Violet Red Bile Agar with Glucose (VRBG) EP/USP/ISO

Technique

For plate inoculation, follow the laboratory's standard methods or the applicable norms (spiral plating method, ecometric methods, streak plating, dilution banks, spread plating with Drigalski rod, etc.).

Violet Red Bile Dextrose Agar is widely used in the analysis of food, medicines and cosmetics. It is particularly indicated for the recovery of bacteria which have been damaged during preparation. In such cases, a progressive enrichment is recommended in TSB and subsequently in EE Broth. The enriched culture can be inoculated in tubes or on Violet Red Bile Dextrose Agar plates. For a count of enterobacteria, follow the technique described for Violet Red Bile Agar.

Results can be read after 24 hours of incubation at 37 ± 1 °C. Enterobacterial colonies are an intense purple colour surrounded by a clearer zone. If enterococci colonies eventually develop, they will be small and pink coloured.

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

LC-911, 90 mm plates

Results can be read after 24 hours of incubation at 35 ± 2.0 °C. Enterobacterial colonies are an intense purple colour surrounded by a clearer zone. If enterococci colonies eventually develop, they will be small and pink coloured.

LC-4524, contact plates

Contact plates are used in the microbiological control of disinfection and cleaning of surfaces. They act simultaneously as a sampler and incubation culture medium without the need for any other intermediate steps.

The plates come in a form appropriate for this function and can be used with different culture media depending on the type of microbe that needs to be controlled. On average the plates provide a contact surface of approximately 25 cm².

To use, remove the cover and gently press the culture medium on the surface to be controlled, ensuring contact between the two surfaces. The contact plate is removed and covered with the lid to prevent air contamination. It is advisable that the lid is secured with adhesive tape and the bottom labelled with the sampling data (place, date and time).

If the sample surfaces are rough, the contact plates will not make good contact, even when the pressure is increased. In these cases it is advisable to delineate a sample surface area of 25 cm² and rub this area vigorously with a wet sterile swab and then rub the swab over the contact plate.

If verifying the effectiveness of a cleaning or disinfection process, contact plates should be used within two hours after the end of the process, ensuring that the sample surface is dry. It is advisable to always include positive controls, sampling the area before disinfection or dirty areas beside the disinfected area.

The technician will determine the frequency of sampling and disinfection according to performance criteria. Apply the agar directly onto the surface to be monitored ensuring that the pressure is distributed over the whole plate for 10 seconds. Clean the surface where the sample was collected in order to remove any traces of agar.

The inoculated plates are incubated at 35 ± 2 °C for 24 ± 2 h.

Note: Contact plates are used for monitoring the microbiological contamination of surface and air inside cleanrooms, isolators, RABS, food industries and hospitals.

LC-4670 and LC-5158, bottles

This medium can be used as a presumptive medium for E. coli (by fluorescent reaction) if MUG is added before sterilization.

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

Melt the medium contained in the bottles in a water bath (100 °C) or in a microwave oven, avoiding overheating, before pouring into Petri dishes when cooled to room temperature.

Once solidified on a flat surface, spread the plates by streaking methodology or by spiral method. Incubate the plates right side up aerobically at 35 ± 2.0 °C for 24 h.

(Incubation times longer than those mentioned above, different inoculation methods or different incubation temperatures may be required depending on the sample, on the specifications, etc. This medium can be inoculated directly or after any enrichment broth.)

After incubation, enumerate all the reddish-violet colonies that have appeared on the surface of the agar, with a red-violet halo due to bile salts precipitation.

Presumptive isolation of E. coli or coliforms must be confirmed by further microbiological and biochemical tests.

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, that enables to differentiate total coliforms and faecal coliforms.

Physical/chemical control

Microbiological control

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

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

Aerobiosis. Incubation: 30-35 °C, reading at 24 h (E.P.) / 37 ± 1 °C, reading at 24 h (ISO).

Note: results ATCC 8739/6538/9027 (30-35 °C) and ATCC 8739/25922/19433/14028 (37 °C).

LC-911, LC-4524, LC-4670 and LC-5158: microbiological control

LC-911 and LC-4524: inoculate with 10-100 CFU according to the harmonized Pharmacopoeias or with 10⁴-10⁶ CFU for selectivity.

LC-4670 and LC-5158: 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: 30-35 °C, reading at 24 h (E.P.) / 37 ± 1 °C, reading at 24 h (ISO).

Note (LC-911, LC-4670 and LC-5158): results ATCC 8739/6538/9027 at 30-35 °C, rest at 37 °C.

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.

• EUROPEAN PHARMACOPOEIA 8.0 (2014) 8th ed. § 2.6.13. Microbiological examination of non-sterile products: Test for specified microorganisms. Harmonised Method. EDQM. Council of Europe. Strasbourg.
• ISO 21528-1:2004. Microbiology of food and animal feeding stuffs - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 1: Detection and enumeration by MPN technique with pre-enrichment.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• MOSSEL, D.A.A. (1985) Media for Enterobacteriaceae. Int. J. Food Microbiol. 2:27-35.
• MOSSEL, D.A.A., H. MENGERINK & H.H. SCHOLTS (1962) Use a Modified MacConkey Agar Medium for the selective growth and enumeration of all Enterobacteriaceae. J. Bact. 84:381.
• MOSSEL, D.A.A., M. VISER & A.M.R. CORNELISSEN (1963) The examination of foods for Enterobacteriaceae using a test of the type generally adopted for the detection of salmonellae. J. Appl. Bact. 26:444-452.
• MOSSEL, D.A.A. & M.A. RATTO (1970) Rapid detection of sub-lethally impaired cells of Enterobacteriaceae in dried foods. Appl. Microbiol. 20:273-275.
• PASCUAL ANDERSON, Mª R. (1992) Microbiología Alimentaria. Díaz de Santos, S.A. Madrid.
• USP 33 - NF 28 (2011) <62> Microbiological examination of non-sterile products: Test for specified microorganisms. Harmonised Method. USP Corp. Inc. Rockville. MD. USA.