thoroughly wash hands upon entering
and leaving lab.
Keep work areas uncluttered and clean
Minimize splashes and aerosols
Disinfect work surfaces daily
DECONTAMINATION
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Sterilization
Disinfection
CHEMICAL DECONTAMINATION
General Lab Use There will always be a
container of disinfectant on the back table for
disposal of contaminated material such as
slides, swabs, etc.
DISINFECTION
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.
DECONTAMINATION
Sterilization: The use of physical or
chemical procedures that destroy all
microbial life forms, including highly
resistant bacterial endospores.
Autoclave: Pressurized steam at 15 psi
and 121oC for an average of 20 min (10 –
40 min depending on bulk and load)
IN CASE OF A SPILL
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Get me, do not pick up glass!
Wear disposable gloves
Cover large spill with paper towels and soak with
5% household bleach and allow to stand for at
least 5 minutes
Small spill - wipe with paper towel soaked in 5%
bleach
Discard contaminated towels in bleach container.
Wipe down the area with clean towels soaked in a
same dilution of household bleach
WHAT NOT TO DO IN A MICRO LAB
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Don’t play with
anything!!!!
Also don’t sleep…picture
illustrates both.
WHAT NOT TO DO IN A MICRO LAB
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It’s a micro lab.
We work with bacteria.
Don’t lie down on the
floor.
WHAT NOT TO DO IN A MICRO LAB
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This was not ok in
genetics.
It’s def not ok now.
Don’t tell anybody about
this.
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
Petri dishes and proper growth media
Incubators
Autoclave (pressure cooker)
Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant
provided in lab.
GROWTH MEDIA
Broth—liquid media
Agar plates
Agar slants
Media Types
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General purpose
Enriched
Selective
Differential
Media Types
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General purpose:
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Enriched
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fastidious organisms—organisms that are pickier, more difficult
to grow.
Selective:
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Supports growth of most non fastidious organisms
Tryptic Soy Agar
Favors the growth of one type of microorganisms and inhibits
the growth of others
Saboraud Dextrose Agar (SDA)
Differential Media:
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Distinguishes between different groups of bacteria on the basis
of biochemical characteristics
ASEPTIC TECHNIQUE
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Required for all microbiology preparations to
assure that contaminants are not introduced.
On a personal note, aseptic technique assures
that infectious agents are not spread to you,
fellow students, or the laboratory surfaces.
GENERAL RULES OF MICROBIOLOGY LABORATORY
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The inoculating loop is usually used for making transfers of bacterial
cultures (see next few slides for technique).
Allow the loop to cool sufficiently so that any organisms to be tested
will not be killed by the hot wire, but do not allow the loop to
contact anything during the cooling period or contamination will
result.
Learn to remove and replace the caps or lids efficiently without
setting them on the countertop or leaving the cover off too long.
After the transfer is completed the loop must be sterilized again.
Follow the procedure outlined on the following slides to prevent
splattering of infectious materials.
It is probably easier to work while sitting down.
Attention to details and practice will allow you to work both rapidly
and accurately.
How to hold an Inoculating Loop
FLAMING A LOOP
FLAMING A LOOP
FLAMING A LOOP
PROCEDURE
In this lab, we will practice transferring
bacteria from an agar slant and a broth culture
to a TSA plate.
 We will be using a technique called spot
inoculation.
 This will not give us isolated colonies.
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get isolated colonies, we will do the streak plate
technique later in the semester.
STREAK PLATE
STREAK PLATE GOOD AND BAD
PROCEDURE FOR TRANSFERRING MICROORGANISMS FROM A SLANT
TO A PLATE
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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 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 already-flamed inoculating loop onto the slant
and remove some bacteria. You do not need much!!!!
5. Flame the mouth of your culture tube again and replace
the cap. Place it in your rack .
6. Pick up the plate in your non dominant hand
8. Move the loop in a small circle in one quadrant of the
plate.
9. Put the plate back on its top.
10. Flame the loop.
PROCEDURE FOR TRANSFERRING MICROORGANISMS FROM BROTH
TO A PLATE
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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 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 already-flamed inoculating loop into the broth.
5. Flame the mouth of your culture tube again and replace
the cap. Place it in your rack .
6. Pick up the plate in your non dominant hand
8. Move the loop in a small circle in one quadrant of the
plate.
9. Put the plate back on its top.
10. Flame the loop.
FLAMING TUBES
TRANSFERRING MICROORGANISMS FROM SLANT TEST TUBES
TRANSFERRING MICROORGANISMS FROM SLANT TEST TUBES
Fig. 3.12.b
Fig. 3.12.c