GN BROTH

Cat. no. K01 GN Broth, 15x103mm Tube, 6ml 20 tubes/box
Cat. no. K39 GN Broth, 16x125mm Tube, 10ml 20 tubes/box
Cat. no. R76BX GN Broth, 12x80mm Tube, 4ml 100 tubes/box

INTENDED USE

Hardy Diagnostics GN (Gram-Negative) Broth is recommended for the selective enrichment of Shigella and Salmonella spp. in clinical and non-clinical specimens.

SUMMARY

GN Broth was originally developed by Hajna to improve the recovery of Salmonella and Shigella from clinical and non-clinical specimens. Hajna recommended specimens be enriched in GN Broth 1-6 hours prior to plating on a solid medium.(7) Hajna's formulation employed an increased amount of mannitol over dextrose. This formulation produced an accelerated growth of Salmonella and Shigella while limiting the growth of Proteus spp. and Pseudomonas aeruginosa. Inhibitory chemicals in the medium allow normal fecal flora to be maintained in a prolonged lag phase. The Shigella and Salmonella are less inhibited and enter a log or stimulated phase of growth during the first few hours of incubation.

Casein and meat peptones provide amino acids and other nitrogenous substances to support bacterial growth. Dextrose and mannitol supply the energy source. The pH of the medium is maintained by phosphate buffers and osmotic equilibrium is maintained by sodium chloride. Gram-positive microorganisms and early multiplication of coliforms are both inhibited by sodium citrate and sodium deoxycholate.

Direct inoculation of rectal swabs on plating media, compared to plating after 6-8 hours of incubation on GN Broth, was first reported by Craft and Miller.(9) Their results showed that more isolates of Shigella were obtained when using the GN Broth. Taylor and Schelhart reported that a greater frequency of isolation of Salmonella and Shigella was obtained when enrichment with GN Broth was used as opposed to direct plating.(8) Taylor and Harris compared various enrichment broths for their ability to support the growth of Shigella species.(10) They reported GN Broth more satisfactory than Silliker Broth, Selenite Broth or Tetrathionate Broth for propagation and recovery.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 10.0gm
Peptic Digest of Animal Tissue 10.0gm
Sodium Chloride 5.0gm
Sodium Citrate 5.0gm
Dipotassium Phosphate 4.0gm
Mannitol 2.0gm
Monopotassium Phosphate 1.5gm
Dextrose 1.0gm
Sodium Deoxycholate 0.5gm

Final pH 7.0 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or 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

Specimen Collection: Consult listed references for information on specimen collection.(1-4,6) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Specimens should be delivered to the laboratory within 2-3 hours. Special attention is required for stools. They should be collected early in the course of the disease and need to be cultured within two hours after collection. Due to their delicate nature, Shigella species are best recovered by inoculating the media directly at the bedside. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Method of use:

1. Place 1.0gm of feces or 1ml of liquid stool in tube. Swab specimens may be inserted directly into the broth.

2. Emulsify the specimen thoroughly.

3. Incubate aerobically for six to eight hours at 35ºC.

4. Place one to two drops of the incubated broth onto selective plate media, such as MacConkey or Hektoen Enteric Agar and streak for isolated colonies.

5. Incubate aerobically at 35ºC.

6. Examine for pathogens in 18-24 hours.

INTERPRETATION OF RESULTS

Culture analysis is made from the media to which the enriched specimen is subcultured. Consult listed references for the interpretation of growth and other identification tests to identify growth of organism in the medium to which subculture has been made.(1-6)

LIMITATIONS

GN Broth, due to its low concentration of deoxycholate, is partially inhibitory to E. coli and other coliforms. The coliforms will eventually begin to overgrow the pathogens. Subculturing within eight hours after initial inoculation is necessary for optimal recovery of pathogens.

GN Broth does not encourage the growth of Shigella dysenteriae.

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
Shigella sonnei
ATCC ® 9290
I 24hr 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
I 24hr 35°C Aerobic Growth
Salmonella enterica
ATCC ® 14028
I 24hr 35°C Aerobic Growth

Note: GN Broth is inoculated with organism, incubated for 6-8 hours, then subcultured to MacConkey.

User Quality Control

PHYSICAL APPEARANCE

GN Broth should appear clear, and 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. IIsenberg, 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. Hajna, A.A. 1955. Public Health Lab.; 13:83.

8. Taylor, W.I. and D. Schelhart. 1968. Applied Microbiology; 16:1383.

9. American Journal of Clinical Pathology; 26:411, 1956.

10. American Journal of Clinical Pathology; 44:426, 1965.


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

012617gr