MacConkey Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

MacConkey Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology and Uses

Introduction of MacConkey Agar

MacConkey agar(MAC) uses for the culture of gram-negative bacteria and therefore Enterobacteriaceae belonging bacteria grow well on this medium and coliforms also enjoy this medium. MacConkey Agar is a modification of Neutral Red Bile Salt Agar developed by MacConkey. It was one of the earliest culture media for the cultivation and identification of enteric organisms from clinical specimens as well as food and water.

It is a selective, differential, and indicator medium because of the following properties-

  • Selective due to bile salts that inhibit gram-positive bacteria and select gram-negative bacilli.
  • The indicator medium is due to having neutral red incorporated in it.
  • Differential medium is due to separate whether lactose fermenter or non-lactose fermenter bacteria.
  • The above picture is showing the lactose and non-lactose fermenter colony of bacteria.

Principle of MacConkey Agar

MacConkey Agar is recommended for use as a selective and differential medium for the isolation of gram-negative bacilli including coliform organisms and enteric pathogens, on the basis of lactose fermentation. Peptones (meat and casein)  and pancreatic digest gelatin and provide the essential nutrients, vitamins, and nitrogenous factors required for the growth of microorganisms. Lactose monohydrate is the fermentable source of carbohydrates. 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 here as neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Agar is the solidifying agent.

Composition of MacConkey Agar

(Himedia)

Ingredients       Gms / Litre

  • Peptones (meat and casein):  3.0
  • Pancreatic digest of gelatin: 17.0
  • Lactose monohydrate:  10.0
  • Bile salts:  1.500
  • Sodium chloride: 5.0
  • Crystal violet:  0.001
  • Neutral red:  0.03
  • Agar: 13.5
  • Distilled water: 1000 ml

pH after sterilization( at 25°C) 7.1±0.2

Preparation of MacConkey agar

  1. Suspend 49.53 grams of the dehydrated medium in 1000 ml purified/distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes i.e. validated cycle.
  4. Cool to 45-50°C.
  5. Mix well before pouring into sterile Petri plates.
  6. Leave for drying.

Storage and Shelf life of MacConkey Agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test procedure ( specimen/organism inoculation)

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Inoculate and streak the specimen as soon as possible after collection.
  3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  4. Streak for isolation with a sterile loop.
  5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
  6. Examine colonial characteristics.

Colony Characteristics of various organisms in MacConkey Agar

Lactose positive (pink colonies): Lactose fermenting species will grow pink colonies. Lactose fermentation will produce acidic by-products that lower the pH and this turns the pH indicator pink. e.g. Escherichia coli, Enterobacteria, Klebsiella
Lactose negative (white colonies): Gram-negative bacterial species will still form colonies, but colonies will have a white appearance as there will be no change in pH in the absence of lactose fermentation. e.g. Salmonella, Proteus, Yersinia, Pseudomonas
No colonies: Gram-positive bacteria will not form any colonies on the MacConkey medium. e.g. Staphylococcus, Enterococcus, Micrococcus
Slow or weak Lactose positive: Weak lactose fermenters will form colonies slower than the rest. e.g.  Serratia, Citrobacter
Mucoid (sticky, wet colonies): Encapsulated bacteria produce capsules using lactose. This gives sticky, wet-appearing colonies and mucoid colony-forming species are Klebsiella, and Enterobacter.

Keynote: It is of various types for the purpose of uses like

  1. MacConkey agar without bile salt- It uses both gram-negative and gram-positive bacteria
  2. McConkey agar with bile salt- Selective for gram-negative bacteria but Enterococcus species may grow.
  3. MacConkey agar with bole sat and crystal violet: Strict selective medium for gram-negative bacteria that also inhibits Enterococcus species due to having crystal violet in its composition.
  4. The amount of medium for preparation also varies slightly from manufacturer to manufacturer. e.g. Himedia 49.53 gm for 1 liter, whereas Oxoid 51.5 gm and Hardy Diagnostics 52.49 gm.
  5. MacConkey Agar with Sorbitol is to be used as a selective and differential medium for the detection of enterohemorrhagic Escherichia coli O157:H7.

Uses of  MacConkey agar  (MAC)

  1. MacConkey Agar is recommended for use as a selective, differential, and indicator medium for the isolation of gram-negative bacilli including coliform organisms and enteric pathogens.
  2. It is used in the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria.
  3. It is used for the isolation of coliforms and intestinal pathogens from clinical specimens as well as food and water samples.

Limitations of MacConkey Agar

  1. Colony characteristics only provide presumptive identification and thus biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for final identification.
  2. The concentration of bile salts in MacConkey Agar is relatively low in comparison with other enteric plating media. The parallel use of more selective media for gram-negative enterics, such as Hektoen enteric agar (HEK, HE or HEA)or Xylose lysine deoxycholate (XLD) agar is recommended in order to increase the chances of pathogen isolation.
  3. Some strains of the organism may be encountered that grow poorly or fail to grow on this medium
  4. Some strains of Proteus may swarm on this medium.
  5. Serial inoculation may be required to assure adequate isolation of mixed flora samples.
  6. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce the growth and recovery of a number of strains of Gram-negative bacilli.

Some MacConkey Agar Related Videos

#Klebsiella growth on MacConkey agar, blood agar, and chocolate agar as shown below-

#E. coli on MacConkey agar, blood agar, and Chocolate agar as shown below-

#Acinetobacter on MacConkey agar as shown below-

#Mucoid strain of Pseudomonas aeruginosa on MacConkey agar as shown below-

6 different types of Pseudomonas colonies may be observed-

  • Type 1:Large and leafy
  • Type 2: smooth, circular, and coliform type
  • Type 3: Rough
  • Type 4: rugose
  • Type 5: Mucoid due to exopolysaccharide as shown in the video
  • Type 6: Dwarf and smallest

#Pseudomonas typical colony on MacConkey agar as shown below-

#Proteus growth on blood agar and MacConkey agar as shown below-

#Salmonella growth on MacConkey agar, blood agar, and chocolate agar as shown below-

#Sorbitol MacConkey agar-Sorbitol MacConkey agar is used for Screening Haemorrhagic/Verotoxigenic/Shiga toxigenic Escherichia coli which is Sorbitol non-fermenter but in video fermenter so it is not Haemorrhagic Escherichia coli as shown below-

#OMG what dangerous bacteria ||Over growth of Mucoid Klebsiella on MacConkey agar-This is the bacterium that kills neonates in NICU due to its infection in the blood called bacteremia. This is Klebsiella pneumoniae pan drug-resistant bacteria as shown below-

#Shigella spp. biochemical and its Growth on various media( XLD agar, MacConkey agar, and S-S agar) as shown below-

Further Readings on MacConkey Agar 

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  7. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CRITN-MacConkeyAgar.htm
  8. https://www.ncbi.nlm.nih.gov/books/NBK557394/
  9. http://www.himedialabs.com/TD/M081B.pdf
  10. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0115&cat=&c=UK&lang=EN

 

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