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Laboratory Exercise 11
PHYSICAL AND CHEMICAL FACTORS AFFECTING GROWTH

Objectives: At the end of the experiment, the student must be able to:
1. Determine the effects of several chemical and physical factors on the growth of different
microorganisms;
2. Classify different bacterial species according to their response to various temperature,
pH, oxygen and salinity/osmolarity.

Materials: Cultures of E. coli, S. aureus, alcohol lamps, loops, NA Slants (modified)

Procedure:
A. Effect of Temperature
1. Inoculate 3 NA slants with E. coli or S. aureus.
2. Incubate the first tube at the refrigerator (4°C), the second at room temperature (28°C),
the third at the incubator and the last one at the oven (55°C).
3. Examine the slants for growth after 24 hours of incubation. Record your results as
follows: 0 = no growth, 1+ = slight growth, 2+ = moderate, 3+ = abundant. Determine
temperature classification of each bacterial species.

B. Effect of pH
1. Inoculate S. aureus into agar slants of different pH (pH 5, 7, 9).
2. Incubate the tubes at 35°C for 24 hours.
3. Examine the slants for growth after incubation. Record your results, as in A-3. Determine
the optimum pH requirement for growth of each organism.

C. Effect of Osmolarity of the Environment

1. A. Inoculate S. aureus into agar slants of different salt concentrations (0%, 5% and
10%).
B. Inoculate S. aureus into agar slants of different glucose concentrations (0%, 10%,
25%).
2. Incubate the tubes at room temperature for 24 hours.
3. Examine the slants for growth after incubation. Record your results, as in A-3. Determine
the optimum glucose and salt concentration required for growth of each organism.

Study Questions:
 Short but concise answers must be placed after every question.
 Cite your references.

1. How does temperature, pH, and salinity/osmolarity affect microbial growth?

2. If a bacterial culture is incubated at a temperature that is above the maximum growth
temperature of that organism, what effect will that have on its cellular enzymes?
3. What is the pH tolerance of bacteria compared to yeast?
4. How does osmolarity of the environment affect microbial growth?
5. Compare isotonic, hypotonic, and hypertonic solutions and the effects they have on bacterial
cells.
6. Differentiate the following: obligate anaerobe, obligate aerobe, facultative anaerobe,
aerotolerant anaerobe, microaerophile. Give examples of microorganisms on each.
7. At the molecular level, why are strict aerobes unable to grow in the absence of oxygen?
8. Since humans require oxygen in cellular respiration, we are classified as aerobic organisms.
Therefore, how are obligate anaerobes such as Clostridium tetani (causative agent of
tetanus) and Clostridium difficile (causes diarrhea) able to infect the human body?