Laboratory Procedure MFLP-42
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Health Products and Food Branch
Canadian Food Inspection Agency 3650 36th St. N.W.,
Health Products and Food Branch (HPFB), Health Canada, 1001, St-Laurent ouest,
R. A. Szabo
Microbiology Evaluation Division, Bureau of Microbial Hazards,
Health Products and Food Branch,
Health Canada, Postal Locator: 2204E,
Ottawa, Ontario, K1A 0L2
This method is applicable to the isolation, identification and enumeration of the Bacillus cereus group (with limitations as described in the method) in foods in accordance with the requirements of Sections 4 and 7 of the Food and Drugs Act. The revised method replaces MFLP-42, dated April, 2003.
The method has been shown to produce satisfactory results with naturally-contaminated meats, vegetables, dairy products, cereals and dried foods (8.5).
The Bacillus cereus group is widely distributed in nature, consisting of B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis. B. mycoides, B. pseudomycoides, and B. weihenstephanensis are relatively straightforward to differentiate from the other members of the B. cereus group and are thus not considered further in this document.
B. cereus is not easily distinguished from other closely related organisms in the B. cereus group. B. thuringiensis is thought to be nearly identical to B. cereus, with the exception of the production of the protein toxin crystals, which are encoded by the cry genes. The difficulties that may be encountered in identifying carriage or expression of cry makes distinguishing B. cereus and B. thuringiensis laborious and complicated. B. anthracis is non-motile and non-hemolytic. Atypical strains of B. cereus are variable in expression of motility and hemolysis and further testing may be necessary to identify the isolates. Consider the source of the sample when identifying the isolates as B. cereus. Only B. cereus and B. thuringiensis are likely to occur naturally in food products.
B. cereus is commonly found in a variety of foods and when it grows to high numbers in a food (> 106/g), sufficient enterotoxin may be produced resulting in foodborne illness. This method determines the presence of B. cereus group isolates by plating known quantities of (dilutions of) a food sample onto a selective agar. After incubation, presumptive B. cereus colonies are selected and subjected to confirmatory testing. From the results obtained, the number of presumptive B. cereus per g or mL of the food is calculated.
See Appendix A of Volume 3 (8.4)
See Appendix B of Volume 3 (8.4)
The following media and reagents (1-5) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3, and Section 9 for the formula of individual media.
1) Peptone Water diluent (PW)
2) Citrate solution, 2%, warmed to 45°C (for cheese)
3) Trypticase Soy Broth (TSB)
4) Nutrient Agar plates
5) Polymyxin Pyruvate Egg Yolk Mannitol Bromthymol Blue Agar (PEMBA Medium)
6) Blood Agar plates (TSB agar with 5% sheep blood)
7) Sporulation broth (9.1) or TSA-MnSO4 agar (optional)
8) Staining solutions (optional): Malachite Green, 5% aqueous solution; Safranin, 0.5% aqueous solution; Sudan Black B, 0.3% in 70% ethanol; Xylol
9) Basic fuchsin, 0.5% aqueous solution OR TB Carbol-fuchsin ZN stain (Difco) [protein toxin crystals]
10) Methanol [protein toxin crystals]
11) BC Motility Medium (9.2)
12) Rapid identification kits (optional)
13) Control cultures, ATCC or equivalent
14) Blender, stomacher or equivalent
16) Incubators capable of maintaining 30 and 35°C
Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions:
7.1 Handling of Sample Units
7.1.1. During transport, with the exception of shelf-stable products, keep the sample units refrigerated (0-5°C) or frozen depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
7.1.2. Analyze the sample units as soon as possible after receipt at the laboratory.
7.2 Preparation of Dilutions
7.2.1 To ensure a representative analytical unit from a solid sample, combine portions from several locations within each solid sample unit.
7.2.2 If the sample unit is a liquid or a free-flowing solid (powder), thoroughly mix each sample unit by shaking the container.
7.2.3 Prepare a 1:10 dilution of the food by adding aseptically 11 (10) g or mL (the analytical unit) to 99 (90) mL of diluent (Table 1). Shake, blend or stomach according to the type of food as indicated in Table 1.
7.2.4 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.
7.2.5 Mix for the minimum time required to produce a homogeneous suspension to avoid overheating; blending or stomaching time should not exceed 2 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface.
7.2.6 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.
7.2.7 Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer.
7.2.8 Shake all dilutions (as in 7.2.6) immediately prior to making transfers to ensure uniform distribution of the microorganisms present.
7.3 Enumeration of Presumptive B. cereus
126.96.36.199 Dry PEMBA plates in a bio-hood or laminar flow hood immediately before using. Agitate each dilution to resuspend material that may have settled during preparation. Plating should be carried out within 15 min of preparing the dilutions.
188.8.131.52 Solid foods
(i) If fewer than 1,000 B. cereus group per g are expected: spread 0.2 mL of the 1:10 dilution evenly over the surface of one of each of ten selective agar plates (PEMBA).
(ii) Routinely, or if counts higher than 1,000 B. cereus group per g are expected: spread 0.2 mL of each dilution on each of duplicate PEMBA plates
184.108.40.206 Liquid sampes:
If the sample units are liquid, 0.2 mL of the undiluted analytical unit may be spread on each of duplicate PEMBA plates.
220.127.116.11 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 readily absorbed by the medium, the plates may be placed in an upright position in an incubator for up to 1 h.
18.104.22.168 Invert the plates and incubate at 35°C for 24 ± 2 h.
22.214.171.124 Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature.
126.96.36.199 Examine the plates for presumptive B. cereus. Count the number of presumptive B. cereus colonies present (Section 7.3.3). Re-incubate the plates at room temperature for an additional 24 h and re-examine.
7.3.3 Counting Colonies and Recording Results
188.8.131.52 Count colonies immediately after the incubation period. Look for the following 2 types of presumptive B. cereus colonies on PEMBA:
Type 1: Uneven margins, fimbriate or slightly rhizoidal, 2 to 5 mm in diameter, turquoise to peacock blue (intensity variable) in colour with flat ground glass surface and surrounded by a grey to turquoise halo of dense precipitate (egg yolk reaction) which may become peacock blue after 48 h incubation.
Type 2: Colonies similar to type 1 but with no surrounding halo of precipitation.
184.108.40.206 Counting the Ten Plates of the 1:10 Dilution (Solid Food Only)
220.127.116.11(a) If the number of all presumptive B. cereus colonies per plate is fewer than 20, add separately the counts for each type from all ten plates and record as the respective presumptive count. This is the count of one of the two types per 2 mL (0.2 g of food) (B). Multiply the 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.
18.104.22.168(b) If the number of all presumptive B. cereus colonies is greater than 20 per plate but the total count of the two types does not exceed 200, select two plates at random, count separately the colonies of each type and compute the respective average presumptive count per plate (per 0.2 mL) (A/2). Multiply each count by 50 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.
22.214.171.124(c) If the number of presumptive B. cereus colonies on some of the ten plates is < 20, but on others is ≥ 20, proceed as in 126.96.36.199(a) above.
188.8.131.52 Counting of Duplicate Plates (Any Dilution)
184.108.40.206(a) Select plates containing 20-200 presumptive B. cereus colonies per plate consisting of the combined counts of the two types. An alternate counting range of 10-100 or 10-150 may be used, as these ranges are recommended in other standard methods due to the spreading nature of Bacillus colonies.
220.127.116.11(b) 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.
18.104.22.168(c) If plates from more than one dilution are used, the counts are to be averaged as shown below (Sec. 22.214.171.124)
126.96.36.199(d) If no plates containing 20-200 presumptive B. cereus are 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.
188.8.131.52(e) When an estimated count contributes to an average count, this average itself becomes an estimated value.
184.108.40.206 Averaging of Counts Over Two Dilutions
220.127.116.11(a) If plates from two consecutive decimal dilutions contain counts within the range of 20-200 presumptive B. cereus colonies per plate, the counts on all four plates should be used to arrive at the average count. Inasmuch as the two different types are to be counted separately and it is quite possible that individual counts may be < 20, although the combined counts are within range, estimates and true values would have to be combined in order to arrive at an average value. This can be avoided by using the following formula:
Average colony count/g or mL = Total number of colonies counted / Volume used per dilution (1/dilution1 + 1/dilution2)
For an example of counting colonies see Table II.
18.104.22.168(b) If no presumptive B. cereus colonies are obtained, record presumptive counts as < 5 per g or mL for the ten plates of the 1:10 dilution, or < 2.5 x the dilution factor for duplicate plates.
7.4.1 Selection of Colonies
22.214.171.124 From the plates counted, a number of each colony type observed is selected as follows:
a) When the total count per type for all the plates of a dilution is less than five, pick all colonies of that type.
b) 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.
7.4.2 Screening for B. cereus / B. thuringiensis
It is recommended that suspect colonies be streaked onto non-selective agar (Nutrient or Blood agar) for purity. Inoculate 5 mL of Trypticase-soy broth (TSB) with suspect colonies, as well as appropriate controls, and incubate for 18 h at 30°C.
126.96.36.199 Motility (8.3)
Inoculate BC motility medium (BCMM) by stabbing down the centre of the tube with a 3 mm loopful of a 24 h culture suspension. Incubate tubes for 18 to 24 h at 30°C and examine for type of growth along the stab line. Most strains of B. cereus and B. thuringiensis are motile by peritrichous flagella, and produce diffuse growth out into the medium away from the stab. Among members of the B. cereus group, B. anthracis and all but a few strains of B. mycoides are non-motile.
188.8.131.52 Rhizoid growth (8.3)
Inoculate a pre-dried nutrient agar plate by touching the medium surface near the centre with 2 mm loopful of culture. Let the inoculum be absorbed, and incubate the plate in an upright position for 24 to 48 h at 30°C. Check the plate for rhizoid growth characterized by root or hairlike structures which may extend several cm from the point of inoculation. This type of growth is typical for B. mycoides species. B. cereus strains produce rough irregular colonies that should not be confused with rhizoid growth.
184.108.40.206 Hemolytic activity
After incubation of broth, divide a blood agar plate into 6 to 8 equal segments. Label each segment and inoculate one or more segments near the centre by gently touching the agar surface with a loopful of incubated broth.
Let inoculum be absorbed, and incubate plates for 24 h at 30°C. Check plates for hemolytic activity.
B. cereus is usually strongly beta hemolytic. B. thuringiensis and B. mycoides are often weakly beta hemolytic with production of complete hemolysis only underneath the colonies. B. anthracis is usually non-hemolytic. Aging cultures may demonstrate weak gamma hemolysis. Take proper precautions if a non-hemolytic colony is isolated.
220.127.116.11 Use of a rapid identification system such as VITEK or API may be useful to confirm that the isolate is B. cereus or B. thuringiensis with a high degree of confidence. However, systems such as Vitek will not differentiate these two species (personal communication, Shirley Fredette, Technical Consultant, BioMerieux, 2002).
18.104.22.168 Isolates that are motile, do not exhibit rhizoid growth and are hemolytic have a high probability of being B. cereus or B. thuringiensis. Strongly hemolytic strains are likely B. cereus. To confirm the presence of B. cereus, the following test for protein toxin crystals can differentiate B. cereus from B. thuringiensis.
22.214.171.124 Protein toxin crystals
Inoculate nutrient agar slants with 3 mm loopfuls of 24 h TSB culture suspensions, including positive controls of B. thuringiensis known to produce protein toxin crystals. Incubate slants 24 h at 30°C and then at room temperature 2-3 days. Prepare smears with sterile distilled water. Air-dry and lightly heat-fix. Place slide on staining rack and flood with methanol. Let stand 30 s, pour off methanol, and allow slide to air-dry. Return slide to staining rack and flood completely with 0.5% Basic fuchsin or TB carbolfuchsin ZN stain (Difco). Heat slide gently from below until steam is seen. Wait 1-2 min and repeat this step. Let stand 30 s, pour off stain, and rinse slide thoroughly with clean tap water. Dry slide without blotting and examine under oil immersion for presence of free spores and darkly stained tetragonal (diamond-shaped) toxin crystals. Crystals are usually smaller than spores. Toxin crystals are usually abundant in a 3- to 4-day-old culture of B. thuringiensis but cannot be detected by the staining technique until lysis of the sporangium has occurred. Therefore, unless free spores can be seen, cultures should be held at room temperature for a few more days and re-examined for toxin crystals. B. thuringiensis usually produces protein toxin crystals that can be detected by the staining technique either as free crystals or parasporal inclusion bodies within the exosporium. B. cereus and other members of the B. cereus group do not produce protein toxin crystals.
126.96.36.199 Confirm with staining procedure as outlined below if necessary. It is recommended that a sporulation step be included before following this procedure.
7.4.3 Sporulation Procedure (Optional)
188.8.131.52 Inoculate a prepared flask of sporulation broth with one isolated presumptive B. cereus colony from PEMBA. Place on a stir plate (without heat), loosen the cap and stir moderately at room temperature for five days. Stain as outlined in 7.4.4.
184.108.40.206 Alternately, streak presumptive colony onto TSA-MnSO4 agar. Incubate at room temperature for 2-3 days. Stain as outlined in 7.4.4.
7.4.4 Staining Procedure (Optional)
220.127.116.11 Prepare smears on glass microscope slides from the centre of colonies selected.
18.104.22.168 Air dry the smears and fix with minimal flaming.
22.214.171.124 Place the slides on a staining rack and flood with 5% w/v Malachite Green.
126.96.36.199 Heat slides with a gentle flame until vapour can be seen to rise. Continue for 3 min taking care not to boil the staining solution on the slides.
188.8.131.52 Wash slides well with cold tap water; blot dry.
184.108.40.206 Flood slides with 0.3% w/v Sudan Black B in 70% ethanol. Allow to sit for 15 minutes.
220.127.116.11 Wash slides well with cold water; blot dry.
18.104.22.168 Flood slides with xylol for 5 seconds.
22.214.171.124 Wash slides with cold tap water; blot dry.
126.96.36.199 Flood slides with 0.5% aqueous Safranin for 30 seconds.
188.8.131.52 Wash slides with cold tap water and allow to dry in an upright position.
184.108.40.206 Vegetative cells of B. cereus stain red and generally have a characteristic `boxcar' appearance 4-5 µM long and 1.0-1.5 µM wide with square ends and rounded corners usually appearing as chains. Spores stain pale to mid-green and lipid globules are black. Vegetative cells displaying: i) central or paracentral spores not obviously swelling the sporangium and ii) lipid globules, confirm the isolates as B. cereus Group.
7.4.5 Calculations and Reporting (See also Table 2)
For each of the two types of cultures, record the total number of presumptive B. cereus per g or mL of food (N). Total number of presumptive B. cereus per g or mL equals the sum of the number of presumptive B. cereus types 1 and 2 (NT=N1+N2).
No. presumptive B.cereus type 1 per g or mL (N) = (No. of colonies of presumptive B. cereus (P)/ No. colonies tested (G)) x presumptive count type 1 (C)
Same for type 2.
8.1 AOAC. 2001. Bacteriological Analytical Manual (Online) Chapter 14, Bacillus cereus. USDA, Center for Food Safety and Applied Nutrition.
8.2 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
8.3 Harmon, S.M. 1982. New Method for Differentiating Members of the Bacillus cereus Group: Collaborative Study. J. Assoc. Off. Anal. Chem. 65:1134-1139.
8.4 Health Canada. 2003. Compendium of Analytical Methods.
8.5 Holbrook, R. and Anderson, J.M., 1980. An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Can. J. Microbiol. 26: 753-759.
8.6 Vilas-Bôas GT, Peruca AP, Arantes OM. 2007. Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Can J Microbiol. 53: 673-87.
|Yeast extract||30.0 g|
|Manganese sulphate (MnSO4)||3.0 g|
|Distilled water||1.0 L|
Add ingredients to 1L of distilled water and bring to a boil to dissolve. Dispense 100 mL into 500 mL erlenmeyer flasks. Autoclave at 121°C for 15 minutes.
|Yeast extract||2.5 g|
|Distilled water||1 L|
Heat to dissolve and dispense into tubes (2 mL into 13 X 100 mm tubes is suggested). Autoclave 10 minutes at 121°C. Final pH 7.4 ± 0.2. For best results store at room temperature for 2 to 4 days before use to prevent growth along the side of the medium.
|basic fuchsin||0.5 g|
|distilled water||80 mL|
Dissolve 0.5 g basic fuchsin in 20 mL of alcohol and dilute to 100 mL with water. Filter solution if necessary thru fine paper to remove excess dye particles. Store in tightly stoppered container.
|Type of Food Product||Preparation||Treatment|
|A stomacher may also be used to provide the initial blend.|
|Liquids||pipette directly into petri plate and/or peptone water diluent||shake|
|Viscous and non-miscible liquids||weigh into peptone water diluent||blend|
|Water soluble solids||weigh into peptone water diluent||shake|
|Cheese||weigh into previously warmed (45°C) sterile 2% sodium citrate (Na3C6H5O7.2H 2O) solution||blend|
|Spices||weigh into peptone water diluent||shake|
|powders, meat and other solids||weigh into peptone water diluent||blend|
|Total No. of Colonies of one of the two Types on Duplicate Plates "A"||No. of Isolates Tested "G"||No. of Isolates of Presumptive B. cereus "P"||Total No. of Colonies of one of the two Types per g or mL "C" C= 1/2AxDx 5||No. of Presumptive B. cereus from one of the two Types per g or mL "N" N= (P/G)xC|
Dilution factor = 100
For duplicate plates, 0.2 mL per plate (5.4.5). Divide by 2 since "A" represents the total count of one of the two types on two duplicate plates.
|Fewer than 5(e.g. 4)||All (4)||2||1,000||500|
|More than 5(e.g. 18)||5(5)||4||4,500||3,600|
Calculate N1 and N2 for each colony type to obtain total number of presumptive B. cereus. (NT) per g or mL NT= N1 + N2
e.g. if N1 = 1,000 and N2 = 100
NT = 1,000 + 100 = 1,100/g
Report total number of presumptive Bacillus cereus per g or mL of food to two significant figures.
If the ten plates of the dilution are counted (220.127.116.11(a)); C=Bx10x0.5, where B is the total count of one of the two types on all ten plates.
If the two of the ten plates of the 1:10 dilution are counted (18.104.22.168(b)); C=1/2Ax10x5