Microbiological Examination of Cocoa and Chocolate

Health Protection Branch - Ottawa

Official Method MFO-11
November 30, 1981

---

1. APPLICATION

This method shall be used for the determination of the bacteria of the genus Salmonella in cocoa and chocolate, in accordance with Section B.04.010 and Section B.04.011, 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 product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or, (b) the quantity of the same kind of product 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2.1.4 Analytical Unit: That amount of product withdrawn from the sample unit for analysis.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 ten sample units drawn at random from each lot, shall be taken.

2.2.2 Each sample unit shall contain 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. When the lot consists of containers smaller than 100 g, a sample unit will consist of more than one container (e.g., four 25 g containers in each sample unit).

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 ten sample units shall be analyzed individually or as one or more composite(s) 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 at the laboratory.

3.2 Preparation of media

The following media, 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 Prepare a 10% w/v of non-fat-dry-milk solution in distilled water containing brilliant green at a final concentration of 1:50,000. Sterilize at 121°C and cool to 45°C.

3.3.2

a. Weigh 25 g (the analytical unit) from each sample unit into a separate blender jar and add 225 ml of non-fat-dry-milk solution, blend for the minimum time required to produce a homogeneous suspension and transfer the contents to a sterile container. To avoid overheating, blending time should not exceed 2.5 min.

b. The analytical units may be composited. Suspend the composite units in nine times their weight of non-fat-dry-milk solution and proceed as in 3.3.2.a.

3.3.3 Check the pH of each blended analytical or composite unit. If the pH is outside the range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.

3.3.4 Inoculate 10ml of the non-fat-dry-milk- solution prepared in 3.3.1, above, 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.

3.3.5 Incubate the inoculated pre-enrichment broth(s) and the controls at 35°C + 0.5^o for 18-24 hr. In no circumstances shall 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 broth and TBG broth for 24 + 2 hr at 35°C + 0.5^o and at 43°C + 0.5^o, 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 inhibits Salmonella serotypes other than S. typhi unless it is refrigerated at 4°C 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 plates at 35°C + 0.5^o 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 plates at 35°C + 0.5^o for 24 + 2 hr.

3.6.3 Observe the MA plates after the incubation period. Typical Salmonella colonies are lactose-negative and 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 35°C + 0.5^o 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 I. If additional 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 suggestive of Salmonella, then bacteria of the genus Salmonella are considered to be absent from the analytical or composite units 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 2-8°C 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 35°C + 0.5^o 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 2-8°C 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 (from the slant area not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) 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-test culture mixture of the negative control with a sterile needle or loop.

Tilt the 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. 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 (autoagglutination).

3.8.2 Testing with Somatic Grouping Antisera

It is preferable to test the culture against somatic grouping antisera whenever possible; however the culture may be sent to a typing centre for identification. The majority of the 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 possessing 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, mark 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 biochemical 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 the 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 area.

f. Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-test culture mixture of the negative control with a sterile needle or loop.

Tilt the 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 grouping 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 (auto-agglutination).

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 cocoa or chocolate sampled shall be considered in compliance with Section B.04.010 or Section B.04.011, of the Food and Drug Regulations when bacteria of the genus Salmonella are not found in any of the ten sample units analyzed (individually or as composites).

Table I

Minimal Biochemical Screening Media

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

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