CIN (Cefsulodin_Irgasan-Novobiocin) Agar

Purpose: isolation and characterization of Yersinia enterocolitica from most other gram
negative bacteria able to grow on CIN medium

1. selective (high): due to presence of inhibitors

2. differential: ability to ferment mannitol with resultant acidity in presence of
neural red indicator

Fermentable carbohydrates present: Mannitol 2%

pH indicator: Neutral Red

• acid: pH 68: red color
• Alkaline: pH 8.0: yellow color

Inhibitors
1. Irgasan
2. Antimicrobics: Cefsulodin and novobiocin
3. Sodium desoxycholate and Crystal Violet (CV)

Inhibits:
1. highly selective inhibition of Escherichia coli, Klebsiella pneumoniae, Proteus
mirabilis, and Pseudomonas aeruginosa
2. Gram-positive bacteria

Quality control:
• Positive control: Yersinia enterocolitica
• Negative control: Escherichia coli, Klebsiella pneumoniae, Pseudomonas
mirabilis, Pseudomonas aeruginosa, Staphylococcus spp

Incubation:
1. 32C, 24hrs
2. 22C, 48 hrs

Storage:
1. Medium: Room temperature, 22-25C; plates inverted (lid down)
2. shelf life: 6-9 days

Interpretation:
• Y. enterocolitica and other Yersinia sppp. (Y. intermedia, Y. frederiksenii, and Y.
Kristensenii
a. Deep red centes witgh shaprt borders (irregular or entire) surrounded by
an outer zone which is usually translucent
b. Pigmentation due to mannitol fermentation (acid)

Precaution: colony size of Y. enterocolitica is larger on CIN than on MacConkey agar

Pigmentation is stronger and more complete at 22C with 48 hrs incubation than 35C for
24hrs

Clinical specimens for possible Yersinia isolation should be delievered to the laboratory
immediately after collection and cultured as soon as possible. Reffrigerate specimen if it
cannot be delivered immediately to the laboratory. Food and other samples of sanitary
significance should be collected and transported according to recommended procedures.
Prior to CIN inoculationk,k enrichment procedures are necessary for recovery of
Y.enterocolitica

Note: y. intermedia, Y. frederiksenii, and Y kristensenni grow equally well on CIN

media; all exhibit same colony morphology as Y. enterocolitica

CAL agar/Broth (cellobiose-Areginine-Lysine Agar)

Purpose:
• Agar: isolation and characterization of Yersinia enterocolitica from fecal
specimens; organism is biochemically similar to other Enterobacteriaceae
• Broth: Enumeration of Y. enterocolitica from water and other liquid specimens

Fermentable carbohydrate present: Cellobiose, 0.35%

pH indicator: Neutral red

• Acid: pH 6.8: red color
• Alkaline: pH 8.0; yellow color

Inhibitor: sodium deoxycholate

Inhibits: Gram positive bacteria

Quality control:
1. Positive Control: Yersinia enterocolitica
2. Negative Control: Escherichia coli

Incubation: aerobically 25C, 36-40 hrs; optimal growth temperature for Y. enterocolitica
and also avoid overgrowth by other enterics

Storage
1. refrigerator, 2-8C plates inverted
2. Shelf life: 6-8 weeks
Interpretation:
• Agar: Y. enterocolitica differentiation based on three characteristic colony types;
also observe plates for a color reaction
• Cellobiose fermnation:
i. Positive: acid pH: burgundy red rolored colonies
 ii. Negative: alkaline pH; clear, white orange-colorless colonies
• Y. enterocolitica colony types
i. Diameter: 0.5 – 2.5mm; bright burgundy colnies
ii. Diameter: 0.5 – 1.0mm; colonies clear with pinpoint red centers
iii. Diameter: 0.5; clear colonies
• Broth:
• Positive: turbid pink color with growth
• Negative: medium remains light orange in color

Occasional strain of Y. enterocolitica fails to ferment collobiosse and produces clear

colonies; always subculture a suspected yersinia colonies onto TSI media for further
biochemical testing

Streptococcus faecalis usually inhibited but if present sparse and found only in heavily
inculcated areas of plate

Pates should be red initially at 36-40 hours; other organism which ferment cellobiose and
decarboxylate arginine or lysine initially produce red colored colonies at 25C, but after
36hr, basic pH conditions brought about by decarboxylation of amino acids changes color
to white and orange

Chart from: the microbiology bench companion by J. Michael Miller

Yersinia frederiksenii Environmental, rare in humans
Yersinia intermedia Environmental, rare in humans
Yersinia kristensenii Environmental, rare in humans
Yersinia pestis
Yersinia pseudotuberculosis Mesenteric lymphadenitis, diarrhea, septicemia
Yersinia ruckeri Causes a disease in trout; rare in human