Download Microbe Handling in the General Biology Lab

Microbe Handling in the
General Biology Lab
A Guide for Biology Faculty
What are microbes?
„ Microorganisms such as bacteria, yeast and
other fungi, viruses, and protozoans
Bacteria:
Escherichia coli
Fungus:
Saccharomyces
Protozoan:
Trypanosoma
Why do we need special handling for
microbes?
„ Some microbes can cause infectious
diseases
„ Special protocols must be implemented to
minimize the risk to students, faculty, and
staff
Potential Routes of Transmission
„ Contact with skin and mucous membranes
„ Ingestion
„ Inhalation
„ Inoculation
Precautions to Reduce Transmission
„ Wear personal protective equipment
„ Strictly prohibit eating and drinking in the lab
„ Minimize aerosols and wear respirators
when needed
„ Take special precautions with sharps
What types of microbes can be
handled in a general biology lab?
„ The Centers for Disease Control divides
microbes into 4 Biosafety Levels based on
their ability to cause disease
„ Only organisms designated BSL-1 may be
used in GB lab
During which specific GB labs will
microbes be used?
„ Unit 9
„ Saccharomyces (yeast) for fermentation
„ Unit 14
„ Environmental samples obtained by students
„
„
Staphylococcus epidermidis, Escherichia coli,
and Bacillus megaterium
„
„
Growth and characterization
Gram staining
Pond water samples
„
Microscopy
What are the general safety guidelines when
working with BSL-1 organisms?
„ Absolutely no food or drink in lab
„ Students should practice sterile technique
„ All microbial waste should be discarded in
orange BIOHAZARD bags
What specific steps need to be taken with
Units 9 & 14?
„ Unit 9: None
„ Unit 14: Special precautions for:
Obtaining environmental samples
„ Gram staining
„
„ Required Safety Equipment for Unit 14
„
Disposable gloves
Obtaining Environmental Samples
„ Step 1: Put on gloves
„ Step 2: Obtain samples with sterile cotton
swabs and streak onto agar plate
Obtaining Environmental Samples
„ Step 3: Dispose of gloves and swabs in
Orange BIOHAZARD bag
„
DO NOT THROW IN TRASH!
Obtaining Environmental Samples
„ Step 4: Tape agar plates shut on both sides
and place upside-down in incubator
Obtaining Environmental Samples
„ Step 5: After incubation, Instructor should
seal plates with parafilm before returning to
students for observation
„
The students SHOULD NOT OPEN the sealed
petri plates
Obtaining Environmental Samples
„ Step 6: Dispose of sealed plates in
BIOHAZARD waste
Gram Staining
„ Students will be provided with BSL-1
laboratory strains of Bacillus megaterium,
Staphylococcus epidermidis, and Escherichia
coli for Gram staining
„
The students SHOULD NOT Gram stain their
environmental samples
Gram Staining
Overview:
Gram +
Gram Fixation
Crystal Violet
Iodine
Decolorization
Safranin
Gram Staining
Preparing bacterial smears
„ Step 1: Put on gloves
„ Step 2: Sterilize the inoculating loop
SCC/PRC
CPC
Gram Staining
„ Note: DO NOT leave loops unattended in
incinerators
Gram Staining
Preparing bacterial smears (cont)
„ Step 3: Obtain a small amount of bacteria on
sterile loop and place in a drop of water on
the slide
Gram Staining
Preparing bacterial smears (cont)
„ Step 4: Sterilize loop again
„ Step 5: Allow slide to air dry completely
Gram Staining
Preparing bacterial smears (cont)
„ Step 6: Heat fix slide
CPC
SCC/PRC
Gram Staining
„ Why heat fix?
Kills the microbes
„ Makes microbes more permeable to stains
„ Fixes microbes to the slide
„
Gram Staining
Step 1: Apply Crystal Violet
Step 2: Rinse with dH2O
Step 3: Apply Iodine
Step 4: Rinse with dH2O
Gram Staining
Step 5: Decolorize with Ethanol
Step 6: Rinse with dH2O
Step 7: Apply Safranin
Step 8: Rinse with dH2O
Gram Staining
„ Step 9: Blot slide with bibulous paper
„ Step 10: View Slide under Microscope
„
Use 100X Oil Immersion lens
Gram Staining
Results:
Gram Negative
Gram Positive
Gram Staining
„ Dispose of glass slides in SHARPS container
„ Dispose of gloves in BIOHAZARD waste
Questions?
„ Please Ask!