TTC 1% Supplement

Description

This dye is colorless in the oxidized form and red when reduced by live microorganisms, due to formation of formazan, an insoluble red pigment which is kept inside granules in the cells. Depending on TTC concentration, it may have a very little inhibition of microbial growth in Gram-positive organisms.

Technique

Aseptically reconstitute 1 vial with 5 mL of sterile distilled water. Mix gently until complete dissolution and aseptically add 2.5 mL to 1000 mL of TTC Chapman Agar (LC-1076), autoclaved and cooled to 45-50 °C. Mix well and distribute into sterile containers.

When the TTC is required to be added to other media, like KF Streptococcal Agar (LC-1034) or KF Streptococcal Agar with Bromocresol Purple (LC-1101), refer to the specific instructions of the medium for the quantity of TTC 1% Supplement that should be added.

Instructions for use

For the detection and enumeration of Escherichia coli and coliform bacteria in water samples:

• Filter two samples of water over two different membranes and incubate on TTC Chapman Agar (LC-1076) at 36 ± 2 °C and 44 ± 4 °C respectively for 21 ± 3 hours.

Typical colonies have the appearance as follows:

• E. coli and Citrobacter spp. present yellow colonies with orange-colored center.
• Enterobacter spp. forms red colored colonies and dark yellow with orange-colored center. The medium is yellow.
• Klebsiella spp. form red colored or yellow colonies, but without center. The medium is yellow.
• Lactose non-fermentative bacteria grow with purple colonies and change the medium to blue.

Klebsiella and Enterobacter species can also produce yellow-green colonies.

The results will always refer to counts per 100 mL of sample, considering if it has been necessary to make dilutions.

• Count as lactose-positive bacteria the colonies that present a yellow development of the medium under the membrane.
• Subculture the characteristic colonies obtained, in non-selective agar and Tryptophan Culture Broth (LC-1237).
• Carry out the oxidase test and incubate the tubes of Tryptophan Culture Broth at 44 ± 0.5 °C for 21 ± 3 hours.
• Indole production is determined by adding a few drops of Kovac's Reagent (LC-5205) to the incubated Tryptophan Culture Broth tubes. A positive test is indicated by the development of red color in the reagent layer.
• The colonies that are oxidase negative will be considered as coliform bacteria and the colonies that are oxidase negative and indole positive will be considered as E. coli.

Reconstitute the original freeze-dried vial by adding 5 mL of sterile distilled water.

Note: each vial is sufficient to supplement 2 L of Chapman Agar.

Physical/chemical control

Microbiological control

Reconstitute 1 vial as indicated in Composition; shake and dissolve completely.

Add 1 vial to 2 L of medium base. DO NOT HEAT once supplemented.

Cool the medium to 50 °C and pour into MF dishes.

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

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.

• Kennor, G.A., Clark, H.F. and Kabler, P.W. (1961) J. Appl. Microbiol. 9: 15-20.
• Slanetz, L.W. and Bartley, C.H. (1957) J. Bact. 74: 591-595.
• Chapman, G.H. (1947) J. Bact. 53: 504.
• Nordic Committee on Food Analysis (1968) Leaflet 68.