Dichloran Glycerol Agar Base

Intended Use

Recommended for selective isolation of xerophilic moulds from food samples. The composition & performance criteria are in accordance with ISO 21527-2 and 11133:2014 (E) /Amd. :2020

Composition**

ISO specification - Dichloran Glycerol Medium Base

Final pH (at 25°C) 5.6±0.2

Glycerol anhydrous 220.000

Dichloran Glycerol Medium Base M11291

Final pH (at 25°C) 5.6±0.2

**Formula adjusted, standardized to suit performance parameters

$ - Equivalent to Casein enzymatic digest

Directions

Suspend 15.77 gram (the equivalent weight of dehydrated medium per litre) in 500 ml purified/ istilled water. Heat to boiling to dissolve the medium completely. Add 110 grams of glycerol (Analytical Reagent Grade). Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Mix well and pour into sterile Petri plates.

Principle And Interpretation

Dichloran Glycerol Medium was formulated by Hocking and Pitt (1) and is recommended for isolation and enumeration of xerophilic moulds from dried and semidried foods. The glycerol at 18% (w/w) lowers the water activity (aw) from 0.999 to 0.95 (1) without causing any problem. This restrictive characteristic makes the medium especially suitable for foods. This medium is also recommended by ISO (2, 3).

Tryptone provides carbon, nitrogen, vitamins and minerals. Dextrose is a carbohydrate source. Phosphate buffers the medium. Magnesium sulfate provides divalent cations and sulfate. Dichloran is an antifungal agent, added to the medium to reduce colony diameters of spreading fungi. Chloramphenicol is included to inhibit the growth of bacteria present in environmental and food samples. Inhibition of growth of bacteria and restriction of spreading of more-rapidly growing moulds aids in the isolation of slow-growing fungi by preventing their overgrowth by more-rapidly growing species.

Type of specimen

Food samples

Specimen Collection and Handling

Process 40 gm of food sample in a stomacher by adding 200 ml of 0.1% Peptone Water (M028). Shake periodically for 30 minutes with 0.1% Peptone Water for powdered products. Dilute the sample to 1:10 in 0.1% Peptone water and spread on plate. Count the number of Xerophilic colonies per gram of food. The medium can also be used as general medium for the isolation of yeasts and moulds from foodstuffs (2). 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. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.

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 yellow homogeneous free flowing powder

Gelling Firm,comparable with 1.5% Agar gel

Colour and Clarity of prepared medium Medium amber coloured, clear to slightly opalescent gel forms in Petri plates

Reaction Reaction of 3.16% w/v aqueous solution 22 grams of glycerol after sterilization. pH : 5.6±0.2

pH 5.40-5.80

Cultural Response

Productivity: Cultural characteristics observed with added 22 gram of glycerol after an incubation at 25±1°C for upto 5 days. Recovery rate is considered as 100% for fungal growth on Reference medium - Sabouraud Dextrose Agar

Selectivity: Cultural characteristics observed with added 22 gram of glycerol after an incubation at 25±1°C for upto 5 days.

Key: (*) Corresponding WDCM numbers.

$ - Formerly known as Bacillus subtilis subsp. spizizenii

Storage and Shelf Life

Store between 15-25°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 inorder 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 (4,5).

Disposal

1. Hocking A.D. and Pitt J.I., 1980, J. Appl. Environ. Microbiol., 39:488.
2. Microbiology of food and animal feeding stuffs- Horizontal method for the enumeration of yeasts and moulds-Part 2: Colony count technique in products with water activity less than or equal to 0.95 ISO 21527-2:2008
3. Microbiology of food, animal feeding stuffs and water- Preparation, production, storage and performance testing of culture media, EN ISO 11133:2014 (E) /Amd. :2020
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
5. 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.