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MacConkey Agar, Granulated

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GMH081
Recommended for selective isolation and differentiation of coliforms from pharmaceutical products in accordance with the microbial limit testing by harmonised methodology of USP/EP/BP/JP.

Intended Use

Recommended for selective isolation and differentiation of E.coli and other enteric bacteria from pharmaceutical products in accordance with the microbial limit testing by harmonized methodology of USP/EP/BP/JP.

Composition

Ingredients g/L
Gelatin peptone # 17.000
HMC peptone ## 3.000
Lactose monohydrate 10.000
Sodium chloride 5.000
Bile salts 1.500
Neutral red 0.030
Crystal violet 0.001
Agar 13.500
pH after sterilization (at 25°C) 7.1±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Pancreatic digest of gelatin

## Equivalent to Peptones (meat and casein)

Directions

Suspend 49.53 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/distilled water. Boil for 1 minute with constant stirring. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes or as per validated cycle. Avoid overheating. Cool to 45-50°C. Mix well before pouring into sterile Petri plates. The surface of the medium should be dry when inoculated.

Principle And Interpretation

MacConkey Agar is the earliest selective and differential medium for cultivation of coliform organisms (1,2). Subsequently MacConkey Agar and Broth have been recommended for use in microbiological examination of foodstuffs (3) and for direct plating / inoculation of water samples for coliform counts (4). This medium is also accepted by the Standard Methods for the Examination of Milk and Dairy Products (5). It is recommended in pharmaceutical preparations and is in accordance with the harmonized method of USP/EP/BP/JP (6-9).

Gelatin peptone and HMC peptone provide the essential nutrients, vitamins and nitrogenous factors required for growth of microorganisms. Lactose monohydrate is the fermentable source of carbohydrate. The selective action of this medium is attributed to crystal violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Sodium chloride maintains the osmotic balance in the medium.

After enrichment of Escherichia coli in MacConkey Broth (MH083), it is then subcultured on MacConkey Agar. Gram-negative bacteria usually grow well on the medium and are differentiated by their ability to ferment lactose. Lactose fermenting strains grow as red or pink and may be surrounded by a zone of acid precipitated bile. The red colour is due to production of acid from lactose, absorption of neutral red and a subsequent colour change of the dye when the pH of medium falls below 6.8. Lactose non-fermenting strains, such as Shigella and Salmonella are colourless and transparent and typically do not alter appearance of the medium. Yersinia enterocolitica may appear as small, non-lactose fermenting colonies after incubation at room temperature.

Type of specimen

Pharmaceutical samples, Food and dairy samples; Water samples.

Specimen Collection and Handling

For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per guidelines (6-9).

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3,5). For water samples, follow appropriate techniques for sample collection and processing as per guidelines (4).

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 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. Though the medium is recommended for selective isolation, further biochemical and serological testing must be carried out for further confirmation.
  4. The surface of the medium should be dry when inoculated.

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 coloured granular medium

Gelling
Firm comparable with 1.35% Agar gel.

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

pH
6.90-7.30

Cultural Response

Growth Promotion is carried out in accordance with the harmonized method of ICH (USP/EP/BP/JP). Cultural response was observed 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 <=100 cfu (at 30-35°C for 18-72 hours).

Organism Inoculum (CFU) Growth Observed Lot value (CFU) Recovery Colour of colony Incubation period
Growth Promoting + Indicative
Escherichia coli ATCC 8739 (00012*) 50-100 luxuriant 25-100 >=50% pink-red with bile precipitate 18-72 hrs
Additional Microbiological testing
Escherichia coli ATCC 25922 (00013*) 50-100 luxuriant 25-100 >=50% pink to red with bile precipitate 18 -24 hrs
# Klebsiella aerogenes ATCC 13048 (00175*) 50-100 luxuriant 25-100 >=50% pink to red 18 -24 hrs
Enterococcus faecalis ATCC 29212 (00087*) 50-100 fair-good 0-10 <=10% colourless to pale pink 18 -24 hrs
Salmonella Typhimurium ATCC 14028 (00031*) 50-100 luxuriant 25-100 >=50% colourless 18 -24 hrs
Staphylococcus aureus subsp. aureus ATCC 6538 (00032*) >=10³ inhibited 0 0% >=24 hrs
Staphylococcus aureus subsp. aureus ATCC 25923 (00034*) >=10³ inhibited 0 0% >=24 hrs
Salmonella Enteritidis ATCC 13076 (00030*) 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
Salmonella Paratyphi A ATCC 9150 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
Salmonella Paratyphi B ATCC 8759 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
Salmonella Typhi ATCC 6539 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
Salmonella Abony NCTC 6017 (00029*) 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
## Proteus hauseri ATCC 13315 50-100 luxuriant 25-100 >=50% colourless 18-24 hrs
Shigella flexneri ATCC 12022 (00126*) 50-100 fair to good 15-40 30-40% colourless 18-24 hrs
Staphylococcus epidermidis ATCC 12228 (00036*) >=10³ inhibited 0 0% >=24 hrs
Corynebacterium diphtheriae type gravis >=10³ inhibited 0 0% >=24 hrs

Key :-

  • (*) Corresponding WDCM numbers
  • (#) Formerly known as Enterobacter aerogenes
  • (##) Formerly known as Proteus vulgaris

Storage and Shelf Life

Store between 10- 30°C in a tightly closed container and the prepared medium at 20 - 30°C. For better performance ot is advised to store the plates 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 (10,11).

More Information
Product Name MacConkey Agar, Granulated
SKU GMH081
Product Type Granulated
Physical Form Granular
Origin Animal
Packaging type HDPE
References 1.Murray P. R, Baron E, J., Jorgensen J. H., Pfaller M. A., Yolken R. H., (Eds.), 8th Ed., 2003, Manual of Clinical Microbiology,ASM, Washington, D.C.
2.Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed.,APHA Inc., Washington, D.C.
3.Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination ofFoods, 5th Ed., American Public Health Association, Washington, D.C.
4.FDA Bacteriological Analytical Manual, 2005, 18th Ed., AOAC, Washington, D.C.
5.Baird R.B., Eaton A.D., and Rice E.W., (Eds.), 2015, Standard Methods for the Examination of Water andWastewater, 23rd ed., APHA, Washington, D.C.
6.The United States Pharmacopoeia, 2018, The United States Pharmacopeial Convention, Rockville, M.D.
7.Williams, (Ed.), 2005, Official Methods of Analysis of the Association of Official Analytical Chemists, 19th Ed., AOAC,Washington, D.C8.Medrek T. F and Barnes Ella M., 1962, J. Appl. Bacteriol., 25(2),159-1689.Barnes Ella M. and Shrimpton D. H., 1957, J. Appl. Bacteriol., 20(2),273-285.10.Thornley Margaret J., 1957, J. Appl. Bacteriol., 20(2), 273-285.
11.Eddy B. P., 1960, J. Appl. Bacteriol., 23(2).216-249.
12.MacConkey A., 1905, J. Hyg., 5:333.
13.MacConkey A., 1900, The Lancet, ii:20.
14.British Pharmacopoeia, 2016, The Stationery office British Pharmacopoeia.1
5.Isenberg, H.D. Clinical Microbiology Procedures Handb0ook. 2nd Edition
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