TRYPTOPHAN BROTH

INTENDED USE Tryptophan broth allow the culture of microorganisms presenting no particular growth requirements. This medium is used most often in water analyses for the identification of Escherichia coli by indole production.

PRINCIPLES Under aerobic conditions, Escherichia coli degrades tryptophan to indole by through the action of a tryptophanase. The indole produced is revealed by Kovacs reagent.

PREPARATION Suspend 16,0 g of dehydrated medium (BK163) in 1 liter of distilled or demineralized water. Slowly stir until complete dissolution. Dispense in tubes by adding 3 to 5 mL per tube. Sterilize in an autoclave at 121C for 15 minutes.

INSTRUCTIONS FOR USE - For the identification of Escherichia coli, transfer a loop of a typical culture from the selective solid media used to a tube of Tryptophan broth prepared as described, or into a tube of ready-to-use medium BM076. - Incubate at (44.0 0.5)C in a temperature-controlled water bath for (21 3) hours if application of the standard NF EN ISO 9308-1 is respected. - Incubate at 37C for 24 hours if the procedure outlined in standards ISO 6785/IDF 93, NF EN ISO 16654, NF EN ISO 6579 and/or NF EN ISO 21567 are applied.

RESULTS After incubation of the inoculated tubes, the presence of indole is revealed by the presence of a red color in the alcohol phase of the Kovacs reagent that has been added in quantities of 0.2 to 1.0 mL per tube. NOTE : Kovacs reagent : p-dimethylaminobenzaldehyde ........... 5 g Amyl alcohol .................................. 75 mL Hydrochloric acid ........................... 25 mL

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- Dissolve p-dimethylaminobenzaldehyde in normal amyl alcohol in a temperature-controlled water bath at 60C. Cool and slowly add HCl drop by drop, keeping the recipient in cold water. Store between 2-8C in a tightly sealed, inactinic container. - The reaction is immediate : indole positive : red ring indole negative : brownish ring (color or the reagent)

TYPICAL COMPOSITION (can be adjusted to obtain optimal performance) For 1 liter of medium : - Tryptone .........................................................................................10.0 g - L-Tryptophan....................................................................................1.0 g - Sodium chloride ...............................................................................5.0 g pH of the ready-to-use medium at 25C : 7.5 0.2.

QUALITY CONTROL - Dehydrated medium : cream powder, free-flowing and homogeneous. - Prepared medium : clear, limpid amber solution. - Typical culture response after 21 hours of incubation at 44C (XP CEN ISO 11133-2, qualitative method) : Microorganisms Escherichia coli Escherichia coli Salmonella Typhimurium 1 Escherichia coli
1

Growth (turbidity) good, score 2 good, score 2 good, score 2 good

Indole production positive positive negative positive

ATCC 25922 ATCC 8739 ATCC 14028 RIVM WR1

according to NF T 90-461, qualitative method

STORAGE / SHELF LIFE Dehydrated medium : 2-30C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Media in tubes : 6 months at 2-8C. Ready-to-use media in tubes : - Store between 2-25C, shielded from light. - The expiration date is indicated on the label.

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PACKAGING Ready-to-use media in tubes : - 50 x 3 mL Dehydrated media : - 500 g bottle

Code

BM07608 BK163HA

PHOTO SUPPORT : Product reference : BK163HA, BM07608 Media used for : Confirmation of E. coli through indole production.

Salmonella spp.

Escherichia coli

Negative control

Escherichia coli (indole positive ; center tube) Tryptophan broth Ref : BK163HA Incubation : 24 hours / 37C (NF EN ISO 16654) Characteristics : Indole production in E. coli is characterized by a red ring on the surface of the broth after addition of Kovacs reagent. Negative indole remains colorless to yellow.

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BIBLIOGRAPHY
NF EN ISO 9308-1 (T 90-414). Septembre 2000. Qualit de leau. Recherche et dnombrement des Escherichia coli et des bactries coliformes. Partie 1 : Mthode par filtration sur membrane. ISO 6785 / IDF 93. Mai 2001. Lait et produits laitiers. Recherche de Salmonella spp. NF EN ISO 16654 (V 08-032). Juillet 2001. Microbiologie des aliments. Mthode horizontale pour la recherche des Escherichia coli O157. NF EN ISO 6579 (V 08-013). Dcembre 2002. Microbiologie des aliments. Mthode horizontale pour la recherche des Salmonella spp. NF EN ISO 21567 (V 08-411). Mars 2005. Microbiologie des aliments. Mthode horizontale pour la recherche de Shigella spp.

*Benchmark value refers to the expected value under standard laboratory conditions following manufacturers instructions. It is provided as a guide only and no warranty, implied or otherwise is associated with this information.

The information provided on the package take precedence over the formulations or instructions described in this document. The information and specifications contained in this technical data sheet date from 2009-02-17. They are susceptible to modification at any time, without warning. Code document : BK163/A/2001-09 : 6.

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