Test Method Plate Count Swab

Animal Health Research Institute
Alex food inspection lab
Enumeration of microorganisms using pour plate technique

for swabs
Code No. Test Procedure ( )

Version No. 2
Issue Date 1/ 8/2022
No. of Pages 9
Preparation Revision Approval

Name Dr\ayman aamr Dr\mohamed Dr\ahmed aiad
elsendiony
Position Head unit of Quality manager Technical manager
microbiology unit
Sign
Date
) ‫( جدول التعديالت‬
‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫مراجعة‬ ‫إصدار‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬
CONTENTS
Version : 1 Issue date: 09/01//2019 Page 1/9

1. Purpose:
2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
7. Procedure.
8. Retention And Disposal.
9. Method Validation.
10. Test Report
11 Assuring the test result
12. Appendices.
13 Distribution List.
1. Purpose:
Enumeration of microorganisms for swabs .
2. Scope:

A horizontal method for the enumeration of microorganisms. by counting the

colonies growing in a solid medium after aerobic incubation at 300c .
.(iso :4833-1: 2013 /AMD :2022)and iso 18593:2018
3. Applicability.
This procedure shall be applied by all personnel who authorized to
Carry out this test.
4. Environment.
 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 and 45-50% humidity
5. Equipments and Media.
5.1. Equipments
 Autoclave
 Sterilized oven
 Incubator capable of operating at 30 ±1°C.
 Stomacher, Stomacher bags
 Analytical balance
 pH meter
 colony counter
 Glassware (test tubes\ 1ml pipettes, Petri dishes90mm to100mm and
flask not greater than 500ml)
 Vortex
 Water bath
 Lamina flow

 Microscope
 Pipette aid
 swabs
5.2 Culture media
 Diluent (peptone salt solution).
 Plate count agar .
 Overlay medium.
6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put any thing in or near your mouth
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 oC
 Operation should not be carried out in direct sunlight.
 Mouth pipetting is prohibited.
7. Procedure.
7.1 swabbing , initial suspension and dilutions
• Label the swab tube with sufficient detail.

• Remove the swab stick from the sterile wrapping.

• Moisten the tip, if required, by immersing it in a tube containing dilution liquid.
• Press the tip of the tube against the inside wall of the tube to remove excess fluid.
• Select the area to be swabbed and apply a suitably sized template for example (10
x 10 cm ²)
• Press the tip of the swab onto the surface and streak in two directions at right angles
within the template whilst rotating the swab stick between thumb and forefinger.
• Put the swab back in the tube with the dilution material and aseptically break or cut
off the stick at 100 ml of peptone water saline .
 Prepare serial dilutions by using peptone water saline .
7.2 inoculation and incubation

 Take two sterile Petri dishes .Transfer to each dish, by means of a
sterile pipette 1 ml of the initial suspension .
 Take two other sterile Petri dishes. Transfer to each dish, by means
of another sterile pipette .1 ml of the 10−1 dilution (liquid product) or
1 ml of the 10−2 dilution (other products).
 If necessary, repeat the procedure with the further dilutions, using a
new sterile pipette for each decimal dilution.
 If appropriate and possible, select only the critical dilutions steps (at
least two consecutive decimal dilutions) for the inoculation of the
Petri dishes that will give colony counts of between 15 and 300
colonies per plate.
 Pour about 12 ml to 15 ml of the plate count agar at 44 °C to 47 °C
into each Petri dish. The time elapsing between the end of the
preparation of the initial suspension (or of the 10−1 dilution if the
product is liquid) and the moment when the medium is poured into
the dishes shall not exceed 45 min.
 Carefully mix the inoculums with the medium by rotating the Petri
dishes and allow the mixture to solidify by leaving the Petri dishes

standing on a cool horizontal surface.

 After complete solidification, and only in the case where it is
suspected that the product under examination contains
microorganisms whose colonies will overgrow the surface of the
medium, pour about4 ml of the overlay medium at 44 °C to 47 °C
onto the surface of the inoculated medium. Allow to solidify as
described above.
 Invert the prepared dishes and place them in the incubator at 30 °C
± 1 °C for 72 h ± 3 h. Do not stack the dishes more than six high.
Stacks of dishes should be separated from one another and from the
walls and top of the incubator
7.3 counting of colonies

 After the specified incubation period , count the colonies on the
plates , using the colonycounting equipment if necessary.
 Examine the dishes under subdued light. It is important that pinpoint
colonies should be included in the count, but it is essential that the
operator avoid mistaking particles of undissolved or precipitated
matter in dishes for pinpoint colonies.
 Examine doubtful objects carefully, using higher magnification
where required, in order to distinguish colonies from foreign matter.
 Spreading colonies shall be considered as single colonies. If less
than one-quarter of the dish is overgrown by spreading, count the
colonies on the unaffected part of the dish and calculate the
corresponding number of the entire dish. If more than one-quarter is
overgrown by spreading colonies, discard the count.

7.4 Expression of results

Method of calculation
8. Retention And Disposal.

1 - If the materials contain +ve cultures (petri dishes), or
contaminated tools (pipettes) and all materials used for
confirmatory tests must be sterilized by autoclaving at 121
o
C for 20 minutes.
2 - After autoclaving :
a) glass materials, washed by detergent then tap water and
distillated water and leave to dry at room temperature and
sterilized in hot air oven to at least 180 oC for one hour.
b) plastic materials , removed in special container
9. Method Validation .

The test procedure validation is not needed because the procedure is

derived from .iso :4833-1: 2013 /AMD :2022)and iso 18593:2018.
.
10. Test Report .
The final laboratory report has a ( form 20-1)
11. Assuring the quality of test results
The laboratory applied the quality control program to insure the quality
11.1 Internal quality control :
 Spiked samples
 Repeatability
 Duplicate test
12. Appendixes :
Appendix 1 : flow chart of the method
13. Distribution List :
 Technical Manger specialty
 Staff of Microbiological laboratory
Flow Chart Showing Enumeration of Microorganisms Using Pour Plate

Technique


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Animal Health Research Institute

Alex food inspection lab

Enumeration of microorganisms using pour plate technique

Code No. Test Procedure ( )

Preparation Revision Approval

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫مراجعة‬ ‫إصدار‬

Version : 1 Issue date: 09/01//2019 Page 1/9

Version : 1 Issue date: 09/01//2019 Page 2/9

A horizontal method for the enumeration of microorganisms. by counting the

Version : 1 Issue date: 09/01//2019 Page 3/9

5.2 Culture media

 Diluent (peptone salt solution).

 Plate count agar .

Version : 1 Issue date: 09/01//2019 Page 4/9

• Remove the swab stick from the sterile wrapping.

7.2 inoculation and incubation

Version : 1 Issue date: 09/01//2019 Page 5/9

standing on a cool horizontal surface.

7.3 counting of colonies

Version : 1 Issue date: 09/01//2019 Page 6/9

7.4 Expression of results

8. Retention And Disposal.

b) plastic materials , removed in special container

Version : 1 Issue date: 09/01//2019 Page 7/9

The test procedure validation is not needed because the procedure is

Flow Chart Showing Enumeration of Microorganisms Using Pour Plate

Version : 1 Issue date: 09/01//2019 Page 8/9

Version : 1 Issue date: 09/01//2019 Page 9/9

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