Bacillus cereus is an aerobic sporeformer that is commonly found in soil, on vegetables, and in many raw and processed foods. Consumption of foods that contain large numbers of B. cereus (10⁶ or more/g) may result in food poisoning, especially when foods are prepared and held for several hours without adequate refrigeration before serving. Cooked meat and vegetables, boiled or fried rice, vanilla sauce, custards, soups, and raw vegetable sprouts have been incriminated in past outbreaks (1). Two types of illness are 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 (4,5). The second, which is characterized by an acute attack of nausea and vomiting that occurs within 1-5 h after a meal; diarrhea is not a common feature in this type of illness.

Although certain physiological and cultural characteristics are necessary for identifying B. cereus (4), its enterotoxigenicity indicates whether a suspect strain may be a public health hazard. Evidence shows that diarrheal toxin is a distinct serological entity; in vitro methods that use specific antibodies have been developed to detect the toxin in culture fluids. The evidence for the emetic toxin, however, is still incomplete. This chapter presents a method for the routine culturing of suspect Bacillus spp., using a semisolid agar medium and a serological procedure (the microslide gel double diffusion test) to identify the enterotoxin.

1. Equipment and materials
   1. Test tubes, 25 x 100 and 20 x 150 mm
   2. Petri dishes, 15 x 100 and 20 x 150 mm, sterile
   3. Bottles, prescription, 4 oz
   4. Microscope slides, glass, pre-cleaned, 3 x 1 inch (7.62 x 2.54 cm)
   5. Pipets, sterile, 1, 5, and 10 ml, graduated
   6. Centrifuge tubes, 50 ml
   7. Sterile bent glass spreaders
   8. Electrical tape, 0.25 mm thick, 19.1 mm wide, available from Scotch Brand, 3M Co., Electro-Products Division, St. Paul, MN 55011.
   9. Templates, plastic (Fig. 1)
   10. Silicone grease, high vacuum, available from Dow Corning Corp., Midland, MI 48640
   11. Sponges, synthetic
   12. Wooden applicator sticks
   13. Glass tubing, 7 mm, for capillary pipets and de-bubblers
   14. Pasteur pipets or disposable 30 or 40 µl pipets, available from Kensington Scientific Corp., 1165-67th St., Oakland, CA 94601, if capillary pipets are not available.
   15. Staining jars (Coplin or Wheaton)
   16. Desk lamp
   17. Incubator, 35 ± 1°C
   18. Hot plate, electric
   19. Sterilizer (Arnold), flowing steam
   20. Blender and sterile blender jars (see Chapter 1)
   21. Centrifuge, high speed
   22. Timer, interval

2. Media and reagents
   1. Brain heart infusion (BHI) broth (M24)
   2. Glucose, dextrose anhydrous
   3. Gel diffusion agar, 1.2% (R28)
   4. Nutrient agar slants (M112)
   5. Distilled water, sterile
   6. Phosphate-buffered dilution water (Butterfield's buffer) (R11)
   7. Normal (physiological) saline, sterile (antisera diluent) (R63)
   8. Thiazine Red R stain (R79)
   9. Slide preserving solution for stained slides, 1% acetic acid and 1% glycerol (R69)
   10. No. 1 McFarland standard (R42)
   11. Antisera and reference enterotoxins

3. Preparation of materials and media

   1. BHIG, 0.1%. Adjust BHI broth containing 0.1% glucose to pH 7.4 and dissolve by stirring. Distribute medium in 30 ml portions in 125 ml flasks and autoclave at 121°C for 10 min.

   2. No. 1 McFarland standard. Prepare turbidity standard No. 1 of McFarland nephelometer scale (5). Mix 1 part 1% BaCl₂ with 99 parts 1% H₂SO₄ in distilled water.

   3. 1.2% Gel diffusion agar for gel diffusion slides. Prepare fluid base for agar in distilled water as follows: NaCl 0.85%; sodium barbital 0.8%; merthiolate 1:10,000 (crystalline), available from Eli Lilly and Co., Terre Haute, IN. Adjust pH to 7.4. Prepare agar by adding 1.2% Noble special agar (Difco). Melt agar mixture in Arnold sterilizer (steamer) and filter while hot, in steamer, through 2 layers of filter paper; dispense in small portions (15-25 ml) in 4 oz prescription bottles. (Remelting more than twice may break down purified agar.)

   4. Thiazine Red R stain. Prepare 0.1% solution of Thiazine Red R stain in 1.0% acetic acid.

   5. Preparation of slides. Wrap double layer of electrician's plastic insulating tape around both sides of glass slide, leaving 2.0 cm space in center. Apply tape as follows: Start a piece of tape 9.5-10 cm long about 0.5 cm from edge of undersurface of slide and wrap tightly around slide twice. Wipe area between tapes with cheesecloth soaked with 95% ethanol, and dry with dry cheesecloth. Coat upper surface area between tapes with 0.2% agar in distilled water as follows: Melt 0.2% bacteriological grade agar, and maintain at 55°C or higher in screw-cap bottle. Hold slide over beaker placed on hot plate adjusted to 65-85°C and pour or brush 0.2% agar over slide between 2 pieces of tape. Let excess agar drain into beaker. Return agar collected in beaker to original container for reuse. Wipe undersurface of slide. Place slide on tray and dry in dust-free atmosphere (e.g., incubator). NOTE: If slides are not clean, agar will roll off slide without coating it uniformly.

   6. Preparation of slide assembly. Prepare plastic templates as described by Casman et al. (2) (Fig. 1). Spread thin film of silicone grease on side of template that will be placed next to agar, i.e., the side with the smaller holes. Place about 0.4 ml melted and cooled (55-60°C) 1.2% diffusion agar between tapes. Immediately lay silicone-coated template on melted agar and edges of bordering tapes. Place one edge of template on one of the tapes and bring opposite edge to rest gently on the other tape. Place slide in prepared petri dish (see C-7, below) soon after agar solidifies and label slide with number, date, or other information.

   7. Preparation of petri dishes for slide assemblies. Maintain necessary high humidity by saturating 2 strips of synthetic sponge (about 1/2 inch wide x 1/2 inch deep x 2-1/2 inches long) with distilled water and placing them in each 20 x 150 mm petri dish. From 2 to 4 slide assemblies can be placed in each dish.

   8. Recovery of used slides and templates. Clean slides without removing tape; rinse with tap water, brush to remove agar gel, boil in detergent solution for 15-20 min, rinse about 5 min in hot running water, and boil in distilled water. Place slides on end, using test tube rack or equivalent, and place in incubator to dry. If slides cannot be uniformly coated with hot 0.2% agar, they are not clean enough and must be washed again. Avoid exposure to excessive heat or plastic solvents when cleaning plastic templates. Place templates in a pan and pour hot detergent solution over them; let them soak 10-15 min. Use soft nylon brush to remove residual silicone grease. Rinse sequentially with tap water, distilled water, and 95% ethanol. Spread templates on towel to dry.

   9. Directions for dissolving reagents used in slide gel. The reagents are supplied as lyophilized preparations of enterotoxins and their antisera. Rehydrate antisera in physiological saline. Rehydrate reference enterotoxins in physiological saline containing 0.3% proteose peptone, pH 7.0, or physiological saline containing 0.37% dehydrated BHI broth, pH 7.0. These preparations should produce faint but distinct reference lines in the slide gel diffusion test. The lines may be enhanced (see E-3, below).

4. Procedure for enumeration and selection of B. cereus colonies. For examining food products, use procedures described for detecting B. cereus (see Chapter 14). Test isolates for enterotoxigenicity as described in E, below.

   Production of enterotoxin. Of the methods described for the production of enterotoxin, cultivation of B. cereus in BHIG (0.1% glucose, pH 7.4) is simple and requires no special apparatus other than a shaker. Add loopful of growth from nutrient agar slants to 3-5 ml sterile distilled water or saline. Inoculate BHIG with 0.5 ml of this aqueous suspension, which should contain about 300 million organisms/ml. Turbidity of suspension should be equivalent to No. 1 on McFarland nephelometer scale. Deliver suspension with sterile 1.0 ml pipet. Shake flasks at 3 ± 2°C at 84-125 cycles/ml for 12 h. Good surface growth is obtained after 12 h of incubation. Transfer contents of flasks to 50 ml centrifuge tube. Remove organisms by high speed centrifugation (10 min at 32,800 x g). Examine supernatant for presence of enterotoxin by filling depots in slide gel diffusion assembly, as directed in E, below.

5. Slide gel diffusion test. To prepare record sheet, draw hole pattern of template on record sheet, indicate contents of each well, and give each pattern on record sheet a number to correspond with number on slide.

   1. Addition of reagents (Fig. 2). Place suitable dilution of anti-enterotoxin (antiserum) in central well and place homologous reference enterotoxin in upper peripheral well (if diamond pattern is used); place material(s) under examination in well adjacent to well containing reference enterotoxin(s). Use reference toxins and antitoxins (antiserum), previously balanced, in concentrations that give line of precipitation about halfway between their respective wells. Adjust dilutions of reagents to give distinct but faint lines of precipitation for maximum sensitivity. (See C-9 for directions for dissolving reagents.) Prepare control slide with only reference toxin and antitoxin.

Fill wells to convexity with reagents, using Pasteur pipet (prepared by drawing out glass tubing of about 7 mm od) or disposable 30 or 40 µl pipet. Remove bubbles from all wells by probing with fine glass rod. Make rods by pulling glass tubing very fine, as in making capillary pipets, breaking it into about 2-1/2 inch lengths, and melting ends in flame. It is best to fill wells and remove bubbles against a dark background. Insert rods into all wells to remove trapped air bubbles that may not be visible. Let slides remain at room temperature in covered petri dishes containing moist sponge strips for 48-72 h before examination or for 24 h at 37°C.

2. Reading the slide. Remove template by sliding it to one side. If necessary, clean slide by dipping momentarily in water and wiping bottom of slide; then stain as described below. Examine slide by holding over source of light and against dark background. Identify lines of precipitation through their coalescence with reference line of precipitation (Fig. 3). If concentration of enterotoxin in test material is excessive, formation of reference line will be inhibited; test material must then be diluted and retested. Figure 4, diagram A, shows typical precipitate line inhibition caused by enterotoxin excess in test preparation reactant arrangement in Fig. 2. Figure 5 shows typical line formation. Figure 6 shows a diluted preparation. Occasionally, atypical precipitate patterns that form may be difficult for inexperienced analysts to interpret. One of the most common atypical reactions is formation of lines not related to toxin but caused by other antigens in test material (Fig. 7).

3. Staining of slides. Enhance lines of precipitation by immersing slide in Thiazine Red R strain for 5-10 min, and then examine. Such enhancement is necessary when reagents have been adjusted to give lines of precipitation that are only faintly visible. Use staining procedure described by Crowle (3), modified slightly, when slide is to be preserved. Rinse away any reactant liquid remaining on slide by dipping slide momentarily in water and immersing it for 10 min in each of the following baths: 0.1% Thiazine Red R in 1% acetic acid; 1% acetic acid; and 1% acetic acid containing 1% glycerol. Drain excess fluid from slide and dry in 35°C incubator for storage as permanent record. After prolonged storage, lines of precipitation may not be visible until slide is immersed in water.






Fig. 2. Reagent arrangement for serologic identification of B. cereus diarrheal antigen: 1) antiserum to B. cereus antigen; 2) test preparation; 3) B. cereus toxin reference; 4) and 5) test preparations.





Fig. 3. Microslide gel diffusion test as toxin detection system; Antiserum to B. cereus diarrheagenic antigen is in well 1; known reference enterotoxin is in well 3 to produce reference lines; test preparations are in wells 2 and 4. Interpret reactions as follows: 1) No line development between test preparations and antisera -- absence of B. cereus toxin; 2) coalescence of test preparation line from well 4 with enterotoxin reference line -- presence of enterotoxin in well 4; 3) coalescence of test preparation line from wells 2 and 4 with enterotoxin reference -- presence of enterotoxin in wells 2 and 4.



Fig. 4. Effect of amount of B. cereus enterotoxin in test preparation on development of reference line of precipitation. A, inhibition (suppression) of reference line when 10 and 5 µg enterotoxin/ml, respectively, are used. B-E, precipitate patterns when successively less enterotoxin (test preparation) is used. F, typical formation of reference line of precipitation in slide test control system.






Fig. 5. Diarrheal antigen-antibody line of precipitation with microslide test. Interpretation of reaction: B. cereus culture fluids (right, left, and adjacent to reference toxin) contain diarrheagenic component, indicated by lines of precipitation coalescing with reference line.





Fig. 6. Typical lines of precipitation of diluted B. cereus culture fluids, using microslide test.





Fig. 7. B. cereus enterotoxin: Precipitate patterns in microslide gel diffusion test demonstrate nonspecific (atypical) lines of precipitation caused by other antigens reacting with nonenterotoxin antibodies. Test preparations (wells 2 and 4) are negative for enterotoxin but produce nonspecific lines of precipitation which intersect enterotoxin reference lines of precipitation.

1. Bennett, R. W., and S. M. Harmon. 1988. Bacillus cereus food poisoning. In: Laboratory Diagnosis of Infectious Diseases: Principles and Practice, Vol. I, pp. 83-93. A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Turano (eds). Springer-Verlag, New York.

2. Casman, E. P., R. W. Bennett, A. E. Dorsey, and J. E. Stone. 1969. The micro-slide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6:185-198.

3. Crowle, A. J. 1958. A simplified micro double-diffusion agar precipitin technique. J. Lab. Clin. Med. 52:784.

4. Lancette, G. A., and S. M. Harmon. 1980. Enumeration and confirmation of Bacillus cereus in foods: collaborative study. J. Assoc. Off. Anal. Chem. 63:581-586.

5. McFarland, J. 1907. The nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Am. Med. Assoc. 49:1176.

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