CIN / MACCONKEY WITH SORBITOL AGAR BIPLATE

Cat. no. J49 CIN / MacConkey with Sorbitol Agar, 15x100mm Biplate,
10ml/10ml
10 plates/bag

INTENDED USE

Hardy Diagnostics CIN / MacConkey with Sorbitol Agar is recommended for use in the selective and differential isolation of Yersinia and Aeromonas species (CIN Agar), and Escherichia coli O157:H7 (MacConkey with Sorbitol Agar), from both clinical specimens and food sources.

SUMMARY

CIN Agar (Side I): CIN Agar, originally developed in 1979 by Schiemann, is a highly selective medium designed to isolate Yersinia enterocolitica and Aeromonas spp. The properties of this medium are based on selective chemical agents, antibiotics, dyes, and the basal medium, and it is highly selective against the growth of Escherichia coli , Klebsiella pneumoniae

, Proteus mirabilis , Pseudomonas aeruginosa , Salmonella enterica , Shigella sonnei and Streptococcus faecalis . (6,7) The characteristic deep red center with a transparent margin, or "bull's-eye" appearance of Yersinia and Aeromonas colonies is important for identification, and is due to the presence of mannitol and sodium deoxycholate in the medium. Y. enterocolitica and Aeromonas spp. ferment the mannitol in the medium, producing an acid pH which gives the colonies their red color. The sodium deoxycholate is responsible for the "bull's-eye" phenomenon. Altorfer found that by reducing the concentration of cefsulodin from 15 to 4mg/ml, CIN Agar could also be used to selectively isolate Aeromonas spp., in addition to Yersinia . (11)

MacConkey with Sorbitol (Side II): E. coli O157:H7 is an enteric pathogen that typically causes hemorrhagic colitis and non-bloody diarrhea illnesses. It may be followed by hemolytic uremic syndrome, especially in young children. MacConkey with Sorbitol Agar is recommended for isolation of this organism. Rappaport and Henig first described the formulation of the medium, and it was later confirmed by March and Ratham who reported MacConkey with Sorbitol Agar to have a sensitivity of 100% and a specificity of 85%. (7) This medium has proved to be an inexpensive, rapid, simple yet reliable means for the detection of E. coli O157:H7.

FORMULA

Ingredients per liter of deionized water:*

CIN Agar (Side l):
Mannitol 20.0gm
Peptone 17.0gm
Proteose Peptone 3.0gm
Yeast Extract 2.0gm
Sodium Pyruvate 2.0gm
Sodium Chloride 1.0gm
Sodium Deoxycholate 0.50gm
Sodium Cholate 0.50gm
Neutral Red 30.0mg
Magnesium Sulfate 10.0mg
Irgasan ® 4.0mg
Crystal Violet 1.0mg
Cefsulodin 4.0mg
Novobiocin 2.5mg
Agar 13.5gm

Final pH 7.4 +/- 0.2 at 25ºC.

MacConkey with Sorbitol (Side ll):
Pancreatic Digest of Gelatin 17.0gm
Sorbitol 10.0gm
Sodium Chloride 5.0gm
Pancreatic Digest of Casein 1.5gm
Peptic Digest of Animal Tissue 1.5gm
Bile Salts Mixture 1.5gm
Neutral Red 30.0mg
Crystal Violet 1.0mg
Agar 13.5gm

Final pH 7.1 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult appropriate references to determine correct specimen collection. (1-5)

Method of Use (CIN Agar): The plate should be warmed to room temperature and the agar surface should be dry prior to inoculating. Streak the plate with a suspension of the sample to be tested in a dilution small enough to produce isolated colonies. Incubate at 35ºC. Observe for growth at 18-24 hours. Yersinia and Aeromonas species will grow at 35ºC. within 18-24 hours.

Method of Use (MacConkey with Sorbitol): Inoculate the plates with a suspension of the sample to be tested in a dilution small enough to produce isolated colonies. Incubate plates aerobically at 35ºC. for 24 hours. Examine media macroscopically for typical colonies.

INTERPRETATION OF RESULTS

CIN Agar Colony Morphology: After 24 hours, colonies of Y. enterocolitica and Aeromonas spp. have a deep red center surrounded by a translucent outer zone.

MacConkey with Sorbitol Colony Morphology: E. coli O157:H7 forms colorless colonies, but otherwise typical of that produced by other E. coli species. Any bacteria capable of fermenting sorbitol (including other E. coli species) form pink colonies on MacConkey with Sorbitol Agar.

LIMITATIONS

Citrobacter species, Enterobacter agglomerans , Serratia liquefaciens , Y. frederiksenii , Y. intermedia and Y. kristensenii may grow on CIN Agar and resemble Y. enterocolitica ("bull's-eye" colony morphology), but are easily differentiated by biochemical tests.

Enterobacter cloacae and Serratia marcescens are not inhibited on CIN Agar. However, they usually appear as raised, mucoid colonies with diffuse, pink coloration.

Reading of MacConkey with Sorbitol Agar beyond 24 hours should be avoided since the pink color, seen in colonies that ferment sorbitol, fades.

Other gram-negative organisms are able to grow on MacConkey with Sorbitol Agar. However, colony appearance is generally enough to differentiate these organisms from E. coli O157:H7.

MacConkey with Sorbitol may be used as an aid in the identification of bacteria.

Sorbitol-negative colonies may be presumptively identified as E. coli O157 using our E. coliPRO™ O157 Kit (Cat. no. PL070HD). Further serotyping with H7 antiserum is necessary for definitive identification (Cat. no. 221591).

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
CIN Agar:
Yersinia enterocolitica
ATCC ® 9610
A 24-48hr 35°C Aerobic Growth with red center, transparent border
Aeromonas hydrophila
ATCC ® 7966
A 24-48hr 35°C Aerobic Growth with red center, transparent border
Proteus mirabilis
ATCC ® 12453
B 24-48hr 35°C Aerobic No growth
Enterococcus faecalis
ATCC ® 29212
B 24-48hr 35°C Aerobic No growth
Pseudomonas aeruginosa
ATCC ® 27853
B 24-48hr 35°C Aerobic No growth
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic No growth
MacConkey with Sorbitol:
Escherichia coli O157:H7
ATCC ® 35150
A 24hr 35°C Aerobic Growth; clear colonies seen, no fermentation of sorbitol
Escherichia coli
ATCC ® 25922
A 24hr 35°C Aerobic Growth; pink colonies seen, fermentation of sorbitol

User Quality Control

PHYSICAL APPEARANCE

CIN / MacConkey with Sorbitol Agar, Biplate should appear as follows:

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. March, S.B., S. Ratnam, et al. 1986. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol.; 23:869-872.

7. March, S.B., S. Ratnam, et al. 1988. Characterization of Escherichia coli serotype O157:H7. J. Clin. Microbiol.; 26:2006-2012.

8. Rappaport, F. and E. Henig. 1952. Media for the isolation and differentiation of pathogenic E. coli (serotypes O11 and O55). J. Clin. Path.; 5:361-362.

9. Schiemann, D.A. 1979. Can. J. Microbiol.; 25:1298

10. Schiemann, D.A. 1982. Appl. Environ. Microbiol.; 43:14

11. Altorfer, Regine, et al. 1985. Journal of Clinical Microbiology; Vol. 22, No. 4, p. 478-480. American Society of Microbiology.

ATCC is a registered trademark of the American Type Culture Collection.
Irgasan is a registered trademark of Geigy Chemical Corp.

121415hh