Quality Control

Microbiology
Practical Session
Lesson 1: Enumeration of Microorganisms

Mohamed Mosaad Aboelgheit

mhmd.aboelgheit@gmail.com

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Lesson 1: Enumeration of microorganisms in
food
• Several methods are available for enumeration of microorganisms in
food.
• Methods involving encouraging the microorganisms to multiply in an
agar media is known as culture-based methods.
• Every single bacterium develops as a colony, which can be counted to
calculate the microbial number in the initial sample.
• Agar media used vary depending on the type of microorganisms to be
studied e.g. TSA and PCA for enumerating of all microbes, DG 18 for
yeasts and molds, etc.

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Standard plate count SPC
• Used to estimate the aerobic bacteria present in a sample.
• Also referred to as: Aerobic Plate Count (APC), Total Plate Count (TPC).
• Two widespread techniques:
• Pour Plate Method:
• samples are mixed in a Petri dish with the molten growth media and allowed to solidify.
• Spread Plate Method:
• samples are distributed and spread evenly onto the pre-poured solidified agar surface.

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SPC

1
• Sample Preparation

2
• Serial Dilution

3
• Plating

4
• Incubation

5
• Enumeration

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Sample Preparation
• Food samples may be:
• Solid
• Liquid
• The purpose of sample preparation is to disperse the sample
homogenously so that the microbes inside can be easily released.
• Liquid sample preparation always include diluting of the sample in a
blank solution e.g. saline.
• Solid samples usually require more processing than liquid ones to
achieve an acceptable level of homogenization.

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Sample Preparation: Solid Food
• Materials and apparatus:
1. Balance
2. Beaker
3. Stomacher Bag
4. Forceps / spoon
5. BPW (Buffer Peptone Water) as a diluent
6. Stomacher Blender (Bag Mixer)

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Serial Dilution
When sampling a food product with an unknown concentration of bacteria, it is
necessary to dilute the homogenized sample in order to determine the estimated
bacterial concentration and allow obtaining a countable number of colonies 20 -
300 CFU per plate.

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Enumeration
• Once the sample is plated and incubated, the resulting bacterial
growth is counted (raw colony count)
• Only plates between 20 -300 can be counted. Counts outside this
range should be considered an estimation NOT enumeration.
• Each colony is a representative of a single microbial cell and is called
(CFU).
• Once the raw colony count is done, the dilution factor and the
quantity of sample is incorporated into the count to calculate the
number of microorganisms in the initial sample.

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Enumeration:

Dilution Raw Colony Average Formula for Initial Count

Count Colony Count initial count (CFU/ mL)

10^-2 100, 124 112 112*10^2 1.12* 10^4

10^-3 30, 36

10^-4 150, 170

10^-5 50, 30

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Enumeration:

Dilution Raw Colony Average Formula for Initial Count

Count Colony Count initial count (CFU/ mL)

10^-2 100, 124 112 112*10^2 1.12* 10^4

10^-3 30, 36 33 33*10^3 3.3*10^4

10^-4 150, 170

10^-5 50, 30

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Enumeration:

Dilution Raw Colony Average Formula for Initial Count

Count Colony Count initial count (CFU/ mL)

10^-2 100, 124 112 112*10^2 1.12* 10^4

10^-3 30, 36 33 33*10^3 3.3*10^4

10^-4 150, 170 160 160*10^4 1.6*10^6

10^-5 50, 30

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Enumeration:

Dilution Raw Colony Average Formula for Initial Count

Count Colony Count initial count (CFU/ mL)

10^-2 100, 124 112 112*10^2 1.12* 10^4

10^-3 30, 36 33 33*10^3 3.3*10^4

10^-4 150, 170 160 160*10^4 1.6*10^6

10^-5 50, 30 40 40*10^5 4*10^6

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Pour Plate Method

1
• Sample Preparation

2
• Serial Dilution

3
• Sampling of plates

4
• Pouring the media. Mix and swirl

5
• Allowing to solidify and incubation

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Spread Plate Method

1
• Sample Preparation

2
• Serial Dilution

3
• Sampling of plates

4
• Spreading on surface using a sterile tool

5
• incubation

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Exercise A: instructions (pour plate method)
1. Add 1 ml of sample to 9 ml sterile saline (10^-1)
2. Make serial dilution to the dilution 10^-2
3. Transfer 1 ml of the last dilution (10^-2) to each of two empty petri-dish (in
duplicate)
4. Transfer 1 ml of the 10^-1 dilution to each of another two empty petri-dishes.
5. Pour the media at 45 degree.
6. Mix and swirl
7. Incubate for 3 – 5 days.
8. Count the colonies
9. Enumerate the initial count. Suppose the raw count was 13 & 20 for the 10^-4
dilution and 220 and 180 for the 10^-3 dilution.
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Exercise B: Instructions (Spread Plate
Method)
1. Add 1 ml of sample to 9 ml sterile saline (10^-1)
2. Make serial dilution to the dilution 10^-4
3. Transfer 1 ml of the last dilution (10^-4) to each of two petri-dish containing
culture media (in duplicate) and spread the sample on the media surface evenly.
4. Transfer 1 ml of the 10^-3 dilution to each of another two petri-dish containing
culture media (in duplicate) and spread the sample on the media surface evenly.
5. Incubate for 3 – 5 days.
6. Count the colonies
7. Enumerate the initial count. Suppose the raw count was 13 & 20 for the 10^-4
dilution and 220 and 180 for the 10^-3 dilution.

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