Government of Canada Gouvernement du Canada

Official Method MFO-12

November 30, 1981

HEALTH PROTECTION BRANCH

OTTAWA

MICROBIOLOGICAL EXAMINATION OF MILK POWDER

1. APPLICATION

This method shall be used for the determination of the bacteria of the genus Salmonella in milk powder, in
accordance with Section B.08.014A. of the Food and Drug Regulations.

2. SAMPLING

2.1 Definition of Terms

2.1.1 Lot: A batch or production unit which may be identified by the same code. When there
is no code identification, a lot may be considered as (a) that quantity of milk powder
produced under essentially the same conditions, at the same establishment and
representing no more than one day's production; or, (b) the quantity of milk powder from
one and the same manufacturer available for sampling at a fixed location.

2.1.2 Sample: The sample units (subsamples) taken per lot for analysis.

2.1.3 Sample Unit: Usually a consumer size container of the product, and should consist of
a minimum of 100 g. A sample unit is often referred to as a subsample.

2.1.4 Analytical Unit: That amount of product withdrawn from the sample unit for analysis.

2.2 Collection of Samples

2.2.1 A sample, consisting of 20 sample units drawn at random from each lot, shall be taken.

2.2.2 Each sample unit shall consist of at least 100 g.

2.2.3 Collect original unopened containers wherever possible.

2.2.4 More than one sample unit may be collected from large institutional or bulk containers
when the total number of sample units required exceeds the number of containers in the
lot. A sample unit will consist of more than one container when the lot consists of
containers smaller than 100 g (e.g., four 25 g containers in each sample unit)

Published by: POLYSCIENCE PUBLICATIONS, P.O. Box 1606, Station St-Martin, Laval, Quebec,
Canada H7V 3P9. TEL.: 1-800-840-5870. FAX:(450) 688-1930.
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2.2.5 Employ aseptic techniques in collecting the sample units when sampling from bulk.
Place each collected sample unit into a separate sterile container.

3. PROCEDURE

The 20 sample units shall be analyzed individually or as two or more composites for determining the
presence of bacteria of the genus Salmonella.

The test shall be carried out in accordance with the following instructions.

3.1 Handling of Sample units

3.1.1 Analyze the sample units as soon as possible after they have been received in the
laboratory.

3.2 Preparation of Media

The following media, which are to be prepared and sterilized according to the manufacturer's
instructions, shall be used:

(1) Nutrient Broth (NB)

(2) Selenite Cystine (SC) broth
(3) Tetrathionate Brilliant Green (TBG) broth
(4) Bismuth Sulfite (BS) agar
(5) Brilliant Green Sulfa (BGS) agar
(6) MacConkey Agar (MA)
(7) Triple Sugar Iron (TSI) agar
(8) Lysine Iron (LI) agar
(9) Christensen's Urea (CU) agar
(10) Nutrient Agar (NA)

3.3 Non-selective Enrichment (Pre-enrichment)

3.3.1 Withdraw a 25 g analytical unit from each 100 g sample unit. When a sample unit
consists of more than 1 container mix the contents of each container of the sample unit
aseptically prior to obtaining the 25 g analytical unit. The analytical units may be
composited.

3.3.2 Resuspend the individual analytical units or the composite unit(s) in nine times their
weight of distilled water.

3.3.3 Add brilliant green solution to obtain a final concentration of 1:50,000.

3.3.4 Shake the container to ensure uniform distribution of the microorganisms present, in the
suspension.

3.3.5 Check the pH of each suspended analytical or composite unit. If the pH is outside the
range of 6.0 to 7.0, adjust to 7.0. with either sterile NaOH or HCL.

3.3.6 Inoculate NB with a known culture of Salmonella, and subsequently make transfers to
all other media used in the analysis. This is the positive media control. Set up a
negative control by incubating appropriate uninoculated media during each step of the
analysis.
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3.3.7 Incubate the inoculated pre-enrichment broth(s) and the controls at 35oC + 0.5o for
18-24 hr. In no circumstances should the incubation be prolonged for more than 24 hr.

3.4 Selective Enrichment

3.4.1 Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of SC and TBG
broths using a sterile pipette.

3.4.2 Incubate the SC and TBG broths for 24 + 2 hr at 35oC + 0.5o and at 43oC + 0.5o,
respectively.

3.5 Selective Plating

3.5.1 After the incubation period, streak a loopful of each of the selective enrichment broths
onto BS agar* and BGS agar plates to obtain well-isolated colonies. The broths may
also be streaked onto a third commercially available plating medium.

3.5.1.1 It has been observed that BS agar is inhibitory for Salmonella serotypes other
than S.typhi unless it is refrigerated at 4oC for at least 24 hr before streaking.
The possibility of an inhibitory effect of this medium should be taken into
consideration.

3.5.2 Incubate the plates at 35oC + 0.5o for 24 + 2 hr. It may be necessary to incubate the BS
agar plates for 48 + 2 hr.

3.5.3 Examine the plates after the incubation period for colonies indicative of Salmonella.
On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS
agar, they are usually black, with or without a metallic sheen; with increasing time of
incubation the surrounding medium is gradually blackened. It should be noted however
that lactose and/or sucrose-fermenting strains (e.g., S. arizonae) may develop a
coliform-like (greenish) appearance on BGS agar. A heavy growth of coliforms may
mask the appearance of the Salmonella colonies. On BS agar, some Salmonella types
may form dark brown rather than black colonies.

3.5.4 If there are no colonies indicative of Salmonella on the plates, bacteria of the genus
Salmonella are considered absent from the analytical or composite unit from which the
colonies originated.

3.6 Purification

3.6.1 Streak suspect colonies onto MA plates for purification.

3.6.2 Incubate the agar plates at 35oC + 0.5o for 24 + 2 hr.

3.6.3 Observe MA plates after incubation period. Typical Salmonella colonies are
lactose-negative and will appear colourless. However, lactose-positive biotypes are
known to occur and may appear pink.

3.7 Biochemical Screening

3.7.1 Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates
and inoculate the biochemical media listed in Table 1. Incubate these media at 35oC +
0.5o for 18 - 24 hr. Cap the tubes loosely to avoid the occurrence of erroneous results.

3.7.2 Other media may be used to observe the reactions listed in Table 1. If additional
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biochemical information is desired, other reaction-media or commercially available

diagnostic kits may be used.

3.7.3 If none of the isolates from a particular analytical or composite unit shows biochemical
reactions indicative of Salmonella, then the bacteria of the genus Salmonella are
considered to be absent from the analytical or composite unit from which the isolates
originated. If Salmonella are suspected, proceed to serological testing.

3.7.4 Use TSI or LI agar cultures less than 72 hr old for the serological identification but store
the cultures at 2-8oC if the serological identification is not carried out within 12 hr after
the incubation of the TSI or LI agar cultures.

3.7.5 If the serological identification is not performed within 72 hr after the incubation period,
streak suspect cultures onto NA slants.

3.7.6 Incubate the inoculated NA slants at 35oC + 0.5o for 24 + 2 hr.

3.7.7 Use NA slant cultures less than 48 hr old for the serological identification but store the
cultures at 2-8oC if the serological identification is not carried out within 12 hr after the
incubation of the NA slant cultures.

3.8 Serological Identification

3.8.1 Testing with somatic polyvalent antiserum (Group A to I +Vi)

(a) Mark the following sections on an agglutination plate: C+ (positive control),

C-(negative control), and T (test culture). If several cultures are tested at the
same time, mark several test areas T1, T2, T3, etc.

(b) Place one drop of physiological saline on each of the areas marked T and C+,
and two drops on the area marked C-.

(c) Remove sufficient culture material from a biochemical test medium used (the
slant area not the butt) or from an NA slant, and prepare a heavy suspension
in the test areas and in the negative control area.

(d) For the positive control, use a known Salmonella culture and make a
suspension in the area marked C+.

(e) Prepare the somatic polyvalent antiserum as directed by the manufacturer, and
place one drop onto each of the test suspension areas and onto the positive
control area.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive
control), and the saline-culture mixture of the negative control with a sterile
needle or loop. Title the slides back and forth for 1 min.

(g) Hold the slides against a dark background and observe. Cultures usually
agglutinate within 1 min.

(h) False positive reactions may occur; these can be resolved by further testing
with somatic grouping and with flagellar antisera.

(i) The tests are invalidated if the negative control shows agglutination
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(autoagglutination).

3.8.2 Testing with Somatic Grouping Antisera

It is preferable to test the culture against somatic grouping anterisera whenever

possible; however the culture may be sent to a typing centre for identification. The
majority of Salmonella isolated from foods belong to groups B, C, D, and E. It is
important to recognize that unless a complete set of grouping sera is available some
Salmonella may be missed. In such cases any culture processing the biochemical
reactions indicative of Salmonella should be sent to a typing centre for identification.

(a) Mark the following sections on an agglutination plate: C+ (positive control) C-

(negative control), and T (test culture).

If several cultures are tested at the same time, identify several test areas T1,
T2, T3, etc.

(b) Use a positive control culture for each individual group tested, as in 3.8.1.d.

(c) Place one drop of physiological saline on each of the areas marked T and C+
and two drops on the area marked C-.

(d) Remove sufficient culture material from a bio-chemical test medium (from the
slant area not the butt) or from an NA slant and prepare a heavy suspension in
the test area(s) and in negative control area.

(e) Prepare the grouping antisera as directed by the manufacturer, and place one
drop onto each of the test suspension areas and onto the positive control.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive
controls), and the saline-culture mixture of the negative control with a sterile
needle or loop. Tilt slides back and forth for 1 min.

(g) Hold the slides against a dark background and observe for agglutination.
Cultures usually agglutinate within 1 min.

(h) If the culture-saline-serum mixture has not agglutinated, repeat procedure with
another group antiserum.

(i) If the serological test is positive, the culture shall be sent to a Salmonella typing
centre for serotyping.

(j) The tests are invalidated if the negative control shows agglutination
(autoagglutination).

3.8.3 If all of the serological tests performed on an isolate are negative but the original culture
gave biochemical reactions indicative of Salmonella (see Table 1), that culture shall be
sent to a typing centre for verification.

4. INTERPRETATION

The lot of milk powder sampled shall be considered in compliance with Section B.08.014.A. of the Food and
Drug Regulations when bacteria of the genus Salmonella are not found in any of the 20 sample units
analyzed (individually or as composites).
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TABLE 1
Minimal Biochemical Screening Media

Medium Reaction Observations Reaction shown by

majority of Salmonella

Triple Sugar Lactose and/or Sucrose Positive reaction: Negative

iron (TSI) utilization Yellow slant (some strains may show a
agar Negative reaction: positive reaction)
colour becomes more
intensely red
Dextrose utilization Positive reaction: Positive
yellow butt with or
without gas formation
Negative reaction: colour
of butt remains unchanged
H2S production Positive reaction: Positive
Blackening of butt often Slow H2S producers may be
extending into the slant encountered. If lactose
Negative reaction: positive Salmonella
No blackening are present, the H2S reaction may
be inhibited on TSI agar
Gas formation Positive reaction: Positive
Formation of gas pockets in
the medium
Negative reaction:
No gas pockets in the medium
Lysine Iron H2S production Same as in TSI agar Positive
(LI) agar
Lysine decarboxylase Positive reaction: Positive
butt turns purple
Negative reaction:
yellow butt if dextrose
is utilized
Lysine desaminase Positive reaction: wine Negative
coloured slant
Negative reaction: No
wine coloured slant
Christensen's Production of urease Positive reaction: slant Negative
Urea (CU) pinkish red
agar* Negative reaction: colour
of slant unchanged

*
although lysine deaminase is used to distinguish Proteus from Salmonella, the urease test is a more
reliable indicator for Proteus spp.