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

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. MAC 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 a 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	1.5
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 MAC

1. Suspend 49.53 grams of the dehydrated medium in 1000 ml of 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 MAC

• 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 Medium

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, Enterobacter, 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.

Keynotes: It is of various types on 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 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

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.

Related Pictures

MacConkey medium preparation

MAC plate

MacConkey medium with lactose fermenter (pink) colony, non-lactose fermenter (NLF) colony, and mucoid lactose fermenter (MLF) colony of Gram-negative bacteria

Klebsiella pneumoniae mucoid lactose fermenter colony on MacConkey medium Demonstration

Mixed growth of bacteria on MacCkoney agar of isolated colony

A reddish-brown pigment pyorubin of Pseudomonas aeruginosa of a clinical specimen on MacConkey medium demonstration

Acinetobacter and Klebsiella colony characteristics on Macconkey agar Demonstration

Acinetobacter baumannii-calcoaceticus complex (ABC) colony characteristics on MaCconkey medium

Acinetobacter species colony morphology on MacConkey medium

Aeromonas hydrophila colony characteristics on MacConkey medium

Alcaligenes faecalis colony on MacConkey medium

Beautiful Colony of Klebsiella pneumoniae on MacConkey medium

Heavily mucoid lactose fermenter (MLF) colony of Klebsiella pneumoniae on MacConkey medium

Klebsiella and Acinetobacter growth on MacConkey medium of clinical specimen ICU admitted patient sputum

Cryptococcus on MacConkey medium

E. coli Colony Characteristics on MacConkey medium

E. coli colony morphology on MacConkey medium

Escherichia coli growth on MacConkey medium

Enterobacter aerogenes colony morphology on MacConkey medium

Klebsiella oxytoca growth on MacConkey medium after subculturing from BHI broth

Lactose fermenter and non-lactose fermenter Gram-negative bacteria on MacConkey medium

Lactose Fermenters (LF)-Pink, non-lactose fermenters (NLF)-normal Colour(un-dyed) Gram-negative bacteria on MacConkey medium demonstration

LF and NLF Colonies of Gram-Negative Bacteria on MacConkey Medium

Morganella morganii on MacConkey medium

Mucoid colony of Pseudomonas aeruginosa on MacConkey medium (MAC) and its biochemical tests, oxidase, TSI, MIU, and Urease

Mucoid lactose fermenter (MLF) colony of Klebsiella pneumoniae on MacConkey medium

Mucoid Pseudomonas aeruginosa on MacConkey Medium

Mucoid strain of Pseudomonas aeruginosa colony morphology on MacConkey medium

No growth of Sphingobacterium on MacConkey Medium

Non-lactose fermenter (NLF) colony of Pseudomonas aeruginosa on MacConkey medium of clinical specimen pus

Proteus mirabilis colony characteristics on Macconkey medium showing non-lactose fermenter (NLF) colonies

Proteus vulgaris colony characteristics on MacConkey medium

Proteus vulgaris growth on MacConkey medium

Pseudomonas aeruginosa and Klebsiella pneumoniae colony morphology on MacConkey medium

Pseudomonas aeruginosa mucoid colony on MacConkey medium of sputum sample

Pseudomonas aeruginosa typical colony morphology on MacConkey medium

Salmonella Paratyphi growth on MacConkey Medium

Salmonella Typhi growth on MacConkey medium

Salmonella Typhi and Paratyphi growth on Sorbitol MacConkey medium

Salmonella Typhi, Salmonella Paratyphi, and Shigella flexneri (non-fermenter) colony morphology on Sorbitol MacConkey medium

Serratia marcescens colony morphology on MacConkey medium

Shewanella growth on MacConkey Medium

Shigella flexneri and Salmonella Typhi growth on Sorbitol MacConkey medium

Shigella boydii non-lactose fermenter colonies on MacConkey medium

Shigella flexneri biochemical reactions in TSI, SIM, Citrate, and urea agar plus growth Shigella and Salmonella on SS medium, sorbitol MAC, and XLD medium

Shigella NLF Colony on MacConkey

Sorbitol MacConkey Agar (SMAC)

Vibrio cholerae colony morphology on MAC

Fungal growth on blood agar, MAC, chocolate agar, and SDA demonstration

Further Readings

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