Exercise 23: Pour Plate Counting

What is the purpose of the pour plate technique?

To estimate the number of viable bacteria in a sample by mixing a measured sample with molten agar and counting the resulting colonies.

What are the basic steps of the pour plate technique?

Serially dilute the sample, transfer a measured volume of the appropriate dilution into a sterile Petri dish, add molten agar, mix, allow the agar to solidify, incubate, and count the colonies.

Why is a sample diluted before performing a pour plate count?

To reduce the number of bacteria enough to produce a countable number of colonies.

What is a serial dilution?

A series of stepwise dilutions used to progressively decrease the concentration of microorganisms in a sample.

What does a 10-fold dilution mean?

The sample has been diluted by a factor of 10, so the concentration is one-tenth of the original concentration.

What does a 100-fold dilution mean?

The sample has been diluted by a factor of 100, so the concentration is one-hundredth of the original concentration.

What does a 1,000-fold dilution mean?

The sample has been diluted by a factor of 1,000, so the concentration is one-thousandth of the original concentration.

What does a 100,000-fold dilution mean?

The sample has been diluted by a factor of 100,000, so the concentration is one one-hundred-thousandth of the original concentration.

What is the dilution factor?

The factor by which the original sample has been diluted.

How do you calculate dilution factor?

Dilution factor = original concentration ÷ diluted concentration, or multiply the dilution factors of each dilution step.

If a sample is diluted 10 times and then 10 times again, what is the total dilution factor?

100-fold dilution because 10 × 10 = 100.

If a sample is diluted 10 times five times in a row, what is the total dilution factor?

100,000-fold dilution because 10 × 10 × 10 × 10 × 10 = 100,000.

What is the dilution factor for a 10^-1 dilution?

10-fold.

What is the dilution factor for a 10^-2 dilution?

100-fold.

What is the dilution factor for a 10^-3 dilution?

1,000-fold.

What is the dilution factor for a 10^-4 dilution?

10,000-fold.

What is the dilution factor for a 10^-5 dilution?

100,000-fold.

How do you calculate the original number of cells from a pour plate?

Number of cells in original sample = number of colonies × dilution factor ÷ volume plated in mL.

If 100 colonies grow from a 10^-5 dilution and 1 mL was plated, how many cells were in the original sample?

100 × 100,000 ÷ 1 = 10,000,000 cells/mL, or 1 × 10^7 cells/mL.

If 50 colonies grow from a 10^-4 dilution and 1 mL was plated, how many cells were in the original sample?

50 × 10,000 ÷ 1 = 500,000 cells/mL, or 5 × 10^5 cells/mL.

If 75 colonies grow from a 10^-5 dilution and 0.1 mL was plated, how do you calculate the original concentration?

75 × 100,000 ÷ 0.1 = 75,000,000 cells/mL, or 7.5 × 10^7 cells/mL.

What should you do if the problem gives the dilution as 10^-5?

Use a dilution factor of 10^5, or 100,000.

What should you do if the problem says the sample was diluted 100,000 times?

Use a dilution factor of 100,000.

Why are plates with 30–300 colonies preferred?

They provide enough colonies for reliable counting without being so crowded that individual colonies are difficult to distinguish.

What does TNTC mean?

Too numerous to count.

What does a plate with fewer than 30 colonies indicate?

The count may be less statistically reliable because there are too few colonies.

What does a plate with more than 300 colonies indicate?

The plate may be too crowded to accurately distinguish and count individual colonies.

Why shouldn't you use a plate with too many colonies for the calculation?

Overcrowding can cause colonies to overlap or merge, making the count inaccurate.

Why shouldn't you use a plate with very few colonies for the calculation?

A small number of colonies increases the effect of counting error and makes the estimate less reliable.

What is a colony forming unit (CFU)?

A unit representing a viable microorganism or group of microorganisms capable of producing one visible colony under the given growth conditions.

What does CFU stand for?

Colony-forming unit.

What is a colony?

A visible mass of microbial cells growing on solid media that develops from a viable cell or group of cells.

What is the significance of a colony in a pour plate?

Each countable colony is used as an estimate of one colony-forming unit in the original sample after accounting for dilution.

What is the difference between a colony and a colony-forming unit?

A colony is the visible growth on the plate, while a CFU represents the viable unit that produced that colony.

Does one colony always represent exactly one bacterial cell?

No. A colony may develop from one cell or from a group of cells that were together when plated.

What does CFU/mL mean?

Colony-forming units per milliliter, an estimate of the concentration of viable microorganisms in a liquid sample.

What is the basic formula for CFU/mL?

CFU/mL = number of colonies × dilution factor ÷ volume plated in mL.

When is the pour plate technique advantageous over the streak plate technique?

When you need to estimate the number of viable microorganisms in a sample rather than simply isolate colonies.

What is a major advantage of the pour plate technique?

It allows viable microorganisms to be quantified by counting colony-forming units.

What is another advantage of the pour plate technique?

It distributes microorganisms throughout the agar, allowing colonies to develop both within and on the surface of the medium.

What is a major disadvantage of the pour plate technique compared with streak plating?

The molten agar can expose bacteria to heat, which may injure or kill heat-sensitive microorganisms.

What is another disadvantage of the pour plate technique?

Colonies growing inside the agar can be smaller and more difficult to see or count.

What is another disadvantage of the pour plate technique compared with streak plating?

It requires serial dilution and more materials and steps than a basic streak plate.

What is the main advantage of streak plating compared with pour plating?

Streak plating is simpler and is effective for obtaining isolated colonies from a mixed culture.

What is the main disadvantage of streak plating compared with pour plating?

It is generally not used to accurately quantify the number of viable bacteria in a sample.

If a question asks you to count viable bacteria in a sample, which technique should you choose?

Pour plate technique.

If a question asks you to isolate individual bacterial colonies from a mixed culture, which technique should you choose?

Streak plate technique.

If a plate has 150 colonies, should it be used for counting?

Yes. It is within the ideal range of 30–300 CFU.

If a plate has 25 colonies, should it normally be used for the count?

No. It is below the preferred range of 30–300 colonies.

If a plate has 350 colonies, should it normally be used for the count?

No. It is above the preferred range and may be too crowded to count accurately.

If a plate has 100 colonies and the dilution factor is 10,000, what is the estimated number of cells per mL if 1 mL was plated?

100 × 10,000 ÷ 1 = 1,000,000 cells/mL, or 1 × 10^6 CFU/mL.

If a plate has 200 colonies and the dilution factor is 1,000, what is the estimated number of cells per mL if 1 mL was plated?

200 × 1,000 ÷ 1 = 200,000 CFU/mL, or 2 × 10^5 CFU/mL.

If a plate has 40 colonies and the dilution factor is 100,000, what is the estimated number of cells per mL if 1 mL was plated?

40 × 100,000 ÷ 1 = 4,000,000 CFU/mL, or 4 × 10^6 CFU/mL.

If a plate has 80 colonies from a 10^-4 dilution and 1 mL was plated, what is the original concentration?

80 × 10^4 ÷ 1 = 800,000 CFU/mL, or 8 × 10^5 CFU/mL.

If a plate has 120 colonies from a 10^-5 dilution and 1 mL was plated, what is the original concentration?

120 × 10^5 ÷ 1 = 12,000,000 CFU/mL, or 1.2 × 10^7 CFU/mL.

If 0.1 mL is plated instead of 1 mL, why must the volume be included in the calculation?

The number of colonies came from only 0.1 mL, so the result must be adjusted to express the concentration per 1 mL.

What happens to the calculated CFU/mL when a smaller volume is plated?

The calculated concentration increases because the colony count came from a smaller volume.

What is the overall purpose of dilution in pour plate counting?

To obtain a plate with a countable number of colonies, ideally 30–300 CFU.