Biol 206 Microbiology

Lab 7: Environmental effects on microorganisms

Background:

Physical and chemical agents in the environment may have antimicrobial effects and can be useful in controlling microbial distribution and growth (see appropriate sections in your text or a lab manual). Heat is the best physical agent for destruction of microorganisms and is used extensively to sterilize a great variety of materials. Ultraviolet light is mutagenic and with sufficient exposure radiated cells are incapable of reproduction. A variety of natural and synthetic chemicals inhibit or kill susceptible microorganisms by mechanisms that are characteristic for each chemical. We will examine the effect of several representative agents on some common microorganisms. Use your text as a reference for each topic listed below.

Moist heat:

It is possible to quantitate the response of microorganisms to heat by measuring the time required to kill them at a certain temperature (the Thermal Death Time).

1. Set up a beaker water bath and heat to boiling.
2. Obtain a set of broth tubes with approximately the same amount of medium. Pipette 0.1 ml of a broth culture into the fresh broth and mix gently.
3. Streak a sector of a nutrient agar plate labeled "control" with the mixture.
4. Place the mixture in the boiling water bath for 5, 10, or 20 minutes.
5. Immediately cool the tube in tap water.
6. Streak a labeled sector of the plate with each of the heated mixtures.
7. Invert and incubate the plates at 37 degrees C for 24-48 hr.
8. Record growth (+) or no growth (-) for the control and each time treatment.
9. Determine the TDT for each organism tested.

Ultraviolet light radiation:

We will test the lethal effects and the penetration power of uv light.

1. Obtain three nutrient agar plates and divide them into three parallel sectors. Divide in half perpendicular to the sectors by drawing another line on the bottom. Label each sector with the organism and each half to indicate how it is to be treated.
2. Use a sterile swab to inoculate each sector with a streak of a different organism. The same swab can be used to inoculate the same organism on the other two plates. Discard the swab in the appropriate place.
3. Arrange two of the plates under the uv source. Remove the cover of one and cover tone half of one with a strip of paper. Cover half of another with glass. The third plate will be an unexposed control and should be placed outside the uv source.
4. Expose the plates for 1, 5 or 10 minutes as assigned.
5. Remove the exposed plates, invert and incubate all at 37 degrees C for 24-48 hr.
6. Record the results.

Disinfectants:

A suitable disinfectant must kill microorganisms while it is in contact with them, so they cannot grow again when it is removed. When choosing a disinfectant, factors such as exposure time, concentration required, sensitivity of the organism, and toxicity of the agent for humans or the effect on materials to be disinfected must be considered.

1. Obtain a nutrient plate and label the proper number of sectors.
2. For each chemical agent pipette 5 ml into a sterile tube. Also prepare control tubes (What should control tubes contain?).
3. Add 0.5 ml of an E. coli culture to one and 0.5 ml of a B. cereus culture to the other. Mix gently and note time.
4. At intervals of 2, 5, 10, and 15 minutes, transfer a loopful of the mixture to the labeled sector. Also transfer a control loopful to the control sector.
5. Invert and incubate at 37 degrees C for 24-48 hr.
6. Record the results growth (+) or no growth (-) for each sector for all the chemical agents tested by the class.

Antibiotics:

Antibiotic susceptibility testing of isolated pathogens can indicate specifically which drugs are most effective in their suppression or destruction. In the filter paper disk agar diffusion method a number of small, sterile filter paper disks each impregnated with a different antibiotic are placed on an agar surface previously seeded with an organism to be tested. When the disks are in firm contact with the agar, their antibiotics diffuse into the surrounding medium and come into contact with the growing organisms. Susceptible organisms are inhibited in the zone immediately surrounding the disk.

1. Dip a sterile swab into the culture to be tested, express excess fluid against the inside of the tube and seed the surface of a nutrient agar plate as follows: first streak the whole surface with close, parallel pattern; then rotate the plate about 45 degrees and streak the whole plate again; finally rotate the plate another 90 degrees and streak once more.
2. Deposit different antibiotic disks at different labeled spots.
3. Flame a loop and press each disk gently into full contact with the agar surface.
4. Invert and incubate the plate at 37 degrees C for 24 hr.
5. Measure and record the diameter of the zones of inhibition for each antibiotic and each organism.
6. Interpret the reactions by using the table provided.

Conclusions:

Discuss in your lab notebook the implications of the results you observe.

Questions:

1. What is the Thermal Death Time for each organism tested?
2. Which organism showed the greatest resistance to heat? Was this expected? Why?
3. By what mechanism does uv radiation effect cells?
4. From your observations comment on the penetrating porwer of uv light.
5. Explain why uv radiation therapy is not used for deep-seated tumors.
6. Why are control cultures necessary in evaluating disinfectants?
7. Why are pure cultures used for antibiotic susceptibility testing?
8. What is meant by antibiotic resistance? Susceptibility?
9. What are two major differences between antibiotics and disinfectants?
10. If a laboratory isolates Staph. aureus from 5 patients on the same day, is it necessary to test the antibiotic susceptibility of each isolate? Why?