Bacteria Population Counts

DISCUSSION

As part of daily routine, the laboratory microbiologist often has to determine the number of bacteria in a given sample as well as having to compare the amount of bacterial growth under various conditions. Enumeration (counting of microbes in a sample) is especially important in dairy microbiology, food microbiology, and water microbiology.

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 review of exponential numbers, scientific notation, and dilutions is found in Appendix A.

THE PLATE COUNT (VIABLE COUNT)

The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted (see Fig. 1) and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies (see Fig. 2), the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution.

However, keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample.

FORMULAS AND INFORMATION YOU NEED TO KEEP IN MIND

Total = Previous Volume Transferred

Dilution Dilution New Total Volume

Plate Dilution = (Dilution in the test tube) (Volume Transferred)

CFUs/ml = (Number of colonies on the plate) (Dilution factor of the plate)

Dilution factor = inverse of the dilution

Only the plate with between 30 and 300 colonies is significant. (In other words, you only count the colonies on the plate with 30-300 colonies.)

There are only 2 significant numbers. You report your results in scientific notation.

Fig. 1: Plate count dilution procedure.

Fig. 2: Single Isolated Colonies Obtained During the Plate Count

Example: A plate containing a 1/1,000,000 (or 10^-6) dilution of the original ml of sample shows 150 colonies. 150 represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 (or 10⁶). In scientific notation, we would report that as 1.5 x 10⁸ CFUs/ml.

Recall our formula from the previous page…

CFUs/ml = (Number of colonies on the plate) (Dilution factor of the plate)

Our plate had 150 colonies and a dilution of 10^-6. Therefore, our dilution factor = 10⁶. So, our formula would be calculated as…

CFUs/ml = 150 x 10⁶ = 1.5 x 10⁸

OUR EXPERIMENT

We are going to imagine that we want to determine the bacterial population count of a Tryptic Soy broth culture of Escherichia coli.

MATERIALS

· Culture of E. coli

· 6 test tubes each containing 9.0 ml sterile water

· 3 sterile Petri dishes

· liquid TSA cooled to 96°C,agar plates of TSA

· 7 sterile 5 ml pipettes

· pipette pump

PROCEDURE

1. Aseptically dilute 1.0 ml of a sample of E. coli as shown in Fig. 1 and described below.

a. Remove a sterile 5.0 ml pipette (This is the 1^st pipette you will use.) from the bag. Do not touch the portion of the pipette that will go into the tubes and do not lay the pipette down.

i. From the tip of the pipette to the first line is 0.5 ml.

ii. The largest lines mark whole milliliters.

iii. The medium lines mark 0.5 ml.

iv. The smallest lines mark 0.1 ml.

b. Insert the cotton-tipped end of the pipette into a pipette pump.

c. Insert the pipette to the bottom of the flask, and withdraw 1.0 ml of the sample by turning the pipette pump knob.

i. Draw the sample up slowly so that it isn't accidentally drawn into the filler itself.

d. Dispense the 1.0 ml of sample into the first test tube of 9.0 ml of sterile water either by turning the pipette pump knob or depressing the release trigger.

i. Draw the liquid up and down in the pipette several times to rinse the pipette and help mix the diluted solution.

e. Mix the tube thoroughly by holding the tube in one hand and vigorously tapping the bottom with the other hand.

2. Insert a sterile 5.0 ml pipette (This is the 2^nd pipette you will use. Every time you draw from a new sample or test tube, you need to use a new pipette.) into the pipette pump.

a. Using the same procedure, aseptically withdraw 1.0 ml from the first dilution test tube and dispense into the second dilution tube.

3. Insert a sterile 5.0 ml pipette (This is the 3^rd pipette you will use.) into the pipette pump.

a. Using the same procedure, aseptically withdraw 1.0 ml from the second dilution test tube and dispense into the third dilution tube.

4. Insert a sterile 5.0 ml pipette (This is the 4^th pipette you will use.) into the pipette pump.

a. Using the same procedure, aseptically withdraw 1.0 ml from the third dilution test tube and dispense into the fourth dilution tube.

5. Insert a sterile 5.0 ml pipette (This is the 5^th pipette you will use.) into the pipette pump.

a. Using the same procedure, aseptically withdraw 1.0 ml from the fourth dilution test tube and dispense into the fifth dilution tube.

b. Using the same (the 5^th) pipette, aseptically withdraw 0.1 ml from the fourth dilution test tube and dispense into the 1^st sterile Petri dish.

6. Insert a sterile 5.0 ml pipette (This is the 6^th pipette you will use.) into the pipette pump.

a. Using the same procedure, aseptically withdraw 1.0 ml from the fifth dilution test tube and dispense into the sixth dilution tube.

b. Using the same (the 6^th) pipette, aseptically withdraw 0.1 ml from the fifth dilution test tube and dispense into the 2^nd sterile Petri dish.

7. Insert a sterile 5.0 ml pipette (This is the 7^th pipette you will use.) into the pipette pump.

a. Aseptically withdraw 0.1 ml from the sixth dilution test tube and dispense into the 3^rd sterile Petri dish.

8. Aseptically pour TSA into the 3 Petri dishes.

a. Gently swirl the agar in the dish.

b. Allow the agar to solidify.

c. Place the dishes in the incubator for 24 hours.

9. Calculate the CFUs/ml of the sample.

RESULTS

Plate Count

1. Choose a plate that appears to have between 30 and 300 colonies.

• Sample 1/100,000 dilution plate.

1/100,000 Dilution of Bacterium (10^{- 5})

This plate has over 300 colonies and cannot be used for counting.

We would record TMC (too many to count) for this plate.

(Alternatively, some labs record TNTC – too numerous to count.)

• Sample 1/1,000,000 dilution plate.

1/1,000,000 Dilution of Bacterium (10^{- 6})

This plate has between 30 and 300 colonies and is a suitable plate for counting.

(It actually had 158 colonies. It is hard to count this after it has been photocopied.)

• Sample 1/10,000,000 dilution plate.

1/10,000,000 Dilution of Bacterium (10^{- 7})

This plate has less than 30 colonies and cannot be used for counting.

2. Count the exact number of colonies on that plate using the colony counter (as demonstrated by your instructor).

3. Calculate the number of CFUs per ml of original sample as follows:

CFUs/ml = (Number of colonies on the plate) (Dilution factor of the plate)

Dilution factor = inverse of the dilution

There are only 2 significant numbers.

____________ = number of colonies

____________ = dilution factor of plate counted

____________ = number of CFUs per ml

PERFORMANCE OBJECTIVES

After completing this lab, the student will be able to perform the following objectives:

1. State the formula for determining the number of CFUs per ml of sample when using the plate count technique.

2. When given a diagram of a plate count dilution and the number of colonies on the resulting plates, choose the correct plate for counting, determine the dilution factor of that plate, and calculate the number of CFUs per ml in the original sample.

Plate Count: Practice Problem #1.

A sample of E. coli is diluted according to the above diagram. The number of colonies that grew is indicated on the Petri plates.

a. How many CFUs are there per ml in the original sample?

b. What is the dilution in the last test tube?

c. What is the dilution on the plate with 3 colonies?

d. How many pipettes should be used for this dilution?

Plate Count: Practice Problem #2

10 ml 1 ml 10 ml 1 ml

10 ml 1 ml 0.1 ml

a. What is the dilution in test tube # 1?

b. What is the dilution in test tube # 2?

c. What is the dilution in test tube # 3?

d. What is the dilution in dish A?

e. What is the dilution in dish B?

f. What is the dilution in dish C?

g. How much water should there be in test tube # 4 for a total dilution of 10^-7?

h. How many CFUs/ml did the E. coli sample have?

APPENDIX A: SCIENTIFIC NOTATION AND DILUTIONS

I. SCIENTIFIC NOTATION

When doing scientific calculations or writing, scientific notation is commonly used. In scientific notation, one digit (a number between 1 and 9) only is found to the left of the decimal point. The following examples are written in scientific notation:

• 3.17 x 10³
• 5.2 x 10^-2

Note that exponents (the powers of 10) are used in these conversions.

Multiples of 10 are expressed in positive exponents:

• 10⁰ = 1
• 10¹ = 10
• 10² = 100 = 10 x 10
• 10³ = 1000 = 10 x 10 x 10
• 10⁴ = 10,000 = 10 x 10 x 10 x 10
• 10⁵ = 100,000 = 10 x 10 x 10 x 10 x 10
• 10⁶ = 1,000,000 = 10 x 10 x 10 x 10 x 10 x 10

Fractions of 10 are expressed as negative exponents:

• 10^-1 = 0.1
• 10^-2 = 0.01 = 0.1 x 0.1
• 10^-3 = 0.001 = 0.1 x 0.1 x 0.1
• 10^-4 = 0.0001 = 0.1 x 0.1 x 0.1 x 0.1
• 10^-5 = 0.00001 = 0.1 x 0.1 x 0.1 x 0.1 x 0.1
• 10^-6 = 0.000001 = 0.1 x 0.1 x 0.1 x 0.1 x 0.1 x 0.1

A. Procedure for converting numbers that are multiples of 10 to scientific notation

1. Convert 365 to scientific notation.

· Move the decimal point so that there is only one digit between 1 and 9 to the left of the point (from 365.0 to 3.65).

· 3.65 is a smaller number than the original. To equal the original you would have to multiply 3.65 by 100. As shown above, 100 is represented by 10² . Therefore, the proper scientific notation of 365 would be 3.65 x 10² .

· A simple way to look at these conversions is that you add a positive power of 10 for each place the original decimal is moved to the left. Since the decimal was moved two places to the left to get 3.65, the exponent would be 10² , thus 3.65 x 10².

2. Convert 6,500,000 to scientific notation.

· Move the decimal point so there is only one digit to the left of the point (6,500,000 becomes 6.5).

· To equal the original number, you would have to multiply 6.5 by 1,000,000 or 10⁶. (Since you moved the decimal point 6 places to the left, the exponent would be 10⁶.)

· Therefore, the proper scientific notation of 6,500,000 would be 6.5 x 10⁶.

B. Procedure for converting numbers that are fractions of 10 to scientific notation.

1. Convert 0.0175 to scientific notation.

· Move the decimal so there is one digit between 1 and 9 to the left of the decimal point (0.0175 becomes 1.75).

· To equal the original number, you would have to multiply 1.75 by 0.01 or 10^-2. Therefore, the proper scientific notation for 0.0175 would be 1.75 x 10^-2.

· A simpler way to look at these conversions is that you add a negative power of 10 for each place you move the decimal to the right. Since the decimal point was moved 2 places to the right, the exponent becomes 10^-2, thus 1.75 x 10^-2.

2. Convert 0.000345 to scientific notation.

· Move the decimal point so only one digit (between 1 and 9) appears to the left of the decimal (0.000345 becomes 3.45).

· To equal the original number, you would have to multiply 3.45 by 0.0001 or 10^-4. (Since you moved the decimal point 4 places to the right, the exponent becomes 10^-4.)

· Therefore, the proper scientific notation of 0.000345 is 3.45 x 10^-4.

C. Other examples

· 12,420,000 = 1.242 x 10⁷

· 21,300 = 2.13 x 10⁴

· 0.0047 = 4.7 x 10^-3

· 0.000006 = 6.0 x 10^-6

II. Dilutions: Examples

A. 1 ml of bacteria is mixed with 1 ml of sterile saline. The total ml in the tube would be 2 ml, of which 1 ml is bacteria. This is a 1:2 dilution (also written 1/2, meaning 1/2 as many bacteria per ml as the original ml).

B. 1 ml of bacteria is mixed with 3 ml of sterile saline. The total ml in the tube would be 4 ml, of which 1 ml is bacteria. This is then a 1:4 dilution (also written 1/4, meaning 1/4 as many bacteria per ml as the original ml).

C. 1 ml of bacteria is mixed with 9 ml of sterile saline. The total ml in the tube would be 10 ml, of which 1 ml is bacteria. This is then a 1:10 dilution (also written 1/10 or 10^-1, meaning 1/10 or 10^-1 as many bacteria per ml as the original ml).

D. For dilutions greater than 1:10, usually serial dilutions (dilutions of dilutions) are made. The following represents a serial ten-fold dilution ( a series of 1:10 dilutions):

• The dilution in tube #1 would be 1/10 or 10^-1.
• The dilution in tube #2 would be 1/100 or 10^-2 (1/10 of 1/10).
• The dilution in tube #3 would be 1/1000 or 10^-3 (1/10 of 1/100).
• The dilution in tube #4 would be 1/10,000 or 10^-4 (1/10 of 1/1000).

The dilution factor is the inverse of the dilution. (Inverse means you flip the two numbers of the fraction; with scientific notation you use the positive exponent.)

• For a dilution of 1/2, the dilution factor would be 2/1 or 2.
• For a dilution of 1/4, the dilution factor would be 4/1 or 4.
• For a dilution of 1/10 or 10^-1, the dilution factor would be 10/1 or 10 or 10¹.
• For a dilution of 1/1,000,000 or 10^-6, the dilution factor would be 1,000,000/1 or 1,000,000 or 10⁶.

In other words, the dilution factor tells you what whole number you have to multiply the dilution by to get back to the original 1 ml.