Letheen Agar Modified

Description

In 1965 the methodology was accepted by the AOAC for the antimicrobial assays and extends their use to all the cationic surfactants (detergents). The TAT (Tryptone-Azolectin-Polysorbate) medium, in the Newburger Cosmetic Analysis Manual (2nd ed., 1977), is similar in composition and uses the AOAC formulation. In 1978 the FDA (Bacteriological Analytical Manual, 5th ed., 1978) incorporated it as primary presumptive and enrichment medium for all microbial examinations of cosmetics.

The present formulation appears in the 8th ed. (1998) of the BAM and the notable modifications are the inclusion of sodium chloride providing suitable osmotic pressure and an increased amount of peptones and tissue extracts to promote good growth; these transform this medium into a very rich all-purpose medium suitable for neutralizing almost all preservatives present in samples for examination.

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. 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.

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

Spread the plates by streaking methodology or by spiral method. Incubate the plates right side up aerobically at 35 ± 2 °C for 24-48 h.

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

Each laboratory must evaluate the results according to their specifications.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor if a diluted sample was streaked. Report results as colony forming units (CFU) per mL or g along with incubation time and temperature.

Physical/chemical control

Microbiological control

Melting - pour plates - inoculation: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity).

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

Aerobiosis. Incubation at 32.5 ± 2.5 °C, reading after 24-72 hours.

Sterility control

Incubation 24 h at 30-35 °C and 72 h at 20-25 °C: NO GROWTH.

Incubation 7 days at 30-35 °C: NO GROWTH.

• ASTM Standard E 640-78 (1991) Test Method for the preservatives in water-containing cosmetics. Philadelphia. PA. USA.
• ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London.
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. MD. USA.
• HORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaithersburg. MD. USA.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• LUCAS, I.P. (1977) Microbiological Examination of Cosmetics. Newburger's Manual of Cosmetic Analysis AOAC. Washington. USA.
• US PHARMACOPOEIA (2002) <61> Microbial Limit Tests. 25th ed. US Pharmacopeial Convention. Rockville. MD. USA.
• WEBER, G.R. & L.A. BLACK (1948) Relative efficiency of quaternary inhibitors. Soap and Sanit. Chem. 24:134-139.