EFFECT OF TEMPERATURE ON BACTERIAL GROWTH

Bacteria and fungi can grow across a large spectrum of environmental conditions. Even
though the bacterium may grow well in the human body at 37 C at pH 7 conditions, it may
also be able to withstand out-of-the-ordinary pH, temperature, and osmotic pressure.

Basically, there are 3 large groups of microorganisms with respect to their temperature
preference---mesophiles, thermophiles, and psychrophiles. Animal pathogens are
mesophilic, growing well in the range of 20-45C. Psychrophiles have an optimal range below
mesophiles (even below freezing), while thermophiles grow at temperatures over 45C.
Interestingly, many organisms grow differently in different temperatures, and you have seen
evidence of this already in lab. In an earlier exercise, you should have noticed that the
orange-pigmented Serratia marcescens pigments at 25C but not at 37C. The prodigiosin
pigment, a waste product produced during certain metabolic pathways, is temperature-
dependent. Also, some fungi will change structure at these 2 temperatures. For example,
Candida albicans, the cause of thrush and yeast infections, will produce unicellular yeast
cells at 37C, but at 25C will produce chlamydiospores and hyphal filaments. Although
endospore-forming bacteria such as Bacillus can endure very high temperatures, these
bacteria do not optimally grow in those temperatures, hence the term thermoduric. The spore
wall composition and its dehydrated state make it very resistant to harsh environmental
conditions.

There are 2 parts to this exercise:

 Determination of a bacterium’s optimal growth temperature

 Comparison of the response of sporeforming bacteria and non-sporeforming bacteria
to extreme temperature

Different bacteria will be compared in these exercises, including a sporeforming bacterium

(Bacillus). The other bacteria are non-sporeformers.

OBJECTIVES:

Recognize the effects of temperature on bacterial growth and spore resistance.

Determine optimal growth temperature for a bacterium.
Identify the temperature group based on bacterial growth pattern.

MATERIALS NEEDED: per table

Optimal growth - 5 TSB tubes for your organism (your instructor will assign your table’s
bacteria)

Resistance to Extreme Temperature - 8 TSB

1ml pipets and pi-pumps
2 TSA plates

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2420

PROCEDURE

1. OPTIMAL GROWTH TEMPERATURES

Each table uses 1 assigned bacterium at all temperatures.
a. Inoculate 5 TSB tubes with a loopful of the test organisms. Be sure to label each
tube with temperature and the name of the organism.
b. Incubate each tube at a different temperature:
10oC (fridge), 25oC, 30oC, 37oC, 45oC
c. INTERPRETATION: Determine the optimal temperature at which your bacterium
grows. To do this, you need to quantify the amount of growth by comparing the
turbidity. Carefully shake all 5 tubes and holding them up to the light, determine
the amount of growth based on the following criterion.
- no growth (absolutely clear)
+1 very light turbidity
+2 medium turbidity
+3 heavy turbidity (optimal temperature)

2. RESISTANCE OF CELLS AND SPORES TO EXTREME TEMPERATURES

a. Inoculate 4 tubes of TSB broth each with a 0.1ml inoculum from the E. coli .
b. Inoculate 4 tubes of TSB broth each with a 0.1ml inoculum from the Bacillus
spores.
c. Label the tubes as control, 50, 70, and 100, plus the type of culture on each tube.
d. Place all 8 tubes in the water bath equilibrated to one of the temperatures, or in a
boiling water bath for 100C. Incubate for exactly 10 minutes. Be sure that the
water is deep enough so that the broth of the tube will be totally immersed.
e. After 10 minutes exposure to the temperature, streak out a loopful of each of the
tubes from each temperature on a separate TSA plate and incubate at room
temperature.

control 50C control 50C Use a zig-zag or a straight line

inoculation, but be
70C 100C 70C 100C CONSISTENT between
sections.

E. coli spore suspension of Bacillus

f. INTERPRETATION: Determine the effects of various temperatures on a

vegetative cell suspension and the spore suspension. To do this, you need to
quantify the amount of growth by comparing the amount of growth on the agar
plate.
- no growth
+1 very light growth
+2 medium growth
+3 heavy growth

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LABORATORY REPORT SHEET

QUESTIONS:

1. Fill out the table below by PREDICTING where each type of organism would optimally
grow. Use a + sign to denote growth.

Organism 10 °C 25 °C 30 °C 37 °C 45 °C
Psychrophile
Mesophile
Thermophile
Hyperthermophile

2. Record your results by adding -, +1, +2, or +3 for growth.

Species 10 °C 25 °C 30 °C 37 °C 45 °C Classification based on

Optimal temperature

3. Record the results of the extreme temperature exercise. Record your results by adding -,
+1, +2, or +3 for growth.

Temperature E. coli cells Bacillus spores

Control
50
70
100C

4. How do you determine which temperature is the optimal temperature for an organism?

5. How does extremely high temperature kill vegetative cells?

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