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January 2001
Food poisoning caused by Bacillus cereus may occur when foods are prepared and held without adequate refrigeration for several hours before serving. B. cereus is an aerobic sporeforming bacterium that is commonly found in soil, on vegetables, and in many raw and processed foods. Consumption of foods that contain >106 B. cereus/g may result in food poisoning. Foods incriminated in past outbreaks include cooked meat and vegetables, boiled or fried rice, vanilla sauce, custards, soups, and raw vegetable sprouts. Two types of illness have been attributed to the consumption of foods contaminated with B. cereus. The first and better known is characterized by abdominal pain and diarrhea; it has an incubation period of 4-16 h and symptoms that last for 12-24 h. The second, which is characterized by an acute attack of nausea and vomiting, occurs within 1-5 h after consumption of contaminated food; diarrhea is not a common feature in this type of illness.
Examination of Foods for B. cereus
If the quantity of food to be examined is large, take representative samples of 50 g each from different parts of the suspect food because contamination may be unevenly distributed.
Transport and examine samples promptly without freezing, if possible. If samples must be shipped to the laboratory, pack them in insulated shipping containers with enough gel-type refrigerant to maintain them at 6°C or below. Upon receipt in the laboratory, store the samples at 4°C and analyze as soon as possible. If analysis cannot be started within 4 days after collection, freeze samples rapidly and store at -20°C until examined. Thaw at room temperature and proceed with analysis as usual. Dehydrated foods may be stored at room temperature and shipped without refrigeration.
Enumeration and Confirmation of B. cereus in Foods
Using aseptic technique, weigh 50 g of sample into sterile blender jar. Add 450 ml Butterfield's phosphate-buffered dilution water (1:10 dilution) and blend for 2 min at high speed (18,000-21,000 rpm). Using the 1:10 dilution, make serial dilutions of sample for enumeration of B. cereus as described in D or E, below.
Prepare serial dilutions from 10-2 to 10-6 by transferring 10 ml homogenized sample (1:10 dilution) to 90 ml dilution blank, mixing well with vigorous shaking, and continuing until 10-6 dilution is reached. Inoculate duplicate MYP agar plates with each dilution of sample (including 1:10) by spreading 0.1 ml evenly onto surface of each plate with sterile glass spreading rod. Incubate plates 24 h at 30°C and observe for colonies surrounded by precipitate zone, which indicates that lecithinase is produced. B. cereus colonies are usually a pink color which becomes more intense after additional incubation.
If reactions are not clear, incubate plates for additional 24 h before counting colonies. Select plates that contain an estimated 15-150 eosin pink, lecithinase-producing colonies. Mark bottom of plates into zones with black felt pen to facilitate counting and count colonies that are typical of B. cereus. This is the presumptive plate count of B. cereus. Pick 5 or more presumptive positive colonies from the MYP agar plates and transfer to nutrient agar slants for confirmation as B. cereus. Confirm isolates as B. cereus as described in F and G, below. Calculate number of B. cereus cells/g of sample, based on percentage of colonies tested that are confirmed as B. cereus. For example, if average count obtained with 10-4 dilution of sample was 65 and 4 of 5 colonies tested were confirmed as B. cereus, the number of B. cereus cells/g of food is 65 x 4/5 x 10,000 x 10 = 5,200,000. (NOTE: Dilution factor is tenfold higher than sample dilution because only 0.1 ml was tested).
The MPN technique is recommended for enumerating B. cereus in foods that are expected to contain fewer than 10 B. cereus organisms/g. It may also be preferred for examining certain dehydrated starchy foods for which the plate count technique is inappropriate.
Inoculate 3-tube MPN series in trypticase soy-polymyxin broth, using 1 ml inoculum of 10-1, 10-2, and 10-3 dilutions of sample with 3 tubes at each dilution. (Additional dilutions should also be tested if B. cereus population is expected to exceed 103/g.) Incubate tubes 48 ± 2 h at 30°C and observe for dense growth, which is typical of B. cereus. Streak cultures from positive tubes onto separate MYP agar plates and incubate plates 24-48 h at 30°C. Pick one or more eosin pink, lecithinase-positive colonies from each MYP agar plate and transfer to nutrient agar slants for confirmation as B. cereus. Confirm isolates as B. cereus as described in F and G, below, and calculate MPN of B. cereus cells/g of sample (see Appendix 2) based on the number of tubes at each dilution in which the presence of B. cereus was confirmed.
Pick 5 or more eosin pink, lecithinase-positive colonies from MYP agar plates and transfer to nutrient agar slants. Incubate slants 24 h at 30°C. Prepare Gram-stained smears from slants and examine microscopically. B. cereus will appear as large Gram-positive bacilli in short-to-long chains; spores are ellipsoidal, central to subterminal, and do not swell the sporangium. Transfer 3 mm loopful of culture from each slant to 13 x 100 mm tube containing 0.5 ml of sterile phosphate-buffered dilution water and suspend culture in diluent with Vortex mixer. Use suspended cultures to inoculate the following confirmatory media:
Phenol red glucose broth. Inoculate 3 ml broth with 2 mm loopful of culture. Incubate tubes anaerobically 24 h at 35°C in GasPak anaerobic jar. Shake tubes vigorously and observe for growth as indicated by increased turbidity and color change from red to yellow, which indicates that acid has been produced anaerobically from glucose. A partial color change from red to orange/yellow may occur, even in uninoculated control tubes, due to a pH reduction upon exposure of media to CO2 formed in GasPak anaerobic jars. Be sure to use appropriate positive and negative controls so that a distinction can be made between positive and "false-positive" reactions.
Nitrate broth. Inoculate 5 ml broth with 3 mm loopful of culture. Incubate tubes 24 h at 35°C. To test for nitrite, add 0.25 ml each of nitrite test reagents A and C to each culture. An orange color, which develops within 10 min, indicates that nitrate has been reduced to nitrite.
Tyrosine agar. Inoculate entire surface of tyrosine agar slant with 3 mm loopful of culture. Incubate slants 48 h at 35°C. Observe for clearing of medium near growth, which indicates that tyrosine has been decomposed. Examine negative slants for obvious signs of growth, and incubate for a total of 7 days before considering as negative.
Lysozyme broth. Inoculate 2.5 ml of nutrient broth containing 0.001% lysozyme with 2 mm loopful of culture. Also inoculate 2.5 ml of plain nutrient broth as positive control. Incubate tubes 24 h at 35°C. Examine for growth in lysozyme broth and in nutrient broth control. Incubate negative tubes for additional 24 h before discarding.
MYP agar. This test may be omitted if test results were clear-cut with original MYP agar plates and there was no interference from other microorganisms which were present. Mark bottom of a plate into 6-8 equal sections with felt marking pen, and label each section. Inoculate premarked 4 cm sq area of MYP agar plate by gently touching surface of agar with 2 mm loopful of culture. (Six or more cultures can be tested in this manner on one plate.) Allow inoculum to be fully absorbed before incubating for 24 h at 35°C. Check plates for lecithinase production as indicated by zone of precipitation surrounding growth. Mannitol is not fermented by isolate if growth and surrounding medium are eosin pink. (Yellow color indicates that acid is produced from mannitol.) B. cereus colonies are usually lecithinase-positive and mannitol-negative on MYP agar.
Record results obtained with the different confirmatory tests. Tentatively identify as B. cereus those isolates which 1) produce large Gram-positive rods with spores that do not swell the sporangium; 2) produce lecithinase and do not ferment mannitol on MYP agar; 3) grow and produce acid from glucose anaerobically; 4) reduce nitrate to nitrite (a few strains may be negative); 5) produce acetylmethylcarbinol (VP-positive); 6) decompose L-tyrosine; and 7) grow in the presence of 0.001% lysozyme.
These basic characteristics are shared with other members of the B. cereus group, including the rhizoid strains B. mycoides, the crystalliferous insect pathogen B. thuringiensis, and the mammalian pathogen B. anthracis. However, these species can usually be differentiated from B. cereus by determining specific characteristics typical of each species or variety. The tests described in G, below, are useful for this purpose and can easily be performed in most laboratories. Strains that produce atypical results from these tests require additional analysis before they can be classified as B. cereus.
Tests for differentiating members of the B. cereus group (Table 1)
The following tests are useful for differentiating typical strains of B. cereus from other members of the B. cereus group, including B. mycoides, B. thuringiensis, and B. anthracis.
Motility test. Inoculate BC motility medium by stabbing down the center with 3 mm loopful of 24 h culture suspension. Incubate tubes 18-24 h at 30°C and examine for type of growth along stab line. Motile organisms produce diffuse growth out into the medium away from the stab. Nonmotile organisms produce growth only in and along stab. Alternatively, add 0.2 ml sterile distilled water to surface of nutrient agar slant and inoculate slant with 3 mm loopful of culture suspension. Incubate slant 6-8 h at 30°C and suspend 3 mm loopful of liquid culture from base of slant in a drop of sterile water on microscope slide. Apply cover glass and examine immediately with microscope for motility. Report whether or not isolates tested were motile. Most strains of B. cereus and B. thuringiensis are motile by means of peritrichous flagella. B. anthracis and all except a few strains of B. mycoides are nonmotile. A few B. cereus strains are also nonmotile.
| Table 1. Differential characteristics of large-celled Group I Bacillus species | |||||
| Feature | B. cereus | B. thuringiensis | B. mycoides | B. anthracis | B. megaterium |
|---|---|---|---|---|---|
| Gram reaction | +(a) | + | + | + | + |
| Catalase | + | + | + | + | + |
| Motility | +/-(b) | +/- | -(c) | - | +/- |
| Reduction of nitrate | + | +/ | + | + | -(d) |
| Tyrosine decomposed | + | + | +/- | -(d) | +/- |
| Lysozyme-resistant | + | + | + | + | - |
| Egg yolk reaction | + | + | + | + | - |
| Anaerobic utilization of glucose | + | + | + | + | - |
| VP reaction | + | + | + | + | - |
| Acid produced from mannitol | - | - | - | - | + |
| Hemolysis (Sheep RBC) | + | + | + | -(d) | - |
| Known pathogenicity(e)/characteristic | produces enterotoxins | endotoxin crystals pathogenic to insects | rhizoidal growth | pathogenic to animals and humans | |
| a +, 90-100% of strains are positive. | |||||
| b +/-, 50-50% of strains are positive. | |||||
| c -, 90-100% of strains are negative. | |||||
| d -, Most strains are negative. | |||||
| e See Section H, Limitations of method for B. cereus. | |||||
)
hemolysis surrounding growth. Most B. thuringiensis and B.
mycoides strains are also -hemolytic.
B. anthracis strains are usually nonhemolytic after 24 h incubation.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 somewhat 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.
The method described is intended primarily for use in the routine examination of foods. As noted in F, above, and in Table 1, the confirmatory tests recommended may in some instances be inadequate for distinguishing B. cereus from culturally similar organisms that could occasionally be encountered in foods. These organisms include 1) the insect pathogen B. thuringiensis, which produces protein toxin crystals; 2) B. mycoides, which characteristically produces rhizoid colonies on agar media; and 3) B. anthracis, which exhibits marked animal pathogenicity and is nonmotile. With the exception of B. thuringiensis, which is currently being used for insect control on food and forage crops, these organisms are seldom encountered in the routine examination of foods. The tests described in G, above, are usually adequate for distinguishing the typical strains of B. cereus from other members of the B. cereus group. However, results with atypical strains of B. cereus are quite variable, and further testing may be necessary to identify the isolates. Although a few diarrheal toxin detection kits are commercially available, none are recommended, pending further evaluation. At present, no practical tests for detecting the emetic toxin are available. Until reliable tests are available, cultural tests such as those described in this method must be relied upon for confirming isolates from foods as B. cereus.
Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 14.
