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F. A.S.T.
TEST METHODS TM-
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MANUAL
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LABORATORIES Milk and Dairy Products 0
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Milk Powders 1 of 7

I. Reference: Standard Methods for the Examination of Dairy Products

16th ed. 5.4B1,2(6), b, c(1-4) – Pathogens in Milk and Milk
Products: Salmonella
5.4F1a-c – Pathogens in Milk and Milk Products: Staphylococcus aureus
6.2 – Microbiological Count Methods: Standard Plate
Count (Class O)
7.8 – Coliform and Other Indicator Bacteria: Coliform
Tests with a Solid Medium

II. Responsibility: Microbiologist

III. Scope : This is applicable to instant nonfat dry milk, noninstant nonfat dry milk, dry whole milk
and other milk powders for the determination of standard plate count, coliform count,
Staphylococcus aureus, and detection of Salmonella.

IV. Principle: Known dilutions of sample are allowed to grow on specific media
at suitable temperature and period of time.

V. Apparatus: 1. Autoclave
4. Colony counter
5. Incubator
6. Waterbath

VI. Reagents : 1. Bactident Coagulase (Merck)

2. Baird-Parker Agar
3. Bismuth Sulfite Agar (BSA)
4. Brain Heart Infusion Broth
5. Brilliant Green Dye solution
6. Brilliant Green Water
7. Hektoen Enteric Agar (HEA)
8. Iodine-Potassium Iodide solution
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 Document No:
F. A.S.T.
TEST METHODS TM-
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MANUAL
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9. Lactose Broth
10. Lysine Iron Agar (LIA)
11. Phosphate Buffered Dilution Water
12. Potassium Tellurite Enrichment
13. Standard Methods Agar (SMA)
14. Sterile Distilled Water
15. Tetrathionate Broth
16. Triple Sugar Iron Agar (TSIA)
17. Violet Red Bile Agar (VRBA)
18. Xylose Lysine Desoxycholate Agar (XLDA)

VII. Methodology:

1. PREPARATION OF SAMPLE

1.1 Mix sample thoroughly.

1.22 Aseptically weigh 11.0 grams of sample into 99 ml phosphate dilution
water.
1.3 Shake dilution 25 times in 30 cm arc within 7 seconds. This makes a 1:10
dilution.
1.4 Prepare decimal dilutions of 10-2, 10-3, 10-4, or higher as appropriate.

2.1 STANDARD PLATE COUNT

2.1 Pipet 1 ml of each dilution into separate, duplicate, appropriately marked

petri dishes.
2.2 Pour the inoculated plates with 12 – 15 ml of tempered Standard Methods
Agar and mix thoroughly all samples or dilutions by making about 25
complete up-and-down or back-and-forth movements.
2.31 Allow agar to solidify.
2.4 Invert and incubate plates at 32  1C for 48  3 hours.
2.5 Count plates with 25 – 250 colonies and multiply by corresponding

dilution.
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2.6 Record the computed count and report as colony-forming units ( CFU ) per
gram or per ml sample.

3. COLIFORM COUNT

3.1 Pipet 1 ml of each dilution into separate, duplicate, appropriately marked

petri dishes.
3.2 Pour the inoculated plates with 12 – 15 ml Violet Red Bile Agar and allow
to solidify.
3.3 Overlay an additional 3 – 5 ml of VRBA to inhibit surface growth and
spreading of colonies.
3.4 Allow to set. Invert and incubate at 32º  1C for 18– 24 hours.
3.5 Count purple-red colonies that are 0.5 mm larger in diameter and
surrounded by zone of precipitated bile acids.
3.6 Multiply the colonies counted by the corresponding dilution.
3.7 Record the computed count and report as colony-forming units ( CFU ) per
gram or per ml sample.

4. Salmonella

4.1 Pre-enrichment

4.1.1 Instant/Non-instant Nonfat Dry Milk

4.1.1.1 Aseptically weigh 25 grams into a sterile beaker (250
ml) or other appropriate container.
4.1.1.2 Using a sterile glass or paper funnel (made with tape to
withstand autoclaving), pour the weighed sample gently
and slowly over the surface of 225 ml brilliant green
water contained in a sterile 500 ml Erlenmeyer flask or
other appropriate container. (Prepare brilliant green
water by adding 2 ml of 1% brilliant green dye solution
per 1,000 ml of sterile distilled water.)
4.1.1.3 Allow container to stand undisturbed for 60  5 minutes.

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4.1.1.4 Incubate loosely capped container, without mixing or pH

adjustment, for 24  2 hours at 35ºC.

4.1.2 Dry Whole Milk

4.1.2.1 Aseptically weigh 25 grams of analytical unit to be
added to 225 ml sterile distilled water.
4.1.2.2 Swirl thoroughly by hand and let stand for 60 minutes at
room temperature for 60 minutes, with jar securely
capped.
4.1.2.3 Mix well by swirling and determine pH. Adjust pH if
necessary to 6.8  0.2 with sterile 1N sodium hydroxide
or 1N hydrochloric acid.
4.1.2.4 Add 0.45 ml of a 1% aqueous brilliant green dye
solution and mix well.
4.1.2.5 Loosen jar caps ¼ turn and incubate sample
mixtures 24  2 hrs. at 35C.

4.2 Selective Enrichment

4.2.1 Tighten lid and gently shake incubated sample.
4.2.2 Transfer 1 ml of mixture to 10 ml of tetrathionate broth.
4.2.3 Incubate for 24 ± 2 hours at 35ºC.
4.2.4 Shake tube vigorously and streak a 3-mm loopful of incubated
tetrathionate broth onto BSA, HEA, and XLDA. Prepare BSA
plates the day before streaking, and store them in the dark at room
temperature until streaked.
4.2.5 Incubate plates for 24 ± 2 hours at 35ºC.

4.3 Isolation

4.3.1 Examine the plates for the presence of colonies suspected of being
Salmonella.

4.3.1.1 BSA – Typically brown, gray, or black colonies,

sometimes with a metallic sheen. Surrounding medium
is usually brown at first, but it may turn black in time
with increased incubation, producing the so-called halo

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effect. Some strains may produce green colonies with

little or no darkening of surrounding medium.

4.3.1.2 XLDA – Pink colonies with or without black centers.

Many cultures of Salmonella may have large, glossy
black centers or may appear as almost completely black
colonies. Atypically, a few Salmonella species produce
yellow colonies with or without black centers.

4.3.1.3 HEA – Blue-green to blue colonies with or without black

centers. Many cultures of Salmonella may produce
colonies with large, glossy black centers or may appear
as almost completely black colonies. Atypically, a few
Salmonella species produce yellow colonies with or
without black centers.

5 Staphylococcus aureus

5.1 Preparation of complete medium

5.1.2 Aseptically add 5 ml pre-warmed ( 45 - 50C ) Bacto EY tellurite
enrichment to 95 ml melted base ( Baird-Parker agar ) cooled to
45 – 50C .
5.1.3 Mix well avoiding bubbles, and pour 15 – 18 ml into sterile petri
dishes. The medium must be densely opaque.
5.1.4 Dry plates before use.

5.2 Preparation of sample

5.2.2 Aseptically weigh 11g sample into sterile blender jar.
5.2.3 Add 450 ml phosphate buffered dilution water and homogenize 2
min. at high speed ( 16,000 – 18,000 rpm ).

5.3 Isolation and enumeration

5.3.2 Heat 99 ml of sterile, freshly prepared aqueous 2% sodium citrate
to 40º to 45ºC
5.3.3 For each dilution to be plated, aseptically transfer 1 ml sample

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suspension to 3 plates of Baird-Parker agar, distributing 1 ml of

inoculum equitably to 3 plates ( e.g., 0.4 ml, 0.3 ml, and 0.3 ml ).
5.3.4 Spread inoculum over surface of agar plate, using sterile bent glass
streaking rod.
5.3.5 Retain plates in upright position until inoculum is absorbed by agar
(about 10 min on properly dried plates ).
5.3.6 Invert plates and incubate 45 – 48 hours at 35C.
5.3.7 Select plates containing 20 – 200 colonies, unless only plates at
lower dilutions ( > 200 colonies ) have colonies with typical
appearance of S. aureus.
Colonies of S. aureus are circular, smooth, convex, moist, 2 – 3
mm in diameter on uncrowded plates, gray to jet-black, frequently
with light-colored (off-white) margin, surrounded by opaque zone
and frequently with an outer clear zone; colonies have buttery to
gummy consistency when touched with inoculating needle.
Occasionally from various foods and dairy products, nonlipolytic
strains of similar appearance may be encountered except that
surrounding opaque and clear zones are absent. Strains isolated
from frozen or dessicated foods that have been stored for extended
periods frequently develop less black coloration than typical
colonies and may have rough appearance and dry texture.

5.3.8 Count and record colonies. If several types of colonies are

observed which appear to be S. aureus on selected plates, count
number of colonies of each type and record counts separately.
When plates of the lowest dilution contain < 20 colonies, these
may be used. If plates containing > 200 colonies have colonies
with the typical appearance of S. aureus and typical colonies do
not appear at higher dilutions, use these plates for the enumeration
of S. aureus, but do not count nontypical colonies. Select > 1
colony of each type counted and test for coagulase production. Add
number of colonies on triplicate plates represented by colonies
giving positive coagulase test and multiply by the sample dilution
factor. Report this number as number of S. aureus / g of product
tested.

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5.4 Coagulase Test

5.4.2 Transfer suspect S. aureus colonies into small tubes containing 5
ml BHI broth and incubate for 20 to 24 hours at 37C.
5.4.3 Dissolve the frozen-dried EDTA rabbit plasma in 3 ml of distilled
or demineralized water.
5.4.4 Add 0.3 ml of this solution to a sterile culture tube.
5.4.5 Mix with 0.1 ml of the broth culture.
5.4.6 Incubate at 37C.
5.4.7 Every hour the tube contents are examined for coagulation by
gently slanting the tube (do not shake).
5.4.8 The coagulase test is judged to be positive if more than three
quarters of the tube contents have formed together as lumps.
5.4.9 If the results are negative after 4 to 6 hours, incubate for further 24
hours before making the final judgement.

VIII. Sterility Controls :

Check sterility of agar, dilution water, pipets and air in plating area by pouring control
plates with specific media.

Test known positive and negative cultures simultaneously on specific media.

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