Laboratory Execise 9. Effects of physical factors on survival and growth of microorganisms

OBJECTIVES:

1.     To show the effects of environment on bacterial growth.

2.     To give specific conditions affecting the culture of bacteria in the laboratory.

3.     To compare the results of various changes in pH of the medium on bacterial growth.

4.     To demonstrate the effect of sunlight on bacterial growth.

5.     To demonstrate anaerobic methods of culturing bacteria.

METHODOLOGY

A. Influence of temperature on bacterial growth. Microorganisms tolerate temperature to varying extents. Each bacterium has its minimum, optimum and maximum temperature levels. Exposure to temperature lower than its minimum would inhibit growth of an organism. Exposure to temperature above the maximum denatures proteins that can lead to physical destruction of membranes or disruption of metabolism.

1.     From a 24h- culture of isolate, inoculate 3 nutrient broth tubes

2.     Place one labeled 4^oC in the refrigerator, another tube labeled room temperature and the third labeled 37^oC in the incubator. Incubate for 24h.

3.     Observe relative amount of growth as effected by temperature. Tabulate results.

B. Effect on pH. There exist for the growth of each kind of organism an optimum, minimum and maximum pH requirement. Bacteria in general have optimum pH where growth is most favored at levels close to neutrality while fungi grow best at acidic conditions.

1.     From a 24h culture of bacterium inoculate 3 cultures of nutrient broth.

2.     Add acid or alkali to change the pH of the medium as follows:

a.     pH 4

b.    pH 7

c.     pH 10

C. Effect of ultraviolet radiation                                                                                                                                 Ultraviolet radiation is one of the most effective means of killing microorganisms. UV rays (265 nm) are readily absorbed by DNA. It causes the coupling of pyrimidines leading to deletion mutation. Such mutation can cause death of cells unless the damage is repaired. In the presence of visible light, the enzyme pyrimidine dimerase is activated, splits the pyrimidine dimer and restores nucleic acids to normal.

1. Prepare four NA plates
2. Introduce 0.1ml suspension from a 24h culture isolate.
3. Streak or swab agar surface to induce heavy confluent inoculum.
4. Remove the top cover of the 2-petri plates and expose the plates under UV light for 15 minutes.  Leave the other 2 plates unexposed as the control.
5. Incubate plate 1 in the dark and plate 2 in the light for 24 hours at room temperature. Incubate the first control plate in the dark and the second control plate in the light.    
6. Record the presence and absence of growth of bacteria on the plates.

D. Culture of Anaerobic bacteria

1.     Use 2 butts of thioglycolate medium to reduce oxygen tension. Use candle jar to create the condition of anaerobic growth. Layer mineral oil on the surface of the culture tube from which the oxygen has been expelled from heating.

2.     Inoculate with 24h culture of the isolate. Do the same for agar butt for comparison. 

RESULTS AND DISCUSSION

1. Record results relating the different physicochemical factors to bacterial growth. State what you have proved in this part of the exercise.
2. Why is it necessary to remove the glass cover during the UV exposure? 
3. Discuss the role of thioglycollate medium in anaerobic culture. 

REFERENCES

Laboratory Exercise No. 9

 

Name: Date Performed Rating:

Effects of Physical Factors on

          Survival and Growth of

                Microorganisms