MacConkey
QUALITY CONTROL & PRODUCT INFORMATION: MacConkey II Agar
QUALITY CONTROL PROCEDURES
I. INTRODUCTION
MacConkey II Agar is a selective and differential medium for the detection of coliform organisms and enteric pathogens. Prepared plates of MacConkey II Agar are manufactured from pretested BBL® brand dehydrated base in BBL disposable plastic Stacker® and Space-Saving dishes.
II. PERFORMANCE TEST PROCEDURE
1. Inoculate representative samples with dilutions of the cultures listed below.
a. Streak the plates for isolation. Use an 18 to 24 h broth culture of Enterococcus faecalls diluted to 10(4) to 10(5) CFU/plate. For the remaining organisms, use an 18 to 24 h broth culture diluted to yield i0~ to i0~ CFU/plate.
b. Incubate plates at 35 ± 20C in an aerobic atmosphere.
c. Include Trypticase® Soy Agar with 5% Sheep Blood (TSA) plates as nonselective controls for all organisms.
2. Examine plates after 18 to 24 h for amount of growth, colony size, pigmentation and selectivity.
3. Expected Results
NCCLS Control Organisms (ATCC® Strains)
*Escherichia coil(25922): Growth, pink colonies
*proteus mirabills(12453): Growth, colorless colonies Inhibition of swarming (partial)
*Salmonella choleraesuis(14028) subsp. choleraesuis serotype typhimurium: Growth, colorless colonies
*Enterococcus faecalis (29212): Inhibition (partial)
Additional Strains Utilized
Pseudomonas aeruginosa (10145): Growth, pink to green colonies
Shigella dysenteriae (9361): Growth, pink to green colonies
III ADDITIONAL QUALITY CONTROL
1. Examine plates as described under "Product Deterioration."
2. Visually examine representative plates to assure that any existing physical defects will not interfere with use.
3. Determine the pH potentiometrically at room temperature for adherence to the specification of 7.1
± 0.2.
4. Note the firmness of plates during the inoculation procedure.
5. Incubate uninoculated representative plates at 35 ±
20C for 72 h and examine for microbial contamination.
PRODUCT INFORMATION
IV INTENDED USE
MacConkey II Agar is a selective and differential medium for the detection of coliform organisms and enteric pathogens.
V SUMMARY AND EXPLANATION
At the present time, many culture media are available to the laboratorian for the isolation, cultivation and identification of enteric bacteria. One of the earliest of these was developed by MacConkey and first described as a brief published note.
1 The landmark paper on MacConkey Agar was published in 1905 and contained detailed descriptions of the medium and the bacterial growth patterns obtained.
2 This formulation was devised in the knowledge that bile salts are precipitated by acids and certain enteric microorganisms ferment lactose whereas others do not possess this ability.
Since the publication of the early papers, the MacConkey Agar formula has been modified many times. A compilation of culture media published in 1930 lists ten modifications which were published up to that time.
3 More recent modifications include use of additives (e.g., kanamycin) and the deletion of certain ingredients (e.g., crystal violet, and neutral red4). MacConkey Agar is recommended for use with clinical specimens likely to contain mixed microbial flora, such as urine, respiratory and wound, because it allows a preliminary grouping of enteric and other gram-negative bacteria.
5'6 MacConkey Agar is also utilized in the microbiological examination of foods.
7 The BBL MacConkey II Agar formulation was made available in 1983. It was specially designed to improve the inhibition of swarming Proteus species, to achieve more definitive differentiation of lactose fermenters and nonfermenters, and for the promotion of superior growth of enteric pathogens.
PRINCIPLES OF THE PROCEDURE
MacConkey 1 Agar is a selective and differential medium. It is only slightly selective since the concentration of bile salts, which inhibits gram-positive microorganisms, is low in comparison with other enteric plating media. Crystal violet also is included in the medium to inhibit the growth of gram-positive bacteria, especially enterococci and staphylococci. Differentiation of enteric microorganisms is achieved by the combination of lactose and the neutral red indicator. Colorless or pink to red colonies are produced depending upon the ability of the isolate to ferment the carbohydrate.
REAGENTS
MacConkey II Agar
Approximate Formula* Per Liter Purified Water
Pancreatic Digest of Gelatin 17.0 g
Pancreatic Digest of Casein 1 .5
Peptic Digest of Animal Tissue 1 .5
Lactose 10.0
Bile Salts 1 5
Sodium Chloride 5 0
Neutral Red 0.03
Crystal Violet 0 001
Agar 13.5
*AdJusted and/or supplemented as required to meet performance criteria.
VIII PROCEDURE
Material Provided
MacConkey II Agar
Materials Not Provided
Ancillary culture media, reagents and laboratory equipment as required.
Instructions.
The agar surface should be smooth and moist, but without excessive moisture. Streak the specimen as soon as possible after it is received in the laboratory. The streak plate is used primariiy to isolate pure cultures from specimens containing mixed flora. A nonselective medium should also be streaked to increase the chance of recovery when the popuIation of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Alternatively, if material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge; then streak from this inoculated area. Incubate plates, protected from light at 35 + 20C (do not use C02-enriched atmosphere with MacConkey II Agar) or other appropriate temperature for 18 to 24 h.
RESULTS
After incubation most plates will show an area of confluent growth. Because the streaking procedure is, in effect, a "dilution" technique, diminishing numbers of microorganisms are deposited on the streaked areas. Consequently, one or more of these areas should exhibit isolated colonies of the organisms contained in the specimen. Better isolation is obtained due to the inhibitory action of the medium.
Typical colonial morphology on MacConkey II Agar is as follows:
E. coil: pink to rose-red (may be surrounded by a zone of precipitated bile)
Enterobacter Klebsiella: Mucoid, pink
Proteus: Colorless, swarming in areas of isolated colonies is inhibited
Salmonella: Colorless
Shigella: Colorless
Pseudomonas: Irregular, colorless to pink
Gram-positive bacteria: No growth to slight growth
LIMITATION OF THE PROCEDURE
It has been reported that some Enterobacteriaceae and Pseudomonas aeruginosa are inhibited on MacConkey Agar when incubated in a C02-enriched atmosphere 8
REFERENCES
1. MacConkey, A.T. 1900. Note on a new medium for the growth and differentiation of the Bacillus coil cornmuffs and the Bacillus typhiabdominalis. The Lancet, Part 11:20.
2. MacConkey, A. 1905. Lactose-fermenting bacteria in faeces J Hyg 5:333-379.
3. Levine, M., and H.W Schoenlein. 1930 A compilation of culture media for the cultivation of microorganisms. The Williams & Wilkins Company, Baltimore.
4. MacFaddin, J.F. 1985 Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. I. Williams & Wilkins, Baltimore
5 Baron, E.J., L R Peterson, and S.M Finegold. 1994 Bailey & Scott's diagnostic microbiology, 9th ed. Mosby-Year Book, Inc, St. Louis
6. Farmer, J.J., III. 1995. Enterobacteriaceae: introduction and identification, p. 438-449 In P R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed ), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
7. Vanderzant, C., and D.F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health Association, Washington, D.C.
8. Mazura-Reetz, G., T.R Neblett, and J M. Galperin
1979. MacConkeyagar: CO2 vs. ambient incubation, abstr. C 179. p. 339. Abstr. 79th Annu. Meet. Am. Soc. Microbiol. 1979.
aru~,iuiv DICKINSON
Becton Dickinson Microbiology Systems
Becton Dickinson and Company
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Cockeysville, MD 21030 USA
BBL, Stacker, Trypticase and TSA II are trademarks of Becton Dickinson and Company
ATCC ,s a trademark of the Amer,can Type Culture Collection
©1997 Becton Dickinson and Company
May 1997
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