Lauryl Sulfate Broth (Lauryl Tryptose Broth - LTB) ISO

APHA recommends the use of Lauryl Tryptose Broth for the Most Probable Number Presumptive Test of coliforms in waters, effluent or sewage, also as a confirmatory test of lactose fermentation with gas production for milk samples, and for the detection of coliforms in foods. This broth was elaborated to promote a rich growth and high gas production from small inocula of coliform organisms.

Lauryl Sulfate Broth is recommended by the standards ISO 4831 and ISO 7251 for the detection and enumeration of coliforms and E. coli respectively, by the most probable number technique.

The coliform group is both aerobic and anaerobic, Gram-negative, non-spore forming, and ferments lactose producing acid and gas at 35 °C within 48 hours.

Tryptose in a 2% concentration improves the early growth phase of coliforms when compared to casein peptone. The buffered broth allows slow lactose fermenters to increase gas production in a shorter time.

Tryptose provides nitrogen, vitamins, minerals and amino acids essential for growth. Lactose is a fermentable complex carbohydrate energy source. Potassium phosphates are the buffering agents, and sodium chloride supplies essential electrolytes for transport and osmotic balance. Sodium lauryl sulfate is the selective agent used to inhibit organisms other than coliforms. Sporulating aerobic bacteria are completely inhibited.

Preparation

Suspend 35.6 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes with Durham gas collecting tubes for gas detection. Sterilize in autoclave at 121 °C for 15 minutes.

According to ISO 4831

For the detection and enumeration of coliforms according to ISO 4831:

1. Inoculate the tubes of selective enrichment broth (Lauryl Tryptose Broth) with the test portion.
2. For inocula of 1 mL or less, use single strength LTB medium.
3. For inocula of 10 mL or more, use double strength LTB medium.
4. Incubate at 30 °C or 37 °C (as agreed) for 24 h (double strength medium) or for 24 h and another additional 24 h if neither gas formation nor opacity preventing the detection of gas formation is observed at this stage (single strength medium).
5. From the incubated tubes of double strength LTB and the incubated tubes of single strength LTB showing gas formation or opacity, inoculate a tube of confirmation medium (Brilliant Green Bile Broth 2%, LC-1228) and observe after 24 or 48 h.
6. A tube with gas formation is a positive tube.
7. Count the total number of positive tubes.

According to ISO 7251

For the detection and enumeration of presumptive E. coli according to ISO 7251:

1. Inoculate the tubes of selective enrichment broth (Lauryl Tryptose Broth) with the initial suspension.
2. For inocula of 1 mL or less, use single strength LTB medium.
3. For inocula of 10 mL or more, use double strength LTB medium.
4. Incubate the tubes of LTB at 37 °C for up to 48 h, and examine the gas production after 24 h and 48 h.
5. Each tube of double strength LTB that has given rise to opacity, cloudiness or gaseous emission, and each tube of single strength LTB that has given rise to gaseous emission, is subcultured to a tube containing EC Broth (LC-1522).
6. Incubate the tubes of EC Broth at 44 °C for up to 48 h, and examine the gas production after 24 h and 48 h.
7. Each tube of EC Broth that has given rise to gaseous emission is subcultured to a tube containing Indole-Free Peptone Water (LC-1403) and incubated at 44 °C for 48 h.
8. Tubes showing opacity, cloudiness or gas production in LTB and whose subcultures have produced gas in EC Broth and indole in Peptone Water are considered as positive tubes containing presumptive E. coli.
9. Count the total E. coli by the MPN method.

• ISO 4831 Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of coliforms - Most probable number technique.
• APHA 1998. Standard Methods for the examination of water and wastewater, 20th Edition.
• Association of Official Analytical Chemists. 1995. Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
• Association of Official Analytical Chemists. 1995. Official methods of analysis of AOAC International, 16th ed. AOAC International, Arlington, VA.
• ISO 7251 Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of presumptive Escherichia coli - Most probable number technique.