Enumeration of bacteria  

STUDENT HANDOUT:

• Enumeration of Bacteria Student Handout

LABORATORY REPORT FORM:

• Enumeration of Bacteria Report Form

INTRODUCTION:

Microbiologists often need to enumerate, or count, the number of bacteria in a sample.  Enumeration of bacteria can be used to determine the growth of the bacterial population.  Bacterial growth does not refer to an increase in the size of individual bacteria, but an increase in the number of bacteria in a population.  It is directly related to the growth rate, or reproduction rate of the population, and can give a microbiologist an idea of how quickly the population is increasing or decreasing.  The size of a bacterial population is important in the fields of food industry, the production of probiotics (bacterial supplements that are ingested for health purposes), water and soil sciences, infectious disease, and just basic knowledge of specific bacterial populations.

Microbiologists use a number of ways to estimate the size of a bacterial population.  These include direct and indirect counts.  Direct counts enumerate bacteria either by counting the number of bacterial cells under a microscope or counting the number of colony forming units (CFUs) in a culture. CFUs are aggregates of bacteria growing in a culture that are all derived from one bacterium that asexually reproduce to create the CFU. Indirect counts use surrogate measures of growth to determine bacterial numbers.  Surrogates include turbidity measure (degree of opaqueness in a broth, Figure 1) and the production of a chemical bioproduct such as the amount of acid or protein produced.

Figure 1. Turbidity.  The tube on the left is clear, indicating a lack of microbial growth while the tube on the right is turbid, indicating the presence of microbial growth.

One issue with both direct bacterial cell counts and indirect counts is the number of bacteria in a sample can be overestimated because these counts may include dead cells or products made by now dead bacteria.  Thus, it may be important to determine the number of viable (living) bacteria, and the best way to do that is through direct CFU counts.  One issue with direct CFU counts is that there may well be over a million bacteria in a volume or mass depending on the sample. 

To get more accurate counts when bacteria have large populations, microbiologists use serial dilution to dilute the samples in a controlled way that allows them to calculate back to the original sample size. Serial dilution is a standard method used by scientists that is a stepwise dilution that dilutes samples by a constant.  

The most commonly used constant for serial dilution is diluting by a power of 10 leading to a logarithmic serial dilution (Figure 2).  

Figure 2.  Serial Dilution. Sample is diluted by added 1mL serially from one test tube to the next. Image from Wikimedia Commons.

You can watch a video of how to calculate serial dilutions.

You can watch a video of how to calculate colony forming units.

PRECAUTIONS:

1. Handle the bacterial cultures with care. Wash your hands with soap before and after handling cultures, and wash work surfaces with disinfectant.

2. Clean up spills using disposable plastic gloves, paper towels, and disinfectant. Dispose of all cleanup materials in the biohazard bag.

3. Do not put fingers or any objects near eyes or mouth while working.

4. Wear safety glass and protective gloves through the entire experiment especially when handling any bacteria cultures or instruments that come in contact with bacterial cultures.

EXPERIMENT DAY #1:  Serial dilution and spread plating

OBJECTIVES:

1. Determine how to dilute bacteria in using serial dilution (a logarithmic dilution) to estimate the number of bacteria per mL of sample.

2. Demonstrate good spread plate technique.

MATERIALS (Work in pairs):

1. Nutrient broth culture of Staphylococcus epidermidis grown at 37°C for 24 hours

2. Nutrient agar plates (6)

3. Sterile cotton swabs (6)

4. Plastic/glass test tubes with lids (or caps) (6)

5. Graduated cylinder

6. Sterile mL plastic pipettes (12)

7. 60 mL sterile water

8. Permanent marker

9. Incubator set at 37°C

10. Tape (masking, scotch, or lab tape)

11. Protective gloves

12. Safety goggle/glasses

13. Disposal beaker of disinfectant

14. Biohazard bag

PROCEDURE:

1. Label test tubes using a permanent marker with initials, dates, and dilution factors.  Each tube receives its own unique dilution factor label shown below:

• 10-1  = 1/10 of the original concentration

• 10-2  = 1/100 of the original concentration

• 10-3  =1/1000 of the original concentration

• 10-4  = 1/10000 of the original concentration

• 10-5  = 1/100000 of the original concentration

• 10-6  = 1/1000000 of the original concentration.

2. Using the graduated cylinder, measure out 9mL of sterile water and add it to each test tube.

3. Gently and carefully resuspend the bacteria into the nutrient broth culture test tube by rolling the test tube between your hands until you see a cloudy residue suspended in the broth.

4. Remove the cap of you to your first test tube (10-1). Add 1 mL of your bacteria from your S. epidermidis culture to the test tube labeled 10-1 using a 1 mL plastic pipette.  Replace the cap on the test tube. Dispose of your plastic pipette in the disposable beaker of disinfectant.

5. Gently mix the bacteria into the water by rolling the test tube between your hands.

6. Remove the cap of you to your next test tube (10-2). Add 1 mL of your sample in the test tube labeled 10-1 into the test tube labeled 10-2 using a new plastic pipette.  See Figure 2. Replace the cap on the test tubes.  Dispose of your plastic pipette in the disposable beaker of disinfectant.

Figure 2.  Serial Dilution. Sample is diluted by added 1mL serially from one test tube to the next. Image from Wikimedia Commons.

7.  Gently mix the bacteria into the water by rolling the test tube between your hands.

8.  Remove the cap of you to your next test tube (10-3). Add 1 mL of your sample in the test tube labeled 10-2 into the test tube labeled 10-3 using a new plastic pipette.  See Figure 2. Replace the cap on the test tubes. Dispose of your plastic pipette in the disposable beaker of disinfectant.

9.  Gently mix the bacteria into the water by rolling the test tube between your hands.

10.  Remove the cap of you to your next test tube (10-4). Add 1 mL of your sample in the test tube labeled 10-3 into the test tube labeled 10-4 using a new plastic pipette.  See Figure 2. Replace the cap on the test tubes. Dispose of your plastic pipette in the disposable beaker of disinfectant.

11.  Gently mix the bacteria into the water by rolling the test tube between your hands.

12.  Remove the cap of you to your next test tube (10-5). Add 1 mL of your sample in the test tube labeled 10-4 into the test tube labeled 10-5 using a new plastic pipette.  See Figure 2. Replace the cap on the test tubes. Dispose of your plastic pipette in the disposable beaker of disinfectant.

13.  Gently mix the bacteria into the water by rolling the test tube between your hands.

14.  Remove the cap of you to your next test tube (10-6). Add 1 mL of your sample in the test tube labeled 10-5 into the test tube labeled 10-6 using a new plastic pipette.  See Figure 2. Replace the cap on the test tubes. Dispose of your plastic pipette in the disposable beaker of disinfectant.

15.  Gently mix the bacteria into the water by rolling the test tube between your hands.

16.  Label your petri plates using permanent marker with initials, dates, and dilution factors.  Each plate receives its own unique dilution factor label shown below:

• 10-1  = 1/10 of the original concentration

• 10-2  = 1/100 of the original concentration

• 10-3  =1/1000 of the original concentration

• 10-4  = 1/10000 of the original concentration

• 10-5  = 1/100000 of the original concentration

• 10-6  = 1/1000000 of the original concentration.

17.  Add 0.25 mL of sample from the test tube labeled 10-1 using a new plastic pipette to the center of the petri plate labeled 10-1. Replace the lid of the petri plate.

18.  Create a spread plate by lifting the petri plate slightly like a clam shell. Using a sterile cotton swab, spread the bacteria in a straight line down the plate as shown in Figure 3.  Then in a zig-zag fashion, spread out your inoculation.  Rotate the plate 60° and continue swabbing the plate.  Rotate the plate again 60° and continue swabbing the plate.  Make sure to swab the entire plate leaving no space uninoculated.

Figure 3.  Creating a spread plate.

19.  Tape the lid closed with two small pieces of tape, one on each side.

20.  Repeat Steps 17-19 for 10-2-10-6 samples.

21.  Incubate your plates at 37°C for 24 hours.

QUESTIONS:

1.  Explain how the serial dilution procedure will logarithmically decrease the number of bacteria per sample?

2. What is the purpose of the serial dilution?  How might it aid in enumeration of the number of bacteria in a sample? 

EXPERIMENT DAY #2:  Calculations

OBJECTIVES:

1. Observe the different growth patterns of the petri plates based on dilution factor.

2.  Determine which petri plate contains a countable number of CFUs.

3. Calculate the estimated number of bacteria per mL of broth culture.

MATERIALS (Work in pairs):

1. Serial dilution petri plates contain S. epidermidis

2.  Colony counters (optional)

3.  Permanent marker

4. Protective gloves

5. Safety goggles/glasses

6. Biohazard bag

3.  Choose the petri plate that has been 30-300 CFUs.  Make note of the dilution factor in your Laboratory Report Form.  When recording the dilution factor, record it without the negative sign in the exponent (i.e., 10-2 becomes 102). Put all other plates to the side.  These will not be used for the rest of the experiment.

4.  Count the number of colonies on the plate with 30-300 CFUs. To do this accurately, turn the plate upside down and place a small mark with your permanent marker on each colony that you  count.  Also, for each colony count push down on your colony counter.  This automatically tallies the number of colonies you count.  If you do not have a colony counter, make sure you are keeping an accurate count of your colonies. 

5.  Record the number of colonies on the Laboratory Reporting Form. Also add the volume of the sample plated onto this plate (0.25 mL).

6.  Use the formula below and the Laboratory Reporting Form to calculate the number of bacteria per mL of broth culture. Be sure to use the dilution factor without the negative sign in the exponent.

# of bacteria per ml of sample = (# of CFU x dilution factor) ÷ (mls of sample plated)

7.  Record your calculation on the Laboratory Report Form, in Table 1.

QUESTIONS:

1. Why was the plate with 30-300 colonies used in order to estimate the # bacteria/mL of sample?

2. Why would a microbiologist need to estimate the size of the bacterial population?

3. What do you think would happen to the number of living bacteria in the culture over time?  Why is it likely to change? 

KEY TERMS:

• bacterial growth

• colony forming units (CFUs)

• direct counts

• enumerate

• indirect counts

• growth rate

• logarithmic serial dilution

• probiotics

• serial dilution

• turbidity

• viability

SUGGESTED READINGS:

(One of the earliest journal articles to discuss serial dilutions!)

McCrady, M. H. (1915). The Numerical Interpretation of Fermentation-Tube Results. The Journal of Infectious Diseases, 17(1), 183–212.