Microbiology Lab Techniques and Experiments

Genetic Material/DNA

Variable Cell Count

1. DNA must contain biologically useful information.
2. Must be capable of reproduction = replication = growth.
3. Must be decodable (MRNA) transcription/translation.
4. Must be able to change or show variation (mutate/adapt).

H20 Sample -> 1ml -> A10(2) -> 1ml -> B10(4) -> 1ml -> C10(6)
0.1ml D10(5) F10(7)
1ml E10(4) G10(6)
- Pour 10ml cooled molten agar over the bottom of the plate, close the lid, swirl to mix and allow to solidify.
- Invert & incubate at 37C for 24-48h.
- After incubation, observe.
- Select a plate with 30-300 colonies.
- Count all colonies on the agar surface/embedded in agar.
- Colonies X dilution factor = colonies/ml

Griffith's Experiment

1. Injected living S in mouse - mouse died.
2. Injected living R, the mouse was fine. The mouse's WBC's could destroy bacteria.
3. Heat killed the living S, now we have dead S.
4. Injected heat-killed S, the mouse was fine.
5. Killed bacterium before injection.
6. Mixed heat-killed S & living R, the mouse died.
7. When cultured, living S cells were found.
8. Hypothesis: There must have been a transforming factor that gave heat-killed S the gene to the capsule to the living R.
9. Transforming factor: DNA from heat-killed S.
10. Once heat-killed, DNA & cells start to fall apart. Has to be a competent cell to pull in exogenous DNA.

Antiseptics/Disinfectants

Factors/Considerations

1. Concentration of Agent/Increased concentration may cause rapid death of bacteria. May be toxic to humans. Find the right concentration to destroy bacteria and has little effect on humans.
2. Length of Exposure/Length of time for action to take place. Time for disinfection/antisepsis to be effective.
3. Type of Organism/Asporogenic/Sporogenic; G+/G-; AF+/AF-; Non-capsulated/Capsulated; Age of culture; Continuous/Closed.
4. Environmental Considerations/Temp; pH, location of the organism, fomites, skin, wound.

Moist Heat/Temp/Time/Effectiveness

1. Pasteurization/63-65C 30min/Does not kill all bacteria, especially sporogenic.
2. Boiling/100C 30min/More effective, does not kill sporogenic bacteria.
3. Tyndallization/100C 30min 3days/Kills most bacteria, even some spore formers, but not spores.
4. Autoclave/121C 15lbs Pressure sq inch 15min/Destroys all organisms, including sporogenic ones, and spores.

Growth Requirements

• Grow at various temperatures to determine psychrophilic, mesophilic, thermophilic.
• Grow at various pHs: acidophile, neutrophile, alkalinophile.
• Grow with/without O2: aerobic, anaerobic, facultative anaerobic.
• Test salt, grow at various concentrations to determine halophilic or not.
• Grow with/without sunlight to determine photosynthetic or not.
• Grow with organic & inorganic compounds to determine if heterotrophic or autotrophic.

Disinfectant

• Plate all of the various organisms (pathogenic) from the intended area of use.
• Place a disc of disinfectant on the center of each plate.
• Invert/Incubate 24-48h.
• Observe the place for clear zones. If large areas, the disinfectant worked well. If small, the disinfectant didn't work.

LAB MC/TF

Growth Requirements

Disinfectant