5.1.2: Assessing Effects of Temperature on Microbial Growth Activity

Inoculation and Incubation of Broth Tubes

Label five sterile broths tubes for each of the bacteria to be assessed.

1. Turn on the Bunsen burner and sterilize the inoculating loop by passing it through the flame of the burner.
2. Obtain a bacterial culture. Remove the cap. Pass the mouth of the tube through the flame of the Bunsen burner.
3. Insert the loop into the tube to acquire a sample of the bacterium.
4. Pass the tube through the flame of the Bunsen burner and recap the test tube, and return the tube to the test tube rack.
5. Pick up the broth tube to be incubated at 5°C. Remove the cap and pass the mouth of the tube through the flame of the Bunsen burner.
6. Insert the loop into the tube to introduce the bacterial sample to the media.
7. Pass the mouth of the broth tube through the flame and recap it, and return it to the test tube rack.
8. Sterilize the inoculating loop by flaming the loop in the flame of the Bunsen burner.

Repeat steps 1 - 8 for the tubes that are to be incubated at 25°C, 37°C, 42°C, and 55°C for this same bacterium.

9. Incubate the inoculated broth tubes at the temperature labeled on the tube for 24 hours.

Repeat the above steps for each of the bacteria to be assessed.

Following incubation, you will measure microbial growth and report the data for the growth observed. You will then determine what type of organism each bacterium is based on its temperature preferences, i.e., psychrophile, psychrotroph, mesophile, thermophile.

Two methods have been provided for assessing the microbial growth. The choice of method will depend on what tools are available in your laboratory.

Measuring Microbial Growth:

You will choose your method of analyzing bacterial growth within the broth cultures and then record your data in the provided table.

Spectrophotometric Analysis

If a spectrophotometer is available for analysis, you may use the following steps to obtain your data.

Your instructor will provide instructions for calibrating your spectrophotometer. Calibrate the spectrophotometer using an uninoculated medium. Set the mode to absorbance and the wavelength to 600 nm.

After calibrating the spectrophotometer, you will pipette some of the sample to be measured into a clean cuvette. Make sure to wipe the sides of the cuvette with a Kim wipe to ensure no skin oils or fingerprints interfere with the measurement. Place the cuvette into the spectrophotometer and close the door. Record the absorbance for your sample. Repeat this process until you have measured all of your samples.

Visual Analysis

If a spectrophotometer is available for analysis, you may use the following steps to obtain your data. You will visually examine the sample for growth. You will record your observations based on the following scale.

• - = no growth
• + = minimal growth (minimal turbidity)
• ++ = moderate growth (moderate turbidity)
• +++ = maximal growth (maximum turbidity)

Measuring Microbial Growth: Spectrophotometric Analysis

You will now create a scatter plot from your data using colored pencils. Select a color and place the name of the bacteria in the legend. Then using the same color chart, the data obtained from your experiment. Repeat this for all bacteria assessed.