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TRANSCRIPT
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
Protozoan: Trypanosoma
Bacteria: Escherichia coli
Fungus: Saccharomyces
Why do we need special handling for microbes?
Some microbes can cause infectious diseasesSpecial protocols must be implemented to minimize the risk to students, faculty, and staff
Potential Routes of Transmission
Contact with skin and mucous membranes IngestionInhalationInoculation
Precautions to Reduce Transmission
Wear personal protective equipment Strictly prohibit eating and drinking in the labMinimize aerosols and wear respirators when neededTake 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 diseaseOnly organisms designated BSL-1 may be used in GB lab
During which specific GB labs will microbes be used?Unit 9
Saccharomyces (yeast) for fermentationUnit 14
Environmental samples obtained by studentsGrowth and characterization
Staphylococcus epidermidis, Escherichia coli, and Bacillus megaterium
Gram stainingPond water samples
Microscopy
What are the general safety guidelines when working with BSL-1 organisms?
Absolutely no food or drink in labStudents should practice sterile techniqueAll microbial waste should be discarded in orange BIOHAZARD bags
What specific steps need to be taken with Units 9 & 14?
Unit 9: NoneUnit 14: Special precautions for:
Obtaining environmental samplesGram staining
Required Safety Equipment for Unit 14Disposable 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 smearsStep 1: Put on glovesStep 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 againStep 5: Allow slide to air dry completely
Gram Staining
Preparing bacterial smears (cont)Step 6: Heat fix slide
SCC/PRCCPC
Gram Staining
Why heat fix?Kills the microbesMakes microbes more permeable to stainsFixes 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 8: Rinse with dH2OStep 7: Apply Safranin
Gram Staining
Step 9: Blot slide with bibulous paperStep 10: View Slide under Microscope
Use 100X Oil Immersion lens
Gram Staining
Results:
Gram PositiveGram Negative
Gram Staining
Dispose of glass slides in SHARPS containerDispose of gloves in BIOHAZARD waste
Questions?
Please Ask!