Lysine assay Medium

Intended Use

Recommended for determining lysine concentration by microbiological assay method.

Composition

Final pH ( at 25°C): 6.7±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 10.5 grams in 100 ml of purified / distilled water. Heat to boiling to dissolve the medium completely. Mix well to distribute the slight precipitate evenly. Dispense in 5 ml amounts to each assay tube in increasing amounts of the standard or the unknown and total volume 10 ml per tube is adjusted by addition of distilled / purified water. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes.

Principle And Interpretation

Lysine Assay medium is formulated as described in Kavanagh (3). It contains all the essential growth factors for Pediococcus acidilactici ATCC 8042 except lysine. Exact concentration of lysine in the test material can be calculated by comparing results with standard curve of lysine.

Assay/Procedure: Stock cultures of the test organism Pediococcus acidilactici ATCC 8042, are prepared by stab inoculation into Lactobacilli Agar AOAC. Incubate the cultures at 35-37°C for 16-24 hours. After incubation, centrifuge aseptically and decant the supernatant. The pellet is washed 3-4 times with 10 ml of 0.85% NaCl solution. Then resuspend the cells in 10 ml of 0.85% NaCl solution. Inoculate each tube aseptically with 1 drop of the inoculum.

It is essential that a standard curve be set up for each assay since conditions of autoclaving, temperature of incubation, etc. which influence the standard curve readings, cannot be duplicated exactly from time to time.

Increasing amounts of the standard or the unknown and make up the volume to 10ml per tube containing 5 ml of the rehydrated medium. The growth response is measured turbidometrically.

Type of specimen

Isolated Microorganism

Specimen Collection and Handling

For isolated microorganism samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards. (3)

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

NA

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Off-white to yellow homogeneous free flowing powder

Colour and Clarity of prepared medium: Light amber coloured clear solution, which may contain a slight precipitate.

Reaction: Reaction of 10.5% w/v aqueous solution at 25°C. pH : 6.7±0.2

pH: 6.50-6.90

Cultural Response: Microbiological Assay of Lysine was carried out using Pediococcus acidilactici ATCC 8042 after an incubation at 35-37° C for 16-20 hours.

Storage and Shelf Life

Store dehydrated and prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (1,2).

Reference

1. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
2. Jorgensen, J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
3. Kavanagh F., Analytical Microbiology Academic Press 1963, New York and London.