LAB 3
Bacteria Enumeration

Individual bacteria in a colony

1) Introduction
2) Viable Plate Count
3) Direct Count
4) Turbidity Count
5) Questions

Tables / Graphs

Table 1 Viable Plate Count
Table 2 Direct Count
Table 3 Turbidity Count

Questions

Enumeration of Bacteria (1-5)

Cover Page

Lab 3 Cover Page

1) INTRODUCTION

2) VIABLE (STANDARD) PLATE COUNT

Viable Plate Count (also called a Standard Plate Count) is one of the most common methods, for enumeration of bacteria. Serial dilutions of bacteria are plated onto an agar plate. Dilution procedure influences overall counting process. The suspension is spread over the surface of growth medium. The plates are incubated so that colonies are formed. Multiplication of a bacterium on solid media results in the formation of a macroscopic colony visible to naked eye. It is assumed that each colony arises from an individual viable cell. Total number of colonies is counted and this number multiplied by the dilution factor to find out concentration of cells in the original sample. Counting plates should have 30-300 colonies at least. Since the enumeration of microorganisms involves the use of extremely small dilutions and extremely large numbers of cells, scientific notation is routinely used in calculations.

A major limitation in this method is selectivity. The nature of the growth medium and the incubation conditions determine which bacteria can grow and thus be counted. Viable counting measures only those cells that are capable of growth on the given medium under the set of conditions used for incubation. Sometimes cells are viable but non-culturable.

The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies, the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. The viable plate count method is an indirect measurement of cell density and reveals information related only to live bacteria.
 

CFUs per ml of sample = The number of colonies counted X The dilution factor of the plate counted

In the case of the example above, 150 x 1,000,000 = 150,000,000 CFUs per ml.

At the end of the incubation period, select all of the agar plates containing between 30 and 300 colonies. Plates with more than 300 colonies cannot be counted and are designated "too numerous to count" (TNTC). Plates with fewer than 30 colonies are designated "too few to count" (TFTC).

PROCEDURE
VIABLE PLATE COUNT

We will be testing four samples of water for the Viable Count. The samples include:

1) water from a drinking fountain
2) boiled water from a drinking fountain
3) water from the local river
4) boiled water from the local river

You will need DATA TABLE 1 to input your data and calculate the number of CFU per ml.

DATA TABLE 1 (MS WORD)

DATA TABLE 1 (PDF)

Link for Virtual Lab

***Click Here to Perform Viable Plate Count Virtual Lab

PROCEDURE
1) Take 6 dilution tubes, each containing 9 ml of sterile saline.
2) Dilute 1 ml of a sample by withdrawing 1 ml of the sample and dispensing this 1 ml into the first dilution tube.
3) Using the same procedure, withdraw 1 ml from the first dilution tube and dispense into the second dilution tube. Subsequently withdraw 1 ml from the second dilution tube and dispense into the third dilution tube. Continue doing this from tube to tube until the dilution is completed.

Figure 3.3 Serial dilution

4) Transfer 1 ml from each of only the last three dilution tubes onto the surface of the corresponding agar plates.
5) Incubate the agar plates at 37°C for 48 hours.
6) Choose a plate that appears to have between 30 and 300 colonies.

Figure 3.4 Enumerating a serial dilution

7) Count the exact number of colonies on that plate
8) Calculate the number of CFUs per ml of original sample as follows:

CFUs per ml of sample = The number of colonies X The dilution factor of the plate counted

Figure 3.5 Analyzing a serial dilution

DATA TABLE 1 (MS WORD)

DATA TABLE 1 (PDF)

3) DIRECT MICROSCOPIC CELL COUNT

In the direct microscopic count, a counting chamber with a ruled slide is employed. It is constructed in such a manner that the ruled lines define a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation.

Figure 3.8 Counting chamber graphic

If the bacteria are diluted, such as by mixing the bacteria with dye before being placed in the counting chamber, then this dilution must also be considered in the final calculations.

The formula used for the direct microscopic count is:

# bacteria per cc (ml)
=
The # of bacteria per large double-lined square
X
The dilution factor of the large square (1,250,000)
X
The dilution factor (dye)

PROCEDURE
DIRECT MICROSCOPIC COUNT

We will be testing four samples of water for the Direct Microscopic Count. The samples include:

1) water from a drinking fountain
2) boiled water from a drinking fountain
3) water from the local river
4) boiled water from the local river

You will need DATA TABLE 2 to input your data and calculate the number of bacteria per ml.

DATA TABLE 2 (MS WORD)

DATA TABLE 2 (PDF)

Link for Virtual Lab

***Direct Count Virtual Lab

PROCEDURE
1) Add 1 ml of the sample into a tube containing 1 ml of the dye methylene blue. This gives a 1/2 dilution of the sample.
2) Fill the chamber of a Petroff-Hausser counting chamber with this 1/2 dilution.
3) Place the chamber on a microscope and focus on the squares using 400X.
4) Count the number of bacteria in one of the large double-lined squares. Count all organisms that are on or within the lines.
5) Calculate the number of bacteria per cc (ml) as follows:

The number of bacteria per cc (ml)
=
The number of bacteria per large square
X
The dilution factor of the large square (1,250,000)
X
The dilution factor of any dilutions made prior to placing the sample
in the counting chamber, such as mixing it with dye (2 in this case)

Figure 3.9 Counting Square
 

The large, double-lined square holds a volume of 1/1,250,000 of a cubic centimeter. Using a microscope, the bacteria in the large square are counted. Count all organisms that are on or within the darker double lines.

DATA TABLE 2 (MS WORD)

DATA TABLE 2 (PDF)

4) TURBIDITY COUNT

When you mix the bacteria growing in a liquid medium, the culture appears turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. Within limits, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture. By measuring the amount of light absorbed by a bacterial suspension, one can estimate and compare the number of bacteria present. Spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive.

The amount of absorbance measures what fraction of the light passes through a given solution and indicates on the absorbance display the amount of light absorbed compared to that absorbed by a clear solution.

A standard curve comparing absorbance to the number of bacteria can be made by plotting absorbance versus the number of bacteria per ml. Once the standard curve is completed, any dilution tube of that organism can be placed in a spectrophotometer and its absorbance read. Once the absorbance is determined, the standard curve can be used to determine the corresponding number of bacteria per ml.

PROCEDURE
TURBIDITY COUNT

We will be testing only two samples of water for the turbidity enumeration test. One of the samples has been drawn from a drinking water faucet while the other was taken from the local river. You will need DATA TABLE 3 and a printable version of the STANDARD CURVE CHART to enumerate your samples bacteria.

DATA TABLE 3 (MS WORD)

DATA TABLE 3 (PDF)
 

STANDARD CURVE CHART (MS WORD)

STANDARD CURVE CHART (PDF)

Link for Virtual Lab

***Turbidity Count Virtual Lab

9) Record your values in TABLE 3 for each of the individual samples, along with the dilutions that they came from.

10) Using the standard curve table given below, calculate the number of bacteria per milliliter for each dilution.

DATA TABLE 3 (MS WORD)

DATA TABLE 3 (PDF)

**Review the example of absorbance counts acquired and the determinations of # of bacteria for the dilutions using the STANDARD CURVE CHART given below.  Be sure to keep track of all of the zeros in your calculations of the subsequent calculations for average bacteria per ml.

STANDARD CURVE CHART (MS WORD)

STANDARD CURVE CHART (PDF)

 

Figure 3.16 Standard curve

QUESTIONS

5) QUESTIONS

1) State the formula for determining the number of CFUs per ml of sample when using the plate count technique.
2) State the formula for determining the number of bacteria per ml of sample when using the direct microscopic method of enumeration.
3) State the relationship between absorbance and the number of bacteria in a broth sample.
4) Which method of determining bacterial numbers do you feel is the most accurate and why?
5) Which method of determining bacterial numbers do you feel is the least accurate and why?

Bacterial Enumeration Questions (MS WORD)

Bacterial Enumeration Questions (PDF)

END LAB 3