Microbiology Laboratory

Objectives

Lab exercises from the laboratory manual are scheduled by the instructor and cover
selected objectives described below:

Exercise 1: Introduction to the Microbiology Lab -- Safety: Use and Care of the
Microscope

1. List the specific hazards that one may encounter in the Microbiology Lab and
describe how to protect oneself from each of those hazards.
2. Demonstrate the correct use of a compound light microscope.
3. Diagram the path of light through a compound light microscope.
4. Name and locate the major parts of a compound microscope.
5. Using prepared slides, locate and identify on oil immersion the three basic
morphologies of bacteria

Exercise 9: Microbes in the Environment

1. Culture and grow representative bacteria from various environments in our

building.
2. Describe colonial morphology using accepted descriptive terms.
3. Compare bacterial growth on solid and liquid culture media of the bacteria
isolated from the various environments.

Exercise 3: Aseptic Technique. Preparation of Smears. and Performing Simple

Stains

1. Provide the rationale for aseptic technique.

2. Differentiate among the following: broth culture, agar slant, and agar deep.
3. Aseptically transfer bacteria from one form of culture medium to another.
4. Using the bacteria isolated in Exercise 9 make and fix a smear.
5. List the advantage of staining microorganisms.
6. Explain the basic mechanism of staining.
7. Perform simple direct stains using crystal violet, methylene blue, and safranin.
8. Describe the bacteria stained using oil immersion microscopy.
9. Critique your smears and simple staining techniques.

Exercise 4: Negative Staining

1. Explain the application and mechanism of the negative staining technique.

2. Prepare a negative stain.
Exercise 5: Gram Staining

1. Explain the rationale and procedure for the grarn stain.

2. Perform and interpret gram stains.

Exercise 7: Structural Stains: Endospores. Capsules. and Flagella

1. Using prepared slides, interpret endospore, capsule, and flagella stains.

2. Draw and describe the different flagellar arrangements.
3. Identify the functions of endospores, capsules, and flagella.

Exercise 19: Oxygen and the Growth of Bacteria

1. Identify the incubation conditions for each of the following types of organisms:
aerobes, obligate anaerobes, aerotolerant anaerobes, microaerophiles, and
facultative anaerobes.
2. Describe three methods of culturing anaerobes.
3. Using both reducing media and regular media in an anaerobic pouch and in
aerobic conditions, separate a mixed population of bacteria from soil according to
the organisms'
preferences for atmospheric conditions.

Exercise 20: The Role of Temperature in the Growth of Bacteria

1. Define the following: psychrophile, mesophile, and thermophile.

2. Describe the effect of temperature on the growth of bacteria.
3. Using four different pure cultures of bacteria, determine the optimum temperature
for growth of each organism by subjecting each to incubation at four different
temperatures: 55 degrees C, 35 degrees C, 22 degrees C, and 12 degrees C.

Exercise 21: Effect of Osmotic Pressure of the Growth of Bacteria

1. Define osmotic pressure and explain how it afffects a cell.

2. Using four different microorganisms, determine how osmotic pressure affects the
growth of each organism by subjecting each to growth in increasing amounts of
both NaCl and sucrose.

Exercise 11: Isolation of Bacteria by Dilution Techniques

1. Using a mixed broth culture of several bacteria, isolate the different bacterial
species from one another by using both the streak plate and the pour plate
methods.
2. Prepare and maintain a pure culture from a mixed population of bacteria.

Exercise 12: Special Media for Isolating Bacteria

 1. Differentiate between selective and differential media.
2. Provide an application for enrichment and selective media.
3. Using a mixed broth culture of a mixture of gram + and gram - bacteria, isolate
each type of organism from the other using the appropriate selective media.

Exercise 22: Methods of Control: Phvsical Methods: Heat

1. Compare the bactericidal effectiveness of dry heat and most heat on different
species of bacteria
2. Evaluate the heat tolerance of certain microorganisms.
3. Define and provide a use for each of the following: incineration, hot air oven,
pasteurization, boiling, and autoclaving.
4. Given a number of different microorganisms growing in broth, simulate
pasteurization conditions and attempt to determine the length of time needed to
completely kill each of the different microorganisms.
5. Using your data, evaluate the effectiveness of pasteurization on those organisms.

Exercise 24: Methods of Control: Testing the Effectiveness of Antiseptics and

Disinfectants Using the Fllter Paper Diffusion Method

1. Perform a disk diffusion inhibition assay for disinfectants and antiseptics.

2. Accurately measure a zone of inhibition and describe the information one can get
from a zone of inhibition.
3. Describe the effectiveness of the chemicals you chose to test against the
organisms used in this assay.

Exercise 26: Methods of Control: Effectiveness of Handscrubbing

1. Evaluate the effectiveness of handwashing using a number of different skin

cleansers, including surgical soap.
2. Explain the importance of aseptic technique in the hospital environment.
3. Describe the correct length of time needed for handwashing and the appropriate
soap to use in these situations: in between patients for their routine care; doing an
invasive bedside procedure on a patient; preparing your hands before performing
surgery.

Exercise 34: Survev of the Microbial World: Fungi

1. Characterize and classify fungi.

2. Compare and contrast fungi and bacteria
3. Prepare cultures of and identify common saprophytic molds including
Penicillium, Aspergillus, Pilobolus, Rhizopus, Alternaria, and Fusarium
4. Explain dimorphism and list the systemic dimorphic fungi and the diseases they
cause.

Exercise 8: Identification of a Morphological Unknown

 1. Identify the morphology, staining characteristics, and probable identification of an
unknown organism in pure culture.

Exercise 39: Epidemiology

1. Define the following terms: epidemiology, epidemic, reservoir, and carrier.

2. Describe three methods of transmission.
3. Determine the source of a simulated epidemic.

Exercise 45: Bacteria of the Skin

1. Isolate and identify bacteria from the human skin.

2. Provide an example of normal skin microbiota.
3. List characteristics used to identify the staphylococci.
4. Explain why many bacteria are unable to grow on human skin.

Exercise 46: Bacteria of the Respiratory Tract

1. List representative normal microbiota of the respiratory tract.

2. Differentiate the pathogenic streptococci based on biochemical testing.
3. List a characteristic used to identify Neisseria, Corynebacterium, Mycobacterium,
and Bordetella.

Exercise 49: Bacteria of the Urogenital Tract

1. List bacteria found in urine from a healthy individual.

2. Identify, through biochemical testing, bacteria commonly associated with urinary
tract infections.
3. Determine the presence or absence of Neisseria gonorrhoeae in a G.C. smear.

Exercise 25: Chemical Methods of Control: Antimicrobial Drugs

1. Define the following terms: antibiotic, chemotherapeutic agent, and MIC.

2. Perform an antibiotic sensitivity test.
3. Provide the rationale for the agar diffusion technique.

Exercise 50: Identification of an Unknown from a Clinical Sample

1. Given a culture of bacteria and a description of a patient situation from which that
organism was taken, identify the organism and discuss whether or not that
organism is likely to be causing that situation in that patient.
2. Determine the sensitivity of that organism to chemotherapeutic agents.