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!