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Dehydrated Culture Media

TRYPTONE WATER

Code: CM0087

A liquid medium for the production of indole by micro-organisms.

Typical Formula*

gm/litre

Tryptone

10.0

Sodium chloride

5.0

pH 7.5 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Dissolve 15g in 1 litre of distilled water and distribute into final containers. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Tryptone Water is a good substrate for the production of indole because of its high content of tryptophan and it is more reliable than Peptone Water for this purpose. The ability of certain organisms to break down the amino-acid tryptophan with formation of indole is an important property which is used for the classification and identification of bacteria 1,2.

Technique

Kovac’s reagent:

paradimethylaminobenzaldehyde

5 grams

amyl alcohol

75ml

concentrated hydrochloric acid

25ml

1. Inoculate tubes of Tryptone Water, and incubate for 24-48 hours at 35°C.
2. Add 0.2ml of Kovac’s reagent and shake. Allow to stand for 10 minutes and observe the result.
A dark red colour in the amyl alcohol surface layer constitutes a positive indole test; no change in the original colour of the reagent constitutes a negative test.

Ehrlich reagent:

paradimethylaminobenzaldehyde

4 grams

absolute alcohol

380ml

concentrated hydrochloric acid

80ml

1. Inoculate tubes of Tryptone Water, and incubate for 24-48 hours at 35°C.
2. Withdraw a small portion of the culture and add an equal volume of the reagent. Shake the mixture and allow to stand for a few minutes; a rose colour indicates the presence of indole. If indole is present, this colour change may be accelerated by the addition of saturated potassium persulphate solution.

As an alternative, extract the indole by shaking the cultures with 1ml of ether, allow the mixture to stand for a few minutes and then add 0.5ml of Ehrlich reagent.

Incubation at 44°C for 24 hours has the specific advantage of detecting Escherichia coli, as this is the only organism present in water capable of producing indole at this temperature 3.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at room temperature.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Light straw coloured solution        

Quality control

Positive control:Expected results
Escherichia coli ATCC® 25922*Turbid growth; indole positive   
Negative control:  
Enterobacter aerogenes ATCC® 13048*Turbid growth; indole negative
* This organism is available as a Culti-Loop®

References
1. American Public Health Association(1998) Standard Methods for the Examination of Water and Wastewater. 20th Edn. APHA Inc. Washington DC.
2. Farmer J. J. III et al. (1985) J. Clin. Microbiol. 21. 46-76.
3. DHSS. Report 71 (1982) The Bacteriological Examination of Drinking Water Supplies. HMSO. London.

 
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