TRYPTIC SOY BROTH (TSB) WITH POLYMYXIN B

Cat. no. K180 Tryptic Soy Broth (TSB) with Polymyxin B, 20x150mm Tube, 15ml 20 tubes/box

INTENDED USE

Hardy Diagnostics' Tryptic Soy Broth (TSB) with Polymyxin B is recommended by the AOAC and APHA for the enumeration of Bacillus cereus in foods using the most probable number (MPN) technique.(1,2,4,7) Use of the medium is also preferred for examining certain dehydrated starchy foods when the plate count technique is inappropriate.(7)

SUMMARY

Bacillus cereus is commonly found in nature, on vegetables and in some processed foods. Detection of B. cereus is important for food hygiene because of the organism's hydrolytic activities on foods, the ability of some strains to produce food poisoning toxins and their ability to grow at refrigerated temperatures. B. cereus is the etiological agent in two distinct types of food-poisoning: (1) diarrheal, characterized by abdominal pain with diarrhea 8 to 16 hours after ingestion of contaminated food, and (2) emetic, characterized by nausea and vomiting 1 to 5 hours after eating a contaminated meal; both types are the direct result of unique toxins produced in the food during growth of the bacterium.(3,4) B. cereus spores are resistant to heat and can survive normal cooking procedures. When cooked food is stored improperly, spores can germinate causing vegetative cells to multiply to dangerous levels and produce toxins. Outbreaks of foodborne illness have been associated with the ingestion of boiled and cooked rice, cooked meats, cooked vegetables, starchy foods, custards, soups, sauces and a variety of other food types.(3,7)

The AOAC and APHA recommend the MPN method for enumerating B. cereus in foods; this technique uses either a three-tube or a five-tube dilution series and published tables that provide statistically significant reference combinations.(1,2,4) When results are compared to these tables, the MPN technique provides an estimation of the number of viable cells per gram or milliliter of sample, along with a confidence limit. The technique is most useful on samples expected to contain fewer than 10 microorganisms per gram or on certain dehydrated starchy foods where traditional methods are inappropriate.(4,5,7)

Hardy Diagnostics' Tryptic Soy Broth (TSB) with Polymyxin B contains digests of soybean meal and casein which provide amino acids and other nitrogenous compounds. Sodium chloride helps maintain osmotic equilibrium. A 15% concentration of polymyxin B is added to inhibit the growth of competing gram-negative microorganisms.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 17.0gm
Sodium Chloride 5.0gm
Papaic Digest of Soybean Meal 3.0gm
Potassium Hydrogen Phosphate 2.5gm
Glucose 2.5gm
Polymyxin B Solution, 15% 6.67ml

Final pH 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

If possible, samples should be transported and examined promptly without freezing. If samples must be shipped, pack them in insulated shipping containers using gel-type cold packs to maintain a temperature of 6ºC. or below. Upon receipt, store samples at 4ºC. and analyze promptly. If analysis cannot be initiated within 4 days post collection, freeze samples rapidly and store at -20ºC. until examination; thaw at room temperature and proceed with analysis as normal. Dehydrated foods may be shipped without refrigeration and stored at room temperature prior to analysis.

If the quantity of food for examination is large, take representative samples of 50gm each from different parts of the suspect food to account for uneven distribution. Using aseptic technique, weigh 50gm of food sample into a sterile blender. Add 450ml of Butterfield's Phosphate Buffer solution (Cat. no. U150) to make a 1:10 dilution. Blend on high speed (18,000 to 21,000 rpm) for 2 minutes to mix well. Make serial dilutions for enumeration as follows:

1. Inoculate 3-tube MPN series in Tryptic Soy Broth (TSB) with Polymyxin B, using 1ml inoculum of 10-1, 10-2, and 10 -3 dilutions of sample with 3 tubes at each dilution. Note: Additional dilutions should be used if the B. cereus population is expected to exceed 103 cells/gm.

2. Incubate tubes 48 +/- 2 hours at 30ºC. and observe for dense growth typical of B. cereus.

3. Calculate the MPN of B. cereus cells/gm using the appropriate published tables based on the number of tubes at each dilution in which heavy growth of B. cereus was identified.(1,2,7)

4. Streak cultures from positive tubes onto separate MYP Agar plates (Cat. no. G147) and incubate plates 24 to 48 hours at 30ºC.

5. Pick one or more pink to red, lecithinase-positive colonies from each MYP Agar plate and transfer to Nutrient Agar slants (Cat. no. L20) for confirmation. Proceed with confirmatory methods as listed in the references.(1,2,7)

INTERPRETATION OF RESULTS

Examine tubes for dense growth typical of B. cereus, and calculate the MPN using the appropriate published tables.(7) Cultures can be streaked onto separate MYP Agar plates for further identification and pink to red, lecithinase-positive colonies confirmed using the appropriate confirmatory media as listed in the references.(1,2,4,7)

LIMITATIONS

Upon subculture, all pink to red, lecithinase-positive colonies growing on MYP Agar should be confirmed using the appropriate recommended media for complete identification. (7)

The method described is intended primarily for use in the routine examination of foods as described by the AOAC and APHA.(1,2,4,7) Confirmatory tests outlined in the literature may be inadequate for distinguishing all B. cereus strains from culturally similar microorganisms found in foods. Therefore, results from atypical strains may be variable, and further testing is needed to identify isolates.

At present, commercially available toxin tests are not recommended for identification, pending further evaluation. Until reliable tests become available, cultural methods such as those described and listed in the literature should be relied upon for confirming B. cereus isolates from foods.(1,2,4,7)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, such as Butterfield's Phosphate Buffer (Cat. no. U150), MYP Agar (Cat. no. G147) and Nutrient Agar (Cat. no. L20), incinerators, blenders, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacillus cereus
ATCC ® 13061
A 24hr 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Andrews, W.H. 2001. Committee on microbiology and extraneous materials. General Referee Reports: J. AOAC Intern. ; 84(1):243-250.

4. Association of Official Analytical Chemists. Official Methods of Analysissm, AOAC, Washington, D.C.

5. Lancette, G.A. and S.M. Harmon, 1980. Enumeration and confirmation of Bacillus cereus in foods: collaborative study. J. Assoc. Off. Anal. Chem.; 63(3):581-586.

6. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm.


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