Violet Red Bile Lactose Agar (VRBL) 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.).

The plates are read after 24 hours of incubation at 30 °C.

The size of the colonies ranges from 2 to 5 mm, depending on the amount per plate. If enterococci develop, they will appear small in size and pink coloured. Lactose-fermenting enterobacteria acquire a dark red colour with a clearing zone around them, while lactose non-fermenting ones form colourless colonies.

LC-4532, 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 30 ± 1 °C for 18-24 h.

Note: Contact plates are used for monitoring the microbiological contamination of surface and air inside cleanrooms, isolators, RABS, food industries and hospitals. The double/triple irradiated wrapping ensures that the package itself doesn't contaminate the environment as the first wrapper is removed just before entering the clean area.

LC-4682 and LC-5161, bottles

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. 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 recommended procedure is inoculation directly into Petri dishes, with the molten agar cooled to 45-47 °C. Plates can be read after 24 hours of incubation at 30 °C.

The size of the colonies ranges from 2 to 5 mm, depending on the amount per plate. If enterococci develop, they will appear small in size and pink coloured. Lactose-fermenting enterobacteria acquire a dark red colour with a clearing zone around them, while lactose non-fermenting ones form colourless colonies.

LC-4682 and LC-5161, bottles

Physical/chemical control

Microbiological control

Spiral spreading (LC-4532: inoculate): practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity).

Aerobiosis. Incubation at 30 ± 1 °C during 18-24 h.

LC-4682 and LC-5161, bottles: microbiological control

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

Aerobiosis. Incubation at 30 ± 1 °C during 18-24 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.

• DOWNES, F.P. & K. ITO (2001). Compendium of Methods for the Microbiological Examination of Food. 4th ed. APHA, Washington. DC.
• FIL-IDF. (1998) Standard 73B. Enumeration of coliform bacteria. ICMSF (1978). Microorganisms in Food, University of Toronto Press.
• ISO (1986) Standard 5541-1 Milk and Milk Products. Enumeration of coliforms. Colony count technique at 30 °C.
• ISO (2006) Standard 4832:2006 (E) - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coliforms - Colony-count technique.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products. 16th ed. APHA, Washington. DC.
• PASCUAL ANDERSON, Mª R. (1992) Microbiología Alimentaria. Díaz de Santos, S.A., Madrid.