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BROTHS WITH 15% GLYCEROL

Cat. no. D02 TSB (Tryptic Soy Broth) with 15% Glycerol, 15x45mm Tube, 2ml 10 tubes/box
Cat. no. D04 Brucella Broth with 15% Glycerol, 15x45mm Tube, 2ml 100 tubes/box
Cat. no. D08 TSB (Tryptic Soy Broth) with 15% Glycerol, 15x45mm Tube, 2ml 100 tubes/box
Cat. no. D09 TSB (Tryptic Soy Broth) with 15% Glycerol, 15x45mm Cryovial, 2ml 100 tubes/box
Cat. no. D114 Brain Heart Infusion (BHI) Broth with 15% Glycerol, 15x45mm Tube, 2ml 100 tubes/box
Cat. no. R34 TSB (Tryptic Soy Broth) with 15% Glycerol, 13x100mm Tube, 2ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Brucella, BHI, and TSB Broth with 15% Glycerol are recommended for the preservation of organisms by freezing.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Brucella Broth is composed of peptones, yeast extract, dextrose, sodium bisulfite and sodium chloride. Nitrogenous nutrients, including amino acids, are provided by peptones; yeast extract serves as a source of vitamins; dextrose supplies carbon and serves as an energy source; sodium bisulfite acts as a reducing agent and sodium chloride serves to maintain osmotic equilibrium.

By adding brain tissue to Dextrose Broth, Rosenow discovered a medium useful in the cultivation of streptococci. (9) Formula modifications of the BHI Broth were made by various researchers who found the medium effective in the recovery of dental pathogens. (7-10) The current formulat employs calf brain, which is equivalent in nutritional value but provides a clearer medium. Additionally, a variety of supplements have been added to further enhance the recovery of fastidious microorganisms.

Tryptic Soy Broth is widely used for the cultivation of microorganisms from environmental sources: supporting a wide variety of bacteria and fungi. Tryptic Soy Broth, also known as Soybean-Casein Digest, conforms to the formula given by the U.S. Pharmacopeia. (11) This medium contains digests of soybean meal and casein, which provide amino acids and other nitrogenous substances, making it a highly nutritious medium for a variety of organisms. Sodium chloride is added to maintain osmotic equilibrium. Dextrose is incorporated as an energy source. Dipotassium phosphate is included in the formulation as a buffer to maintain pH.

A 15% concentration of glycerol is added to the broths to maintain viability of microorganisms and to protect cell membranes. Glycerol acts as a cryoprotective agent and allows for long-term bacterial preservation. (4)

FORMULA

Ingredients per 850.0ml of deionized water:*

Brucella Broth with 15% Glycerol
Pancreatic Digest of Casein 10.0gm
Peptic Digest of Animal Tissue 10.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Dextrose 1.0gm
Sodium Bisulfite 0.1gm
Glycerol 150.0ml

Final pH 7.0 +/- 0.2 at 25ºC.

BHI Broth with 15% Glycerol
Calf Brain-Beef Heart Infusion 17.5gm
Pancreatic Digest of Gelatin 10.0gm
Sodium Chloride 5.0gm
Disodium Phosphate 2.5gm
Dextrose 2.0gm
Glycerol 150.0ml

Final pH 7.4 +/- 0.3 at 25ºC.

Tryptic Soy Broth (TSB) with 15% Glycerol
Pancreatic Digest of Casein 17.0gm
Sodium Chloride 5.0gm
Papaic Digest of Soybean Meal 3.0gm
Dextrose 2.5gm
Dipotassium Phosphate 2.5gm
Glycerol

150.0ml

Final pH 7.3 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store product at 2-30ºC. Products should not be used if there are any signs of contamination, deterioration, or if the expiration date has passed. Protect from light, excessive heat, and moisture.

PRECAUTIONS

PROCEDURE

1. Prepare an overnight culture of the organism to be stored on an appropriate non-selective medium, such as TSA (Cat. no. G60) or Blood Agar (Cat. no. A10). Do not use cultures older than 24-48 hours.

2. Using a sterile loop and aseptic technique, transfer several colonies into the vial of broth with 15% Glycerol, resulting in a dense suspension.

3. Shake or vortex well. Let the broth sit at room temperature for approximately 30 minutes before placing into long-term freezer storage.

4. Store at -50 or -70 degrees.

When placed in a -70ºC. freezer, the culture can be stored indefinitely. When stored at -50ºC., the culture should not be kept beyond one year. (4)

Recovery

1. Allow the vial to thaw by placing it at room temperature.

2. Under aseptic conditions, open the vial.

3. Using a sterile loop and aseptic technique, transfer several loop fulls from the vial of Brucella Broth, TSB, or BHI Broth with 15% Glycerol to appropriate plated culture media. Do not refreeze for later use.

See listed references for more information on storage of stock cultures. (1-4)

INTERPRETATION OF RESULTS

Growth of frozen stock culture on solid media indicates the presence of a viable organism.

LIMITATIONS

It may be necessary to perform additional biochemical testing if a mixed population of colonies appears upon propagation of frozen cell suspension.

Organisms from subcultures may be stored as stock strains, however, subcultured strains should be re-characterized at a regular interval to maintain purity. (4)

Data on cultures stores longer term at -20ºC shows impaired recovery. Thus, freezing at -20ºC is not recommended.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922
E 24-48hr 35°C Aerobic Growth upon subculture to TSA after overnight incubation at 35ºC.
Staphylococcus epidermidis
ATCC ® 12228
E 24-48hr 35°C Aerobic Growth upon subculture to TSA after overnight incubation at 35ºC.
Streptococcus pyogenes
ATCC ® 19615
E 24-48hr 35°C Aerobic Growth upon subculture to Blood Agar, 5% after overnight incubation at 35ºC.

User Quality Control

PHYSICAL APPEARANCE

Brucella Broth with 15% Glycerol should appear clear, and amber in color.
BHI Broth with 15% Glycerol should appear clear, and medium amber in color.
Tryptic Soy Broth (TSB), with 15% Glycerol should appear clear, and light amber in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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

7. Falk, C.R., et al. 1939. J. Bacteriol.;37:121.

8. Haden, R.L. 1932. Arch. Internal Med.; 32:828.

9. Hitchens, A.P. 1921. J. Infect. Disease; 29:390.

10. Rosenow, E.C. 1919. J. Dental Research;1:205.

11. The Official Compendia of Standards, 2008. USP27-NF22. United States Pharmacopeial Convention, Rockville, MD.

ATCC is a registered trademark of the American Type Culture Collection.

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