Animal Health Research Institute

Alex food inspection lab

Enumeration of yeasts and moulds

Code No. Test Procedure ( )

Version No. 1
Issue Date 12-1-2020
No. of Pages 9

Preparation Revision Approval

Name Dr ayman amar Dr. M sendiouny Dr.Ahmed Aiad
Position Head of bacteriological Quality manager Technical manager for
unit speciality
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫إصدار مراجعة‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬

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Animal Health Research Institute
Alex food inspection lab

CONTENTS

1. Purpose:
2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
7. Procedure.

8 Expression of results

9. Retention And Disposal.

10. Method Validation.
11. Test Report
12. Assuring the test result
13. Appendices.
14 Distribution List

1. Purpose:
Determination and enumeration of yeasts and moulds in Food and Animal feeding stuffs.
according to in house method based ISO 21527 (2) 2008.

2. Scope:
THIS IS a horizontal method for the enumeration of viable yeasts and moulds in
products intended for human consumption or feeding of animals that have a water activity less
than or equal to 0.95(dry fruits,cackes, james,dried meat , salted fish. Grains. Cereals and cereals
product . flours. Nuts.spices and condiments .etc. by means of the colony count technique at 25
°C 1 °C
3. Applicability.
This procedure shall be applied by all personnel who authorized to carry out this test.

4. Environment.

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Animal Health Research Institute
Alex food inspection lab

 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 and 45-50% humidity

5. Equipments and Media.

5.1. Equipments
 Incubator,
 Total delivery pipettes,
 Water bath
 pH meter
 Bottles, flasks and tubes
 Petri dishes
 Microscope
 Spreaders
 Binocular magnifier.

5.2. Culture media

 0,1 % peptone water broth
 Dichloran-rose bengal chloramphenicol agar (DRBC)
 Dicholoran 18% glycerol agar (DG18)
6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put any thing in or near your mouth
 Do not lay the pippete down, or allow it to touch any thing, until it has been sterilized;
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 C
 Operation should not be carried out in direct sunlight Mouth pipetting is prohibited.

7. Procedure.
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Animal Health Research Institute
Alex food inspection lab

7.1 Test portion, initial suspension and dilutions

Prepare the test portion, initial suspension (primary dilution) and further dilutions in accordance with ISO
6887(all parts), ISO 7218, ISO 8261 and the specific International Standard appropriate to the product
concerned.
Except for specific preparation of the test sample, it is recommended to use 0,1 % peptone water broth
(5.1.3) as diluent. Use a peristaltic homogenizer in preference to a blender or shaker.

7.2 Inoculation and incubation

7.2.1
For product with water activity greater than 0.95 On to one DRBC agar plate , using a sterile pipette ,
transfer 0,1 ml of the test sample if liquid, or 0,1 ml of the initial suspension in the case of other products .
On to a second DRBC agar plate, using a fresh sterile pipette, transfer 0,1 ml of the first decimal dilution
(10−1) dilution (liquid product), or 0,1 ml of the 10−2 dilution (other products).
To facilitate enumeration of low populations of yeasts and moulds, volumes of up to 0,3 ml of a 10−1
dilution of sample, or of the test sample if liquid, can be spread on to three plates.
Repeat these operations with subsequent dilutions, using a new sterile pipette for each decimal dilution.

For product with water activity less than or equal to.95 on to one DG18 agar plate , using a sterile pipette ,
transfer 0,1 ml of the test sample ifliquid, or 0,1 ml of the initial suspension in the case of other products .
On to a second DG18 agar plate, using a fresh sterile pipette, transfer 0,1 ml of the first decimal dilution
(10−1) dilution (liquid product), or 0,1 ml of the 10−2 dilution (other products).
To facilitate enumeration of low populations of yeasts and moulds, volumes of up to 0,3 ml of a 10−1
dilution of sample, or of the test sample if liquid, can be spread on to three plates.
Repeat these operations with subsequent dilutions, using a new sterile pipette for each decimal dilution.

7.2.2 Spread the liquid over the surface of the agar plate with a sterile spreader (6.8) until the liquid is
completely absorbed into the medium.

7.2.3 Incubate the prepared plates (aerobically, lids uppermost, in an upright position in the incubator
at 25 °C ± 1 °C for 5 d. If necessary, leave the agar plates to stand in diffuse daylight for 1 d to 2 d.
It is recommended to incubate the dishes in an open plastic bag in order not to contaminate the incubator in
the event of dissemination of the moulds out of the dishes.
7.3 Counting and selection of colonies for confirmation Read the plates between 2 d and 5 d of incubation.
Select the dishes containing less than 150 colonies/propagules/germs and count these
colonies/propagules/germs. If fast-growing moulds are a problem, count colonies/propagules/germs after 2
d and again after 5 d of incubation.

NOTE 1 Enumeration methods for yeasts and especially moulds are imprecise because they consist of a
mixture of mycelium and asexual and sexual spores. Numbers of colony-forming units depend on the
degree of fragmentation of mycelium and the proportion of spores able to grow on the plating medium.
NOTE 2 Non-linearity of counts from dilution plating often occurs, i.e. 10-fold dilutions of samples often
do not result in10-fold reductions in numbers of colonies recovered on plating media. This has been
attributed to fragmentation of mycelia
and breaking of spore clumps during dilution in addition to competitive inhibition when large numbers of
colonies are present on plates.

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Animal Health Research Institute
Alex food inspection lab

8 Expression of results

Record the colonies of yeasts and the colonies/propagules of moulds separately, if necessary

9. Retention And Disposal.

1 - If the materials contain +ve cultures or contaminated tools (pipettes) and all materials used for
confirmatory tests must be sterilized by autoclaving at 121 ° 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 °C for one hour.

b) Plastic materials, removed in special container.

10. Method Validation.

The test procedure is based on ISO 21527-(1) and(2) 2008. and validation by using reference material to
prove that the test method consistently yields what it is expected using different matrices

11. Test Report.

The final laboratory report has a form 20-1

12. Assuring the quality of test results

The laboratory applied the quality control program to insure the quality

12.1 Internal quality control :

 Spiked samples
 Repeatability
 Duplicate test

12.2 External quality control :

 Proficiency test

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Animal Health Research Institute
Alex food inspection lab

13. Appendixes : examples of water activity according to matrices

14. Distribution List :

 Technical Manger specialty

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