MacConkey Agar
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 Gms / Litre
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
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. Gramnegative 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 homogeneous free flowing powder
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).
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.
