Professional Documents
Culture Documents
OTTAWA
Don_Warburton@hc-sc.gc.ca
Rick A. Szabo
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204A1
Ottawa, Ontario K1A 0L2
Rick_Szabo@hc-sc.gc.ca
1. APPLICATION
This method is applicable to the enumeration of Staphylococcus aureus in foods to determine compliance with
the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an Official Method for a food is
specified, that method shall be followed. This revised method replaces MFHPB-21, dated November 2000,
and Supplements to Method MFHPB-21, dated February 2002, August 2002, and December 2003.
2. DESCRIPTION
The method has been shown to produce satisfactory results with naturally-contaminated meats, fish, poultry,
vegetables, cereals and dairy products, and artificially-contaminated foods in AOAC and HPB studies (8.1-
8.10). This method can be used successfully for the detection of Staphylococcus aureus in other foods, food
ingredients and environmental samples.
3. PRINCIPLE
Certain staphylococci produce enterotoxins which cause food poisoning. This ability to produce enterotoxins,
with few exceptions, is limited to those strains that are coagulase-positive, and/or produce a heat-stable
nuclease (TNase). This method determines the presence of S. aureus by plating known quantities of (dilutions
of) a food sample onto a selective agar. After incubation, presumptive staphylococcal colonies are selected,
and subjected to confirmatory tests. From the results of these tests, the number of S. aureus per g or mL of
the food is calculated. The numbers present may indicate a potential for the presence of enterotoxin, or they
may also indicate a lack of adherence to Good Hygienic Practices.
4. DEFINITION OF TERMS
5. COLLECTION OF SAMPLES
NOTE: The Laboratory Supervisor must ensure that the analysis described in this method is carried out
in accordance with the International Standard referred to as “ISO/IEC 17025:2005 (or latest
version): General Requirements for the Competence of Testing and Calibration Laboratories”.
The media listed below are commercially available and are to be prepared and sterilized according to the
manufacturer's instructions. See also Appendix G of Volume 1 for the formulae of individual media.
NOTE: If the analyst uses any variations of the media listed here (either product that is commercially
available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor
to ensure equivalency.
6.7 A non-selective agar; either Blood agar (BA), Nutrient agar (NA), or Trypticase Soy agar (TSA)
NOTE : Use Staphylococcus broth when investigating food poisoning outbreaks and/or when sample size
is very small. Stomach or macerate sample in 1:10 volume of broth and incubate at 35°C 18-24
h. Streak onto selective agars and proceed as in Section 7.4.
NOTE : It is the responsibility of each laboratory to ensure that the temperature of the incubators or
waterbaths are maintained at the recommended temperatures. Where 35/C is recommended in
text of the method the incubator may be at 35 +/-1.0/ C. Similarly, lower temperatures of 30 or 25
may be +/- 1.0/C. However, where higher temperatures are recommended, such as 43 or 45.5/C,
it is imperative that the incubators or waterbaths be maintained within 0.5/C due to potential
lethality of the higher temperatures on the microorganism(s) being isolated.
6.19.5 Anaerobic utilization of glucose (Phenol red carbohydrate broth with 0.5% glucose, sterile
paraffin oil)
6.19.6 Anaerobic utilization of mannitol (Phenol red carbohydrate broth with 0.5% mannitol,
sterile paraffin oil)
7. PROCEDURE:
Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the
following instructions:
7.1.1 During storage and transport, the following shall apply: with the exception of shelf-stable products,
keep the sample units refrigerated. Sample units of frozen products shall be kept frozen. Thaw frozen
samples in a refrigerator, or under time and temperature conditions which prevent microbial growth
or death.
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7.1.2 Analyze the sample units as soon as possible after receipt at the laboratory.
7.2.2 Clean the surface of the working area with a suitable disinfectant.
7.3.1 Combine portions from several locations within each solid sample unit, to ensure a representative
analytical unit,
or
7.3.2 If the sample unit is a liquid or a free-flowing solid (powder), thoroughly mix each sample unit by
shaking the container.
7.3.3 Prepare a 1:10 dilution of the food by adding aseptically 11(10) g or mL (the analytical unit) to 99(90)
mL of the appropriated diluent (see Table I). Shake, stomach or blend according to the type of food
.
NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions.
7.3.4 Blend/stomach for the minimum time required to produce a homogeneous suspension; to avoid
overheating, blending time should not exceed 2.5 min. With foods that tend to foam, use blender at
low speed and remove aliquot from below liquid/foam interface.
7.3.5 If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc
in approximately 7 sec.
7.3.6 The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. In all
other instances, the analysis should be continued as soon as possible.
7.3.7 Prepare succeeding decimal dilutions in peptone water as required, using a separate sterile pipette
for making each transfer.
7.3.8 Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the
microorganisms present.
7.4.1 Plating
7.4.1.1 Agitate each dilution to resuspend material that may have settled during preparation.
Plating should be carried out within 15 min of preparing the dilutions.
7.4.1.3 The liquid should not be spread right to the edge of the plate, since this causes
confluent growth at the plate-agar interface which is difficult to count.
7.4.1.4 Retain the plates in an upright position until the inoculum has been absorbed by the
medium (approximately 10 minutes on properly dried plates). If the inoculum is not
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readily absorbed by the medium, the plates may be placed in an upright position in
an incubator for up to one hour.
NOTE: It is recommended (but optional) that analysis now include two selective and differential agars (i.e.
Baird-Parker (BP) and one other agar), as per standard methods for other pathogens in this
Compendium. If using only one plating agar, equivalent media can be used in place of BP ONLY
if validation data has been produced that shows equivalency.
7.4.2 Incubation
7.4.2.1.4 MSA - Plates are incubated for 18-24 (to 48 h). Plates should be observed
at 24 to 30 h for possible overgrowth; presumptive colonies may be counted
at this time but the count should be verified at 48 ± 4 h.
7.4.2.1.5 Rapid Staph - Plates are incubated 24-48 h. Plates should be observed at
24 to 30 h for possible overgrowth; presumptive colonies may be counted
at this time but the count should be verified at 48 ± 4 h.
7.4.2.2 Avoid excessive crowding or stacking of plates in order to permit rapid equilibration
of plates with incubator temperature.
Type 1. Convex, entire, shiny black surrounded by clear zones extending into the
opaque medium.
Type 2. Convex, entire, shiny black without well defined clear zones.
Type 3. Dark grey colonies similar to type 1.
Type 4. Dark grey colonies similar to type 2.
For the four types of colonies listed in this section, if obvious morphological
differences or size differences exist within one type, count these types separately
and confirm separately.
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For types 3 and 4 (grey colonies with and without clear zones), only dark grey
colonies are counted. Light grey colonies should not be considered as presumptive
S. aureus colonies.
For all types of presumptive S. aureus colonies, do not count pin point colonies.
Each colony type may show grey-white margins around the colonies and/or opaque
zones (double halos).Black mucoid colonies larger than 2 mm in diameter and
swarmers should not be counted. Such colonies usually belong to the genus
Bacillus.
7.4.3.1.1 Count the colonies of each type and record separately, but add together
to give the total presumptive count.
7.4.3.4 BP-RPF agar - Similar to BP but with a whitish halo around the colonies as S.
aureus converts the plasma fibrinogen to fibrin as a result of coagulase activity.
7.4.3.5 Rapid Staph - Similar to BP but with convex entire, shiny black colonies surrounded
by clear zones extending into opague medium.
7.4.4 Counting of five plates of the 1:10 dilution (solid food only)
7.4.4.1 If the number of all presumptive staphylococcal colonies per plate is fewer than 20,
add separately the counts for each type from all five plates and record as the
respective presumptive count. This is the count of one of the four types per 2 mL (0.2
g of food). Multiply each count by 5, and record as the respective presumptive count
per g of food (C). Add the results, and report as the total presumptive count per g of
food.
7.4.4.2 If the number of all presumptive staphylococcal colonies per plate is between 20 and
200, select two plates at random, count separately the colonies of each type and
compute the respective average presumptive count per plate (per 0.4 mL; which is
equivalent to 0.04 g of food) (A/2). Multiply each count by 25 and record as the
respective presumptive count per g of food (C). Add the results and report as the
total presumptive count per g of food.
7.4.4.3 If the number of presumptive staphylococcal colonies on some of the five plates is
< 20, but on others is $ 20, proceed as in 7.4.4.1. above.
7.4.5.1 Select plates containing 20-200 presumptive staphylococcal colonies per plate
consisting of the combined counts of all types.
7.4.5.2 Compute the average presumptive count per plate for each type (A/2), multiply by
five and by the appropriate dilution factor, and record as presumptive count per g or
mL of food for each type (C). Add the results and report as the total presumptive
count per g or mL of food.
7.4.5.3 If plates from more than one dilution are used, the counts are to be averaged as
shown below.
7.4.5.4 If no plate containing 20-200 presumptive S. aureus is available, estimated counts
may be made on plates giving presumptive counts outside this range. Report results
as estimated counts when results are outside the range of 20-200.
7.4.5.5 When an estimated count contributes to an average count, this average itself
becomes an estimated value.
For confirmation of S. aureus, perform the coagulase test (following manufacturer’s instructions) as an initial
step. Run controls (positive and negative cultures as well as media controls) simultaneously when performing
all confirmation tests.
7.5.1.1 From the replicate plates counted, a number of each colony type observed is
selected as follows to check for culture purity:
When the total count per type for all the plates of a dilution is less than five, pick all
colonies of that type.
When the total count per type for all plates of a dilution is equal to or greater than five
colonies, pick five colonies of that type at random.
7.5.1.2 If the colonies are typical and well isolated proceed to 7.5.1.5. If not well isolated,
MFHPB-21
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streak each colony picked onto a non-selective medium, such as BA, NA or TSA to
obtain discrete colonies.
7.5.1.3 Incubate at 35/C for 24 ± 2 h.
7.5.1.4 If the colonies are typical and well isolated, proceed to 7.5.1.5. Otherwise, make a
smear from the growth of each isolate on the non-selective medium and stain with
a simple stain (e.g., crystal violet). Observe microscopically for the presence of cocci.
If an isolate is not pure, choose another colony at step 7.5.1.2 above and repeat
colony isolation steps above.
7.5.1.5 Transfer inoculum from each colony into a separate tube of BHI broth.
7.5.1.6 Incubate the inoculated BHI broth tubes at 35/C for 18-24 h and observe for growth.
7.5.1.7 Retain BHI broth cultures.
7.5.2.1 Transfer 0.2 mL of each BHI broth culture into sterile 13 x 100 mm tubes containing
0.5 mL certified coagulase plasma. Mix thoroughly.
7.5.2.2 Incubate tubes at 35/C and examine after 1 h and after 4 h. Do not shake tubes
during incubation. Negative tubes should be incubated overnight at room
temperature and rechecked.
7.5.2.3 A firm clot which does not move when the tube is tipped on its side (4+ coagulase
reaction) is considered a positive test for S. aureus; no further confirmation is
required. Run controls (positive and negative cultures as well as media controls)
simultaneously when performing all confirmation tests.
If the coagulase reaction is 3+, 2+ or 1+, perform at least two of confirmation tests
listed in 7.5.3. If two of these tests are positive, then the isolate is considered
S.aureus.
If no clot is observed, it is considered as a negative test for S. aureus; no further
confirmation is required.
Refer to figure 2 for visual explanation.
At least two of the following confirmatory tests may be done keeping in mind that Anaerobic utilization
of glucose and Lysostaphin sensitivity must not be the only two tests performed.
8. REFERENCES
8.1 American Public Health Association (APHA). 1992. Compendium of Methods for the
Microbiological Examination of Foods. APHA. Washington, DC. Chapt. 33. pp:533-550.
8.2 Association of Official Analytical Chemists (AOAC). 1999. Official Methods of Analysis of AOAC
International. 16th. AOAC. Gaithersburg, Md. Vol. I, Chapt. 17. pp:32-34.
8.3 Baird-Parker, A.C. 1962. An improved diagnostic and selective medium for isolating coagulase
positive staphylococci. J. Appl. Bacteriol. 25:12-19.
8.4 Baird-Parker, A.C. and E. Davenport. 1965. The effect of recovery medium on the isolation of
Staphylococcus aureus after heat treatment and after storage of frozen or dried cells. J. Appl.
Bacteriol. 28:390-402.
MFHPB-21
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8.5 Collins-Thompson, D.L., A. Hurst and B. Aris. 1974. Comparison of selective media for the
enumeration of sublethally heated food-poisoning strains of Staphylococcus aureus. Can. J.
Microbiol. 20:1072-1075.
8.6 Crisley, F.D., J.T. Peeler and R. Angelotti. 1965. Comparative evaluation of five selective and
differential media for the detection and enumeration of coagulase-positive staphylococci in foods.
Appl. Microbiol. 13:140-156.
8.7 Koskitalo, L.D. and M.E. Milling. 1969. Lack of correlation between egg yolk reaction in
staphylococci medium 110 supplemented with egg yolk and coagulase activity of staphylococci
isolated from cheddar cheese. Can. J. Microbiol. 15:132-133.
8.8 Rayman, M.K., C.E. Park, J. Philpott and E.C.D. Todd. 1975. Reassessment of the coagulase and
thermostable nuclease tests as means of identifying Staphylococcus aureus. Appl. Microbiol.
29:451-454.
8.9 Selepak, S.T., and F.G. Witelsky. 1985. Inoculum size and lot-to-lot variation as significant
variables in tube coagulase test for Staphylococcus aureus. J. Clin. Microbiol. 22(5): 835-837.
8.10 Sperber, W.H. and S.R. Tatini. 1975. Interpretation of the tube coagulase test for identification of
Staphylococcus aureus. Appl. Microbiol. 29:502-505.
MFHPB-21
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TABLE I
Liquids:
milk, water etc. pipette directly into Petri dishes and/or into peptone water diluent
Solids:
TABLE II
Total No. of Colonies of No. of No. of Total No. of Colonies of No. of S. aureus from
one of the four Types Isolates Isolates one of the four Types per one of the four Types
on Duplicate Plates Tested Confirmed as g or mL. per g or mL
"A" "G" S. aureus "C" "N"
"P" C = 1/2AxD*5** N = (P/G)xC
Calculate N1, N2, N3 and N4 for each colony type to obtain total number of S. aureus. (NT) per g or mL NT =
N1 + N2 + N3 + N4
** For duplicate plates, 0.2 mL per plate. Divide by 2 since "A" represents the total count of one of
the four types on two duplicate plates. Likewise, when 5 plates of the 1:10 dilution are counted,
divide by 5.
Report total number of Staphylococcus aureus per g or mL of food to two significant figures
MFHPB-21
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FIGURE 1
Coagulase test
1. Accuprobe (MFLP-78)
2. Enterotoxin assay (MFLP-47, 67, 68, 69)
3. Latex agglutination
4. Rapid ID kits
5. Anaerobic utilization of glucose
6. Anaerobic utilization of mannitol
7. Lysostaphin sensitivity
8. Thermonuclease test
FIGURE 2
Tube number
Intensity of reaction
(degree of clotting)