T.S.C. Agar Base (Tryptose Sulfite Cycloserine) ISO

Description

Cycloserine does not tolerate temperatures above 100 °C and its stability in solution is variable. Therefore, it is advisable to prepare the exact number of plates that are going to be used.

Technique

To use, the contents of the bottle should be poured into plates. The melting of the culture medium should be carried out according to the manufacturer's instructions, either in a water bath (100 °C) or microwave oven. Add the cycloserine at a concentration of 400 mg/L before pouring the culture medium on the plates or tubes.

Never apply direct heat to melt a medium. The melting temperatures and times depend on the shape of the container, the volume of medium and the heat source. Before melting any medium, loosen the screw cap of the container to avoid breaking the container. The medium should be melted only once and used. Media with agar should not be melted repeatedly as their characteristics change with each remelting. Overheating should be avoided as much as prolonged heating, especially with regard to media with an acidic or alkaline pH. Once melted, pour the plates using aseptic techniques.

To inoculate, follow standard laboratory methods or the applicable norms: spiral plate method, streak plating, ecometric methods, dilution banks, spread plating, etc.

The standard procedure recommends surface inoculation of the samples or their dilutions and, once absorbed, pouring a second layer as a seal for anaerobiosis. After incubation at 44 ± 1 °C for 21 ± 3 h, proceed to enumerate the black colonies that appear on the plate.

Note: the solid media can be melted in different ways: autoclave, bath and, if the customer considers it appropriate, also the microwave. Whenever the microwave option is chosen, it is necessary to take certain safety measures to avoid breaking of the containers, such as loosening the screw cap and putting the bottle or tube in a water bath in the microwave. The fusion temperature and time will depend on the shape of the container, the volume of medium and the heat source. Avoid overheating as well as prolonged heating periods.

LC-4661, 250 mL bottles

The three differential characteristics are sulfite reduction, growth at 46 °C and cycloserine resistance.

A solution of cycloserine in phosphate buffer at pH 8.0 may be prepared (dipotassium phosphate 16.73 g/L and monopotassium phosphate 0.52 g/L) and, if it is kept refrigerated, it can be used for approx. 5 days.

The standard procedure recommends surface inoculation of the samples or their dilutions and, once absorbed, pouring a second layer as a seal for anaerobiosis. After incubation at 44 ± 1 °C for 23 ± 1 h, proceed to enumerate the black colonies that appear on the plate.

Note: an alternative method would be the use of TSC Base Medium + Selective Supplement MUP (25 mg) / 200 mL medium. This reagent allows the identification of Cl. perfringens by its fluorescence.

LC-4728, membrane filtration plates

The three differential characteristics are sulfite reduction, growth at 46 °C and cycloserine resistance.

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

Filter the sample through a 0.45 µm pore membrane and apply it onto the surface of the agar.

Alternatively, a thin layer of molten TSC agar or agar can be used as an overlay on the membrane.

Incubate the plates anaerobically at 44 ± 1 °C for 21 ± 3 h.

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

After incubation, enumerate the colonies with a black iron sulfide precipitate.

Confirmation of characteristic colonies as C. perfringens is required, through further microbiological or biochemical tests.

Add 1 vial of D-Cycloserine Selective Supplement (40 mg).

LC-4728, membrane filtration plates

Physical/chemical control

Microbiological control

Before addition of cycloserine; quality control according to ISO 11133:2014/Amd 1:2018.

Melt medium - prepare plates - spiral spreading: practical range 100 ± 20 CFU; min. 50 CFU (productivity).

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Anaerobiosis. Incubation at 44 ± 1 °C for 21 ± 3 h.

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

A double layer with TSC agar favors the observation of the blackening of the H₂S (+) strains.

LC-4661, 250 mL bottles: physical/chemical control

LC-4728, membrane filtration plates: quality control

Membrane filtration / practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity) / ≥ 10³ CFU (specificity).

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

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Anaerobiosis. Incubation at 44 ± 1 °C for 21 ± 3 h.

A double layer with TSC agar favors the observation of the blackening of the H₂S (+) strains.

Sterility control

Incubation 48 h at 30-35 °C and 48 h 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.
• DIN Standard 10165. Referenz Verfahren fur Bestimmung von Clostridium perfringens. Fleisch und Fleischerzeugnissen.
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed. American Public Health Association. Washington.
• DIRECTIVA 2015/1787/UE de la Comisión por la que se modifica la Directiva 98/83/CE relativa a la calidad de las aguas destinadas al consumo humano (DO L260 de 7.10.2015 pg 6 y ss).
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International Inc. Gaithersburg. MD.
• ISO 7937 (2004) Microbiology of Food and Animal Feeding Stuffs. Horizontal Method for Enumeration of C. perfringens. Colony-count technique.
• ISO 6461-2 (1986) Water Quality - Detection and enumeration of the spores of sulfite-reducing anaerobes (Clostridia) - Part 2: Method by Membrane Filtration.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• ISO 14189 (2013) Water quality. Enumeration of Clostridium perfringens - Method using membrane filtration.
• SMITH, L.D. (1981) Clostridial Anaerobic Infections, in Diagnostic Procedures for Bacterial Mycotic and Parasitic Infections. 6th ed. APHA. Washington.
• UNE-EN ISO 11133 (2014). Microbiología de los alimentos para consumo humano, alimentación animal y agua - Preparación, producción, conservación y ensayos de rendimiento de los medios de cultivo.