HiCrome™ Bacillus Agar Base

Intended use

Recommended for isolation and differentiation between various species of Bacillus from a mixed culture in foods, clinical and non-clinical samples by chromogenic method.

Composition**

Final pH ( at 25°C) 7.1±0.2

**Formula adjusted, standardized to suit performance parameters

#-Equivalent to Meat extract

Directions

Suspend 49.22 grams in 1000 ml purified /distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121ºC) for 15 minutes. Cool to 45-50°C For selective isolation of B.cereus and B.thuringiensis, aseptically add rehydrated contents of one vial of PB Selective Supplement (FD324). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Majority of Bacillus species apparently have little or no pathogenic potential and are rarely associated with disease in humans or lower animals. The principal exception to this are Bacillus anthracis, the agent of anthrax, and Bacillus cereus, but a number of other species, particularly those of the B.subtilis group, have been implicated in food poisoning and other human and animal infections (1). Bacillus cereus causes food poisoning due to consumption of contaminated rice (2,3,4), other starchy foods such as potato, pasta and cheese have also been implicated, eye infections and a wide range of other clinical conditions like abscess formation, meningitis, septicemia and wound infection.

HiCrome™ Bacillus Agar is based on the formulation of MYP Agar formulated by Mossel et al (5) used for enumeration of Bacillus cereus and Bacillus thuringiensis when present in large number in certain foodstuffs.

The medium contains peptone and HM extract, which provide nitrogenous, carbonaceous compounds, long chain amino acids, vitamins and other essential growth nutrients. Mannitol serves as the fermentable carbohydrate, fermentation of which can be detected by phenol red. Mannitol fermenting organisms like B. megaterium yield yellow coloured colonies. The chromogenic mixture present in the medium is cleaved by the enzyme beta-glucosidase found in B.cereus resulting in the formation of blue colonies. B.thuringiensis also grows as blue/green colonies on this medium as B.cereus and B.thuringiensis are biochemically identical, however B.cereus shows flat colonies with distinct blue centers, while B.thuringiensis shows irregular margins. If selective isolation of B.cereus or B.thuringiensis is required aseptically add PB Selective Supplement (FD324).

Type of specimen

Clinical samples - wounds, faeces, etc.; Food and dairy samples; Soil samples

Specimen Collection and Handling

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (6-8).

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (9,10).

For Soil samples follow appropriate techniques for handling specimens as per established guidelines (11).

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

Warning and Precautions

In Vitro diagnostic Use. For professional use only. 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 clinical 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.
3. Slight colour variation may be observed depending upon the utilization of the substrate by the organism.

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: Light yellow to pink homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

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

Reaction: Reaction of 4.92% w/v aqueous solution at 25°C. pH : 7.1±0.2

pH: 6.90-7.30

Cultural Response

Cultural characteristics observed after an incubation at 30°C for 24-48 hours.

Key: (*) - Corresponding WDCM numbers **Formerly known as Bacillus subtilis subsp. spizizenii ( $)-Formerly known as Bacillus megaterium. ( ^)-Formerly known as Bacillus coagulans

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 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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed. American Society for Microbiology, Washington, D.C.
2. Bouza E., Grant S., Jordan C. et al, 1979, Arch. Ophthamol., 97:488.
3. Mortimer P. R. and McCann G., 1974, Lancet, 1043.
4. Wohlgemuth K., Kirkbride C. A., Bicknell E. J. and Ellis R. P., 1972 Am. Vet. Met, Ass. 161:1691.
5. Mossel D. A. A., Koopman M. J. and Jongerium E., 1967, Appl. Microbiol., 15:650.
6. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
7. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
8. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
10. 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.
11. Subba Rao N. S., 1977, Soil Microorganisms and Plant Growth, Oxford and IBH Publishing Co., New Delhi.