Preparing a standard plate count of viable bacteria
Lab 5: Preparing a standard plate count of viable bacteria
A) Read “Techniques for Microbiology” (pg 183), please also use the “Atlas for the Microbiology Laboratory” as your study guide and reference
B) View the below video clip thoroughly to learn how this lab is usually performed:
Additional information: Enumerating Microorganisms: Standard Plate Count or Viable Count
Viable bacteria may be counted by diluting samples, plating the dilutions on solid medium, and counting the colonies that arise. Results are usually expressed as colony-forming units (CFU), since some bacteria may not produce colonies on the medium selected.
Only plates (or replicate plates from the same dilution) with 30-300 colonies are counted. Plates with fewer than 30 colonies give statistically unreliable results, while plates with more than 300 colonies are too crowded to allow all the bacteria to form distinct colonies. Usually, more than one dilution in a series is plated, just to be sure that results in a countable range will be obtained. Ignore dilutions giving results outside of the countable range.
The concentration of bacteria in the original sample is calculated as: CFU ml-1 (or g-1) = (colonies on plate)/(final plate dilution).
Frequently, volumes other than one ml are used to inoculate the plate. For example, 0.1 ml is often used when surface-plating, as larger volumes may not be absorbed by the agar. Plating 0.1 ml of a given dilution is mathematically identical to plating a 1 ml of a further 1:10 dilution. For this reason, the size of the inoculum is usually incorporated with the dilution factor to give the “final plate dilution” (d x i). When 1.0 ml of a 10-4 dilution is plated, the final plate dilution is 10-4. When 0.1 ml of the same dilution is plated, the final plate dilution is 10-4 X 10-1 = 10-5. The formula becomes:
CFU ml-1 (or g-1) = (colonies on plate)/(d x i)
Express answers in scientific notation with two significant figures. Watch for division errors:
1/(4 X 10-6) = 2.5 X 105, NOT 4 X 106, and 1/10-6 = 106, NOT 10-6.
Terms:
Sample: refers to the original liquid that will be tested.
Diluent: the solution that is mixed with the sample (often purified water or nutrient broth).
Concentration: refers to how much of the sample is in a given test. For example, 40 to 1 (1/40) dilution means that for every 40 parts of water, there is 1 part of sample.
Dilutions are given in terms of concentration.
Example:
1 g feces + 99 ml saline = 10-2 dilution (A)
1 ml A + 99 ml saline = 10-2 X 10-2=10-4 dilution (B)
0.1 ml B + 9.9 ml saline=10-4 X 10-2 = 10-6 dilution (C)
1 ml C + 9 ml saline = 10-6 X 10-1 = 10-7 dilution (D)
Triplicate 0.1 ml samples of dilutions C and D are plated. The plates are incubated and the colonies are counted. C gives 320, 300, 298 colonies (average, 306). Ignore dilution C, since it averages more than 300 colonies/plate. D gives 34, 30, 29 colonies (average 31). Use the average number of colonies from dilution D to calculate the CFU/g feces:
CFU/g feces = 31/(10-7 X10-1)=31 X 108 = 3.1 x 109.
The final plate dilution was 10-8 and there were 3.1 x 109 CFU/g fecal material.
d) Questions and inquiries for lab 5 report:
Answer the questions below:
1. The first 2-3 plates from the video were not be selected for a viable count and calculation because: (2.5 pts)
a. They did not contain bacterial growth
b. They were not plated correctly
c. They were contaminated
d. They were confluent and not allow to obtain 30-300 colonies
2. The ultimate purpose of performing a serial dilution, plating the diluted bacterial samples on agar plates, and counting the viable colonies from the plate which contained 30-300 colonies is: (2.5 pts)
a. To find out the original concentration of the bacterial sample used for testing
b. To make the plates easy to count
c. To allow bacterial cells to grow their best
d. To know the morphology of the bacterial cells used for testing
3. When 0.1 ml of the same dilution 10-4 is plated, the final plate dilution is: (2.5 pts)
a. 10-5
b. 10+5
c. 10-3
d. 10+3
4. ________________ may be counted by diluting samples, plating the dilutions on solid medium, and counting the colonies that arise. (2.5 pts)
a. Total count
b. Plate count
c. Viable bacteria
d. All of the above
5) You have collected 4 plates after adequate incubation as shown below:
10-6: 465 colonies, 10-7: 280 colonies, 10-8: 20 colonies, and 10-9: no growth. Please answer the questions:
a) Which plate did you select to calculate the CFU/ml? Why this plate was selected?
b) Calculate the selected plate for counting (CFU/ml)
c) Why is it often necessary to dilute a sample before spreading it on a plate for a plate count?
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