SUDANESE AGROFOOD EXPORT LABORATORIES

(SAFE)

STANDARD OPERATING PROCEDURE FOR

ENUMERATION COAGULASE – POSITIVE STAPHYLOCOCCI IN
FOOD AND ANIMAL FEEDING SAMPLES

(STAPHYLOCOCCUS AUREUS).

SOP.Q. 003

Prepared by Reviewed by Approved by

QA Officer Technical Directorate Laboratories Manager
 SUDANESE AGROFOOD EXPORTS LABORATORIES SOP.No.SAQ.003
(SAFE) Issue No: 1
STANDARD OPERATING PROCEDURE FOR Issue Date: 22.11.2018
ENUMERATION COAGULASE – POSITIVE Page 1/5
STAPHYLOCOCCI (STAPHYLOCOCCUS AUREUS).

Prepared By: Q.A officer Reviewed By: Technical Directorate Approved By: laboratories Manager

1.0 PURPOSE
This procedure is provided to enumeration of Coagulase-positive staphylococci in products
intended for human consumption or feeding of animals.

2.0 PRINCIPLE
2.1 Inoculation of the surface of a solid selective culture medium, using duplicate plates, with a specified
quantity of the test sample if the product is liquid, or with a specified quantity of the initial suspension
in the case of other products.

2.2 Aerobic incubation of the plates at 35 °C or 37 °C) and examination after both 24 h and 48 h

2.3 Calculation of the number of Coagulase-positive staphylococci per milliliter, or per gram, of sample
from the number of typical and/or atypical colonies obtained on plates at dilution levels chosen so as to
give a significant result, and confirmed by a positive Coagulase test result.

3. DEFINITION
Staphylococci: bacteria form colony are black or grey, shining and convex (1 mm to 1.5 mm
in diameter after incubation for 24 h, and 1,5 mm to 2,5 mm in diameter after incubation for
48 h) and are surrounded by a clear zone which may be partially opaque.

4. SCOPE

This method is applicable to products intended for human consumption and for the feeding
of animals.

5. SAFETY PRECAUTIONS
- Sterilize all used media and glassware by autoclaving and disinfect the laboratory table (cabinet) with
ethanol.

- Sterilize all used equipment and media post testing suspect to sample and prior to disposal or reuse.
 SUDANESE AGROFOOD EXPORT LABORATORIES SOP.No.SAQ.003
(SAFE) Issue No: 1
STANDARD OPERATING PROCEDURE FOR Issue Date: 22.11.2018
ENUMERATION COAGULASE – POSITIVE Page 2/5
STAPHYLOCOCCI (STAPHYLOCOCCUS AUREUS).

Prepared By: Q.A officer Reviewed By: Technical Directorate Approved By: laboratories Manager

6. RESPONSIBILITY
- Microbiologist.
7. PROCEDURE
7.1 Weigh 10 g of the test sample directly into the bottle with the diluents.

7.2 To dissolve the test sample, swirl slowly to wet the powder then shake the bottle 25 times, with a
movement of for about 7 seconds.

7.3 Prepare further dilutions. Transfer with a sterile pipette 10 ml of the initial suspension to a bottle
containing 90 ml of sterile diluents (primary dilution). In a routine procedure, transfer 1 ml of primary
dilution to 99 ml of sterile diluents, until reach a 10 –3 dilutions.

7.4 Transfer, by means of a sterile pipette 0.1 ml of the test sample if liquid, or 0,1 ml of the initial
suspension (10–1 dilution) in the case of other products, to each of two agar plates. Repeat the procedure
for the 10–2 dilution and for further decimal dilutions if necessary.

7.5 If, for certain products, it is desirable to count low numbers of coagulase-positive staphylococci, the
limits of detection can be raised by a factor of 10 by inoculating 1,0 ml of the test sample if liquid, or
1,0 ml of the initial suspension for other products, either on the surface of one large agar plate (140
mm) or on the surface of three small agar plates (90 mm). In both cases, prepare duplicates by using
two large plates or six small ones.

7.6 Carefully spread the inoculums as quickly as possible over the surface of the agar plate, trying not
to touch the sides of the dish, using the spreader. Allow the plates to dry with their lids on for about 15
min at laboratory temperature.

7.7 Incubation
Invert the plates prepared and incubate them for 24 h ± 2 h then re-incubate for a further 24 h ± 2 h in
the incubator at 35 °C or 37 °C).

7.8 Selection of plates and interpretation

7.8.1 After incubation for 24 h ± 2 h, mark on the bottom of the plates the positions of any typical
colonies present. Re-incubate all plates at 35 °C or 37 °C) for a further 24 h ± 2 h, and mark any new
typical colonies. Also mark any atypical colonies present. Take for enumeration only those plates that
contain at the maximum 300 colonies with 150 typical and/or atypical colonies at two successive
dilutions. One of the plates shall contain at least 15 colonies. Select for confirmation a given number
 SUDANESE AGROFOOD EXPORT LABORATORIES SOP.No.SAQ.003
(SAFE) Issue No: 1
STANDARD OPERATING PROCEDURE FOR Issue Date: 22.11.2018
ENUMERATION COAGULASE – POSITIVE Page 3/5
STAPHYLOCOCCI (STAPHYLOCOCCUS AUREUS).

Prepared By: Q.A officer Reviewed By: Technical Directorate Approved By: laboratories Manager

in general 5 typical colonies if there are only typical colonies, or 5 atypical colonies if there are only
atypical colonies, or 5 typical colonies and 5 atypical colonies if both types are present, from each
plate). If there are less than 15 typical and/or atypical colonies present on plates inoculated with
undiluted liquid product or the lowest dilution of other products, it is possible to make an estimated
count as described.

NOTE 1 Typical colonies are black or grey, shining and convex (1 mm to 1,5 mm in diameter after
incubation for 24 h, and 1,5 mm to 2,5 mm in diameter after incubation for 48 h) and are surrounded
by a clear zone which may be partially opaque. After incubation for at least 24 h, an opalescent ring
immediately in contact with the colonies may appear in this clear zone.

NOTE 2 Atypical colonies have the same size as typical colonies and may present one of the following
morphologies:

— shining black colonies with or without a narrow white edge; the clear zone is absent or barely visible
and the opalescent ring is absent or hardly visible;

— grey colonies free of clear zone. Atypical colonies are formed mainly by strains of coagulase-positive
staphylococci contaminating, for example, dairy products, shrimps and giblets. They are less often
formed by strains of coagulase-positive staphylococci contaminating other products.

NOTE 3 Other colonies are all the remaining colonies possibly present on the plates that do not show
the typical or atypical appearance described in Notes 1 and 2, and are considered as the background
flora."

7.8.2 To make an estimated count of lower numbers of coagulase-positive staphylococci, retain all
platesthat contain any typical and atypical colonies. Select all such colonies for confirmation within the
limits set out above.

7.9 Confirmation (coagulase test)

From the surface of each selected colony, remove an inoculum with a sterile wire and transfer it to a
tube or bottle of brain-heart infusion broth. Incubate at 35 °C or 37 °C5) for 24 h ± 2 h. Aseptically add
0,1 ml of each culture to 0,3 ml of the rabbit plasma (unless other amounts are specified by the
manufacturer) in sterile haemolysis tubes or bottles, and incubate at 35 °C or 37 °C). By tilting the tube,
examine for clotting of the plasma after 4 h to 6 h of incubation and, if the test is negative, re-examine
at 24 h of incubation, or examine at the incubation times specified by the manufacturer. Consider the
coagulase test to be positive if the volume of clot occupies more than half of the original volume of the
liquid.
 SUDANESE AGROFOOD EXPORT LABORATORIES SOP.No.SAQ.003
(SAFE) Issue No: 1
STANDARD OPERATING PROCEDURE FOR Issue Date: 22.11.2018
ENUMERATION COAGULASE – POSITIVE Page 4/5
STAPHYLOCOCCI (STAPHYLOCOCCUS AUREUS).

Prepared By: Q.A officer Reviewed By: Technical Directorate Approved By: laboratories Manager

As a negative control, for each batch of plasma, add 0,1 ml of sterile brain-heart infusion broth to the
recommended quantity of rabbit plasma and incubate without inoculation. For the test to be valid, the
control plasma shall show no signs of clotting.

7.10 labeling the bottom of dishes by write the dilution factor, date of culture, sample code and product
name.

8. CALCULATION

8.1 Expression of results

For those dishes containing at the maximum 300 colonies, with 150 typical and/or atypical colonies at
two consecutive dilutions, calculate the number of coagulase-positive staphylococci for each dish and
calculate, as a weighted mean from the two successive dilutions, the number N of identified coagulase-
positive staphylococci present in the test sample, using the following equation:

where

is the sum of the Coagulase-positive staphylococcal colonies identified on all the dishes
selected;

V is the volume of inoculums on each dish, in millilitres;

n1 is the number of dishes selected at the first dilution;

n2 is the number of dishes selected at the second dilution;

d is the dilution rate corresponding to the first dilution selected (the initial suspension is a dilution).

8.2 If the two dishes, corresponding to the test sample (liquid products) or the initial suspension (other
products) do not contain any colonies of Coagulase-positive staphylococci and if the inoculation has
been performed with 0,1 ml of sample, report the result as follows (general case of a 0,1 ml inoculums)
 SUDANESE AGROFOOD EXPORT LABORATORIES SOP.No.SAQ.003
(SAFE) Issue No: 1
STANDARD OPERATING PROCEDURE FOR Issue Date: 22.11.2018
ENUMERATION COAGULASE – POSITIVE Page 5/5
STAPHYLOCOCCI (STAPHYLOCOCCUS AUREUS).

Prepared By: Q.A officer Reviewed By: Technical Directorate Approved By: laboratories Manager

— less than 10 Coagulase-positive staphylococci per millilitre (liquid products);

— less than 10/d Coagulase-positive staphylococci per gram (other products), where d is the dilution
rate of the initial suspension.

- If the inoculation has been performed with 1 ml of sample, report the result as follows:

— less than 1 Coagulase-positive staphylococcus per millilitre (liquid products);

— less than 1/d Coagulase-positive staphylococcus per gram (other products).

9. METHOD FOR DATA VALIDATION

We are using reference culture strains.

10. REPORTING OF THE RESULTS

Report the result as the number of coagulase-positive staphylococci per millilitre (liquid products) or
per gram (other products), expressed as a number between 1,0 and 9,9 inclusive multiplied by 10x where
x is the appropriate power of 10x.

11. REFERENCES

- BS EN ISO 6888-1:1999 -– Microbiology of food and animal feeding stuffs = Horizontal method
for
the enumeration of coagulase-positive staphylococci (Staphylococcus aureus) = Part 1: Technique using
Baird-Parker agar medium.
- ISO 7218 - 2007 - Third edition Microbiology of food and animal feeding stuffs — General
requirements and guidance for microbiological examinations.
- ISO 8261- 2001 –Second edition - IDF 122 Milk and milk products — General guidance for the
preparation of test samples, initial suspensions and decimal dilutions for microbiological examination.