Chapman TTC Agar (Tergitol 7 Agar)

Technique

While using the membrane filter technique for the presumptive identification of coliforms in water, it should be kept in mind that the minimum volume to be filtered depends on the type of water being tested. If necessary, dilute with sterile phosphate buffer in order to obtain the number of colonies on the membrane appropriate for counting.

For every water sample two volumes must be filtered over two different membranes and incubated on Chapman TTC Agar at 35 °C and 44 °C respectively.

After 48 hours typical colonies have the appearance as follows:

• Escherichia coli, Citrobacter spp.: yellow with a centred orange nucleus under the membrane filter (MF).
• Klebsiella spp.: brick red or yellow without a nucleus. The medium under the filter (MF) is yellow.
• Enterobacter spp.: dark yellow or brick red with an orange nucleus. The medium is also yellow.
• Non lactose-fermenters: violet or indigo colonies. The medium turns blue.

Most coliforms cannot grow on this medium when incubated at 44 °C, except E. coli, which forms a colony with a characteristic appearance.

Results are always expressed per 100 mL sample including any applied dilutions. Estimation is done by taking typical colonies which have grown at 35 °C as faecal coliforms, together with those grown at 44 °C as E. coli. Nevertheless, according to legislation and despite the medium's selectivity, results can only be considered as presumptive and all coliform colonies have to be confirmed by following the criteria below:

Typical appearance in EMB Agar or Endo Agar Base and characteristic reactions in Kligler Iron Agar.

For the confirmation of faecal E. coli, the following characteristics are used for verification: a motile, Gram-negative bacillus and lactose fermenter with acid and gas production, which gives negative results on the citrate test and positive results on indole production.

Collect, dilute and prepare samples and volumes to be filtered as required according to specifications, directives, official standard regulations and/or expected results.

Note: Incubation times greater than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications.

After incubation, enumerate the yellowish colonies as presumptive E. coli or any other coliform.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per membrane by the inverse dilution factor. Report results as colony forming units (CFU) per mL along with incubation time and temperature.

Confirmation of E. coli detection is required with further microbiological or biochemical tests.

Physical/chemical control

Microbiological control

Membrane filtration / practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU for selectivity.

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Aerobiosis. Incubation at 36 ± 2 °C, reading at 21 ± 3 h.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London.
• CHAPMAN G.H. (1951) A culture medium for detecting and confirming E. coli in ten hours. Am. J. Publ. Hlth 41:1381-1386.
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 3rd ed. APHA. Washington.
• GUINEA, SANCHO, PARES (1979) Análisis Microbiológico de Aguas. Ed. Omega. Barcelona.
• ISO 9308-1:2000 Standard. Water Quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method.
• SPECK, M (Ed.) (1982) Compendium of Methods for the Microbiological Examination of Foods. 2nd ed. APHA. Washington.