Glucose OF Medium

Intended Use

Recommended for the determination of oxidative and fermentative metabolism of carbohydrates by gram-negative bacteria. The composition and performance criteria of this medium are as per the specifications laid down in ISO 21528-1 : 2017, ISO 21528-2:2017, ISO / TS 11059 and ISO 11133:2014 / Amd.2 :2020 (E).

Composition**

Final pH (at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Enzymatic digest of casein

Directions

Suspend 20.38 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Dispense in tubes. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Note : Just before use, heat the medium in boiling water or flowing steam for 15 min to remove oxygen, then cool rapidly to the incubation temperature.

Principle And Interpretation

Hugh and Leifson developed OF Medium to study oxidative and fermentative metabolism of carbohydrates by gram-negative bacteria. This criterion is used during taxonomic studies of Enterobacteriaceae (1). Glucose is the most important carbohydrate for use in OF Basal Medium. Glucose OF Medium is recommended by ISO Committee (2-5).

However, certain organisms may metabolize other carbohydrates even if they are unable to utilize dextrose. Degradation of the carbohydrate to acid is indicated by the pH indicator bromothymol blue which changes its colour to yellow. Oxidative utilization takes place when the medium is exposed to air while fermentative utilization occurs under exclusion of air.

Tryptone in the medium provides the necessary carbon and nitrogen, vitamins etc required for bacterial growth. Phosphate buffers the medium and the low agar concentration determines motility and dispersion of the acid produced on the surface Bromothymol blue acts as the pH indicator. The low concentration of agar permits the determination of motility and aids in the even distribution of any acid produced at the surface of the medium. Motility is observed as diffused zone of flaring out from the line of inoculation. Non-motile organisms grow along the line of inoculation.

Type of specimen

Food samples : meat and meat products

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (2-5). 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

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Other biochemical tests must be performed for confirmation.

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: Cream to greenish yellow homogeneous free flowing powder

Gelling: Semisolid, comparable with 0.3% Agar gel.

Colour and Clarity of Prepared medium: Green coloured clear to slightly opalescent gel forms in tubes.

Reaction: Reaction of 2.03% w/v aqueous solution at 25°C. pH : 6.8±0.2

pH: 6.60-7.00

Cultural Response: Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.

Key :- * Corresponding WDCM Numbers
# Formerly known as Enterobacter aerogenes
^ Formerly known as Pseudomonas aeruginosa

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the 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. Use before expiry date on the label. 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 (6,7).

Reference

1. Hugh R. and Leifson E., 1953, J. Bacteriol. 66:24.
2. Microbiology of the food chain —Horizontal method for the detection and enumeration of Enterobacteriaceae —Part 1: Detection of Enterobacteriaceae ISO 21528-1: 2017
3. Microbiology of the food chain — Horizontal method for the detection and enumeration of Enterobacteriaceae —Part 2: Colony-count technique, ISO 21528-2 : 2017
4. Milk and milk products Method for the enumeration of Pseudomonas spp. ISO/TS 11059:2009 | IDF/RM 225:2009
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
6. 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.