CIN AGAR

Cat. no. G20 CIN Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J49 CIN Agar / MacConkey Agar, 15x100mm Biplate, 10ml/10ml 10 plates/bag

INTENDED USE

Hardy Diagnostics CIN Agar is recommended for use in the selective and differential isolation of Yersinia and Aeromonas species from clinical specimens, environmental samples, and food sources. (6,7)

SUMMARY

Yersinia enterocolitica has been well documented as a causative agent of gastrointestinal infections that invades the intestinal mucosa and lymph nodes. It is a major cause of enteric illness in the northern United States, Canada, and Europe. It may occur either sporadically, or through food or water-borne outbreaks. However, infections have been found at extraintestinal sites as well. Other Yersinia spp. have also been implicated as human pathogens, but are found less frequently than Y. enterocolitica .

CIN (Cefsulodin, Irgasan, Novobiocin) Agar, originally developed in 1979 by Schiemann, is a highly selective medium designed to isolate Yersinia enterocolitica . The properties of this medium are based on selective chemical agents, antibiotics, dyes, and the basal medium. 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. Y. enterocolitica ferments the mannitol in the medium, producing an acid pH which gives the colonies their red color and the "bull's eye" appearance. Sodium deoxycholate, cefsulodin, irgasan, and novobiocin are added as selective agents. Altorfer found that by reducing the concentration of cefsulodin from 15.0 to 4.0mcg/ml, CIN Agar could also be used to selectively isolate Aeromonas spp., in addition to Yersinia . (8)

Studies have proved that CIN Agar is superior to SS (Salmonella-Shigella) Agar and MacConkey Agar in recovery rates of Y. enterocolitica from clinical specimens and food sources. (7)

FORMULA

Ingredients per liter of deionized water:*

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
Cefsulodin 4.0mg
Novobiocin 2.5mg
Crystal Violet 1.0mg
Agar 13.5gm

Final pH 7.4 +/- 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: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult appropriate references to determine correct collection procedures for the specimen to be tested (stool, food, etc.). (1-5)

Method of Use: Allow plates to warm to room temperature. Using standard microbiology procedures, inoculate the plates directly with stool, or with specimen from transport medium or a cold enrichment broth. Streak for isolation. Incubate at 35ºC. for 24 hours or 22-25ºC. for 48 hours. The lower temperature is recommended for primary isolation of Yersinia and will produce colonies with a more distinct "bull's-eye".

INTERPRETATION OF RESULTS

CIN Agar Colony Morphology: After 18-48 hours, colonies of Y. enterocolitica and Aeromonas spp. have a deep red center surrounded by a translucent outer zone.

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.

Characteristic pigmentation is stronger and more complete at 25ºC. and 48 hours of incubation than at 35ºC. for 24 hours of incubation. It has been found that some Yersinia strains may be inhibited at 35ºC. The lower temperature is recommended for primary isolation.

Enterobacteriaceae other than Yersinia may grow on CIN Agar, especially Citrobacter species. Serratia and Citrobacter cannot always be reliably differentiated from Yersinia by colony morphology alone. Therefore, biochemical and serological tests are necessary for confirmation and complete identification of isolates.

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
Yersinia enterocolitica
ATCC ® 9610
A 24-48hr 25°C Aerobic Growth; red center, transparent border
Aeromonas hydrophila
ATCC ® 7966
A 24-48hr 35°C Aerobic Growth; red center, transparent border
Proteus mirabilis
ATCC ® 12453
B 24-48hr 35°C Aerobic Partial to complete inhibition
Enterococcus faecalis
ATCC ® 29212
B 24-48hr 35°C Aerobic Partial to complete inhibition
Pseudomonas aeruginosa
ATCC ® 27853
B 24-48hr 35°C Aerobic Partial to complete inhibition
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

CIN Agar should appear clear, slightly opalescent, and reddish-purple in color.

Y. enterocolitica growing on CIN Agar

Yersinia enterocolitica (ATCC ® 9610) colonies growing on CIN Agar (Cat. no. G20). Incubated aerobically for 24 hours at 35ºC.

A. hydrophila growing on CIN Agar

Aeromonas hydrophila (ATCC ® 7965) colonies growing on CIN Agar (Cat. no. G20). Incubated aerobically for 24 hours at 35ºC.

E. coli inhibited on CIN Agar

Escherichia coli (ATCC ® 25922) growth inhibited on CIN Agar (Cat. no. G20). Incubated aerobically for 24 hours at 35ºC.

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. Schiemann, D.A. 1979. Synthesis of a selective agar medium for Yersinia enterocolitica. J. Microbiol.; 25:1298.

7. Schiemann, D.A. 1982. Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food. Appl. Environ. Microbiol.; 43:14.

8. 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.

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