HiCombi™ Mannitol Salt - Mannitol Salt Agar Plate

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Intended Use

Recommended for selection and subculture of Staphylococcus aureus in accordance with the harmonized method of USP/ EP/BP/JP/IP.

Composition

Ingredients Gms / Litre
Peptone # 5.000
Tryptone ## 5.000
HM Peptone B ### 1.000
Sodium chloride 75.000
D-Mannitol 10.000
Phenol red 0.025
Agar 15.000
pH after sterilization (at 25°C) 7.4±0.2

# Peptic digest of animal tissue

## Pancreatic digest of casein

### Equivalent to Beef extract

**Formula adjusted, standardized to suit performance parameters

Directions

Streak the test inoculum (50-100 CFU) aseptically.

Principle And Interpretation

Staphylococci are widespread in nature, although they are mainly found on the skin, skin glands and mucous membranes of mammals and birds. The coagulase-positive species i.e. Staphylococcus aureus is well documented as a human opportunistic pathogen. The ability to clot plasma continues to be the most widely used and accepted criterion for the identification of pathogenic staphylococci associated with acute infections (13). Staphylococci have the unique ability of growing on a high salt containing media (11). Isolation of coagulase-positive staphylococci on Phenol Red Mannitol Agar supplemented with 7.5%NaCl was studied by Chapman (3). The resulting Mannitol Salt Agar Base is recommended for the isolation of coagulase-positive staphylococci from cosmetics, milk, food and other specimens (13,1,4,6,14). The additional property of lipase activity of Staphylococcus aureus can be detected by the addition of the Egg Yolk Emulsion (FD045). The lipase activity can be visualized as yellow opaque zones around the colonies (7). It is also used in the performance of microbial limit tests for the selective isolation of Staphylococcus. The formulation is in accordance with the harmonization of USP/EP/BP/JP/IP (15,5,2,10,8).

The medium contains HM peptone B, tryptone and peptone which makes it very nutritious as they provide carbon, nitrogen compounds, long chain amino acids, vitamins and other essential growth factors and trace nutrients. Many other bacteria except Staphylococci are inhibited by 7.5% sodium chloride. Mannitol is the fermentable carbohydrate fermentation of which leads to acid production, detected by phenol red indicator.

S.aureus ferment mannitol and produce yellow coloured colonies surrounded by yellow zones. Coagulase-negative strains of S.aureus are usually mannitol non-fermenters and therefore produce pink to red colonies surrounded by red-purple zones. Presumptive coagulase-positive yellow colonies of S.aureus should be confirmed by performing the coagulase test [tube or slide (13)]. Lipase activity of S.aureus can be detected by supplementing the medium with egg yolk emulsion

A possible S.aureus must be confirmed by the coagulase test. Also the organism should be subcultured to a less inhibitory medium not containing excess salt to avoid the possible interference of salt with coagulase testing or other diagnostic tests (e.g. Nutrient Broth)(M002) (12). Few strains of S.aureus may exhibit delayed mannitol fermentation. Negative results should therefore be re-incubated for an additional 24 hours before being discarded (12).

Type of specimen

Pharmaceutical samples

Specimen Collection and Handling

For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per guidelines (2,5,8,10,15). 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. This medium is a selective medium, some strains of Staphylococcus aureus may exhibit a delayed fermentation of mannitol.
  2. Certain other bacteria are also mannitol fermenting other than Staphylococcus, therefore further biochemical testing is required for identification.

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
Sterile Mannitol Salt - Mannitol Salt Agar Plate in 90 mm disposable biplates.

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

pH
7.20-7.60

Quantity of medium
10 ml medium in each side

Sterility Test
Passes release criteria

Growth Promotion Test
Growth Promotion was carried out in accordance with the harmonized method of USP/EP/BPJP/IP, after an incubation at 30-35°C for 18-72 hours. Recovery rate is considered as 100% for bacteria growth on Soybean Casein Digest Agar.

Growth promoting properties
Growth of microorganism comparable to that previously obtained with previously tested and approved lot of medium occurs at the specified temperature for not more than the shortest period of time specified inoculating <=100 cfu(at 30-35°C for <=18 hours).

Indicative properties
Colonies are comparable in appearance and indication reaction to those previously obtained with previously tested and approved lot of medium occurs for the specified temperature for a period of time within the range specified inoculating <=100cfu (at 30-35°C for 18-72 hours).

Inhibitory properties
No growth of the test microorganism occurs for the specified temp for not less than longest period of time specified inoculating >=100cfu (at 30-35°C for >= 72 hours).

Cultural Response

Organism Inoculum (CFU) Growth Observed Lot Recovery value (CFU) Colour of colony Incubation temperature
Growth Promoting + Indicative
Staphylococcus aureus subsp. aureus ATCC 6538 (00032*) 50-100 luxuriant 25-100 >=50% yellow/white colonies surrounded by yellow zone 18-72 hrs
Inhibitory
Escherichia coli ATCC 8739 (00012*) >=103 inhibited 0 0% >=72 hrs
Additional Microbiological testing
Staphylococcus aureus subsp. aureus ATCC 25923 (00034*) 50-100 luxuriant 25-100 >=50% yellow/white colonies surrounded by yellow zone 18-72 hrs
Staphylococcus epidermidis ATCC 12228 (00036*) 50-100 fair - good 15-40 30-40% red 18-72 hrs
Staphylococcus epidermidis ATCC 14990 (00132*) 50-100 fair - good 15-40 30-40% red 18-72 hrs
Proteus mirabilis ATCC 12453 50-100 none-poor 0-10 0-10% yellow 18-72 hrs
Escherichia coli ATCC 25922 (00013*) >=103 inhibited 0 0% >=72 hrs
Escherichia coli NCTC 9002 >=103 inhibited 0 0% >=72 hrs
# Klebsiella aerogenes ATCC 13048 (00175*) >=103 inhibited 0 0% >=72 hrs

Key: (#) Formerly known as Enterobacter aerogenes, (*) Corresponding WDCM numbers

Storage and Shelf Life

On receipt store between 20-30°C 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 (9,13).

Reference

  1. American Public Health Association, 1966, Recommended Methods for the Microbiological Examination of Foods, 2nd Ed, APHA, New York.
  2. British Pharmacopoeia, 2016, The Stationery office British Pharmacopoeia
  3. Chapman G. H., 1945, J. Bacteriol., 50:201.
  4. Davis J. G., 1959, Milk testing, 2nd Ed., Dairy Industries Ltd, London.
  5. European Pharmacopoeia, 2017, EDQM.
  6. Hitchins A. D., Tran T. and McCarron J. E., 1995, FDA Bacteriological Analytical Manual, 8th Ed., AOAC International, Gaithersburg, Md.
  7. Gunn B. A., Dunkelberg W. E. and Creitz J. R., 1972, Am. J. Clin. Pathol., 57:236.
  8. Indian Pharmacopoeia, 2018, Govt. of India, Ministry of Health and Family Welfare, New Delhi
  9. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
  10. Japanese Pharmacopoeia, 2016
  11. Koch P. K., 1942, Zentralbl. Bakteriol. Parasitenkd. Abt. I Orig. 149:122.
  12. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore
  13. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
  14. Silverton R. E. and Anderson M. J., 1961, Handbook of Medical Laboratory Formulae, Butterworths, London.
  15. The United States Pharmacopoeia, 2019, The United States Pharmacopoeial Convention, Rockville, MD.
More Information
Product Name HiCombi™ Mannitol Salt - Mannitol Salt Agar Plate
SKU HB011
Customized Product Available No
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