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LAURYL TRYPTOSE BROTH (LAURYL SULPHATE BROTH)

Code: CM0451

a medium for the detection of coliform organisms in water and waste water, according to the formula of the American Public Health Association

Directions
Dissolve 35.6g in 1 litre of distilled water and distribute into containers with fermentation tubes (Durham). Sterilise by autoclaving at 121°C for 15 minutes.

Description
Lauryl Tryptose Broth provides a selective medium which is used for the detection of coliform organisms in water, dairy products and other foods. The APHA¹ recommend that Lauryl Tryptose Broth should be used for the Most Probable Number (MPN) presumptive test of coliforms in waters, effluent or sewage as a confirmatory test of lactose fermentation with gas production for milk samples ( APHA² ) and for the detection of coliforms in foods ( APHA³ ).

Surface active agents have long been used as the inhibitory ingredients in selective media. MacConkey⁴ introduced bile salts for this purpose and later Albus and Holm³ working with lactobacilli found that sodium ricinoleate exerted a selective action. The development of synthetic wetting agents opened up new fields of investigation. Mallmann and Darby⁶ after comparative tests with a large number of these compounds, showed that sodium lauryl sulphate gave the best results in selective media for the coliform group.

Lauryl Tryptose Broth was designed to promote a rich growth and copious gas production from small inocula of coliform organisms. Aerobic sporing bacteria are completely inhibited. The advantage in using this product is that in addition to giving the fermentation reaction typical of MacConkey Broth it can also be directly tested for the presence of indole. Unlike MacConkey Broth, the medium contains no indicator, but this can be added (if required) after incubation.

Technique
Lauryl Tryptose Broth is recommended for the detection and enumeration of coliform organisms in water and milk products, especially in the control of ice-cream manufacture and in dairy hygiene. A suggested procedure ( Dyett⁷ ) is as follows:

1. Inoculate samples of ice cream into tubes of Lauryl Tryptose Broth in the manner normally employed in the MacConkey test. Examine the tubes after overnight incubation at 35°C and, if no gas is visible, examine again at the end of 48 hours’ incubation.
2. From every tube showing fermentation (primary fermentation), two further tubes of Lauryl Tryptose Broth are inoculated, and these are incubated at 35°C and 44°C respectively. It is advisable that the tube to be incubated at 44°C be warmed in a water bath at this temperature before inoculation.
3. After the two tubes of Lauryl Tryptose Broth have been inoculated for secondary fermentation, test the original primary fermentation tube (which was inoculated directly with ice cream) for indole production. A positive reaction suggests the presence of Escherichia coli and confirmation will be obtained later with the secondary fermentation from the 44°C bath. A negative indole reaction in the primary fermentation tube indicates the absence of E. coli.
4. Check the 44°C incubated secondary broth for fermentation after seven hours. If positive, test for indole production with either Ehrlich or Kovac’s reagent. Due to the lauryl sulphate present, shaking the reagent culture mixture forms a persistent emulsion which interferes with the test. This may be avoided by shaking with ether, which separates rapidly, and then adding Kovac’s reagent to the layer without shaking.
5. If fermentation has not occurred after seven hours, leave the tube overnight at 44°C and test the following day. A positive indole reaction in a broth that has produced gas at 44°C indicates the presence of E. coli.
6. The tube at 35°C is incubated for 24 hours. If no fermentation occurs, the primary fermentation is assumed to be due to organisms other than coliforms. False positives are not uncommon in the primary fermentation tubes, due to fermentation of the sucrose in the added ice cream by organisms other than coliforms.
   

The addition of 4-methylumbelliferyl-ß-D-glucuronide (MUG) (BR0071) to this medium will enhance the detection of E. coli. Lauryl Tryptose Broth with MUG is also available from Oxoid (CM0980). The use of this medium in a Most Probable Number (MPN) technique for enumeration of presumptive Escherichia coli in milk and milk products has been specified in a standard procedure⁸.

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: Straw coloured solution

Quality control

* This organism is available as a Culti-Loop®

Precautions
If stored at 2-8°C the broth will become cloudy or form a precipitate. This should clear at room temperature but gas formation is the criterion of growth not turbidity.

References
1. American Public Health Association (1980) Standard Methods for the Examination of Water and Wastewater. 15th Edn. APHA Inc. Washington DC.
2. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc. Washington DC.
3. American Public Health Association (1976) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington DC.
4. MacConkey A. T. (1938) J. Hyg. 8. 322-334.
5. Albus W. R. and Holm G. E. (1926) J. Bact. 12. 13-18.
6. Mallmann W. L. and Darby C. W. (1941) Am. J. Pub. Hlth. 31. 127-134.
7. Dyett E. J. (1957) Lab. Prac. 6(6). 327-328.
8. ISO Standard 11866-2 Milk and Milk Products - Enumeration of presumptive Escherichia coli - part 2: Most probable number technique using 4- methylumbelliferyl-ß-D-glucuronide.