MacConkey Agar. Dr Adarsh Soman

MacConkey agar is a Selective & Differential media.

To isolate & differentiate gram-negative bacteria of the Enterobacteriaceae family.

1. Presenter : Dr. Adarsh S First Year Post graduate Department of Microbiology ESIC MC & PGIMSR - Bangalore MacConkey agar

2. REFERENCES Jung, B., & Hoilat , G. J. (2022).  MacConkey Medium . StatPearls Publishing. The origin of MacConkey agar . (2019, October 14). Asm.org. https://asm.org/Articles/2019/October/The-Origin-of-MacConkey-Agar Allen, M. E. (n.d.).  MacConkey Agar Plates Protocols . Asm.org. Retrieved March 30, 2023, from https://asm.org/ASM/media/Protocol-Images/MacConkey-Agar-Plates-Protocols.pdf?ext=.pdf Use, I. (n.d.).  MacConkey Agar MH081 . Himedialabs. com. Retrieved March 30, 2023, from https://www.himedialabs.com/eu/coasdstds/index/download/id/MH081/source/tds/lang/EN District Laboratory Practice in Tropical Countries – Monica Cheesbrough Part 2 Cruickshank Text book of Medical Microbiology Mackie & McCartney Practical Medical Microbiology

3. LAYOUT History Composition of MacConkey Agar Principle of MacConkey Agar Uses of MacConkey Agar Preparation of MacConkey Agar Result and Interpretation Colony Morphology on MacConkey Agar Quality Control of MacConkey Agar Modifications of MacConkey Agar

4. Alfred T. MacConkey First solid differential media 1861-1931

5. Selective & Differential media To isolate & differentiate gram negative bacteria of the Enterobacteriaceae family.

6. Peptone Lactose Agar Bile salts Sodium chloride Neutral Red Crystal violet

7. Ingredient Gm/ Litre Peptone # 17 g Polypeptone ## 3 g Lactose 10 g Bile salts 1.5 g Sodium chloride 5 g Agar 13.5 g Neutral red 0.03 g Crystal violet 0.001 g Distilled water 1 L Final pH 7.1 (+/-) 0.2 at 25 Celcius #Equivalent to Pancreatic digest of gelatin ## Equivalent to Peptones (meat and casein)

8. 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.

9. 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. P our about 20- 25ml into sterile  Petri plates   and allow to solidify.

10. After solidification of the plates, label the media plates with the name and date of preparation (Labeling should always be done on the backside of the media plate as lids could be interchanged.)

11. Store inverted (with lids down) at 2-8°C until use The surface of the medium should be dry when inoculated. Shelf life: 4 weeks

12. Peptone Lactose Agar Bile salts Sodium chloride Neutral Red Crystal violet Source of Organic Nitrogen, Minerals & Nutrients Fermentable carbohydrate source Solidifying agent Inhibits Gram positive Bacteria Maintains Osmotic balance pH indicator <6.8 >6.8 Inhibits Gram positive Bacteria

13. PRINCIPLE

14. It also differentiate GNB into Lactose fermenter and Non Lactose Fermenter Lactose fermenter bacterial colony appears PINK Lactose non-fermenter bacterial colony appears Colourless

15. Only Gram negative Bacteria grows on MacConkey agar MacConkey agar is selective for Gram negative bacteria.

16. Uses of MacConkey Agar Medium

17. MacConkey agar is commonly used for the isolation of Gram-negative enteric bacteria. MacConkey is a commonly used media to differentiate members of Enterobacteriaceae. It differentiates between lactose-fermenting and nonfermenting gram-negative rods by the color of colonial growth. MacConkey agar is used for the selective isolation and identification of members of the family  Enterobacteriaceae  from feces, urine, wastewater, and foods

18. Colony Morphology on MacConkey Agar

19. Organism Type Colony characteristics Escherichia coli Lactose fermenter E. coli  gives flat, dry, pink, non-mucoid colonies with a surrounding darker pink area of precipitated bile salts.

20. Klebsiella spp Lactose fermenter Colonies typically appear large, mucoid, and pink, with pink-red pigment usually diffusing into the surrounding agar

21. Citrobacter spp Late lactose fermenter Appear as non-lactose fermenter (NLF) up to 24 hours; however, after 48 hours colonies are light pink.

22. Enterobacter spp Lactose fermenter Pink, mucoid colonies but smaller than  Klebsiella  spp. Serratia spp Late lactose fermenter S. marcescens  may be red-pigmented, especially if the plate is left at 25°C

23. Proteus spp Non-Lactose Fermenter Pale colonies with swarming, characteristic foul smell.

24. Shigella spp Non-Lactose Fermenter except S. sonnei  which is a late lactose fermenter Pale colonies,1-2 mm, flat colonies with jagged edges Providencia spp Non-Lactose Fermenter Colonies are colorless, flat, 2-3 mm in diameter, and do not swarm. Salmonella spp Non-Lactose Fermenter Colorless colonies, convex, 2-3 mm with a serrated margin. Pseudomonas spp Non-Lactose Fermenter Colorless, flat, smooth colonies, 2-3 mm in diameter with greenish to brownish pigmentation. Yersinia spp Non-Lactose Fermenter Colonies may be colorless to peach

25. Quality Control of MacConkey agar

26. Sterility testing Incubate uninoculated plates of MacConkey for 48 hours at 35-37°C and observe for any growth. After 48 hours, the sterility test plate should remain clear. Discard the whole lot if any colonies are seen.

27. Performance testing Inoculate known standard strains on MacConkey agar plates, incubate for 18-24 hours at 35-37°C, and observe for growth and colony characteristics. Organism Used as Colony morphology Escherichia coli  ATCC 25922 Positive control for lactose fermentation Lactose fermenting pink colonies Pseudomonas aeruginosa  ATCC 27853 Positive control for non-lactose fermentation Non-lactose fermenting colonies with or without pigmentation. Streptococcus pneumoniae  ATCC 49619 Negative control No growth

28. Modifications of MacConkey Agar

29. MacConkey Agar without Crystal Violet It is a differential medium but is less selective than MacConkey agar. The lack of crystal violet permits the growth of  Staphylococcus  and   Enterococcus . The medium is also used to separate  Mycobacterium fortuitum  and  M. chelonae  from other rapidly growing mycobacteria.

30. Staphylococcus  spp Lactose fermenter Small pink colonies, 1-2mm in diameter, opaque Enterococcus  spp Lactose fermenter Dark pink to red, very minute, translucent colonies

31. MacConkey Agar, CS (“Controlled Swarming”)   MacConkey agar without crystal violet or salt is used to prevent the swarming of  Proteus  spp. Increse the agar conc. to 5%

32. Sorbitol MacConkey Agar (SMAC) C ontains  sorbitol instead of lactose as fermentable sugar . E.coli  0157 is non-sorbitol fermenting, producing colourless colonies . Most other  E.coli  strains and other enterobacteria ferment sorbitol. Sorbitol-fermenting organisms produce pink colonies. E.coli 0157 : haemorrhagic colitis ( bloody diarrhoea without pus cells) ass. With HUS VTEC- Verotoxin

33. MAC- sorbitol with cefixime & tellurite (CT-SMAC) E.coli  0157 :H7 is non-sorbitol fermenting, producing colourless colonies . Most other  E.coli  strains and other enterobacteria ferment sorbitol. Sorbitol-fermenting organisms produce pink colonies. Better selection than SMAC.