Yersinia Selective Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Yersinia Selective Agar

Yersinia Selective Agar is recommended for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens (feces) as well as non-clinical sample like food samples, feces, etc. Image source:https://www.fishersci.com/

Principle of Yersinia Selective Agar

The mannitol of the medium helps to differentiate between mannitol fermenting and non-fermenting bacteria. Microbes that ferment the sugar mannitol acidify the medium. It causes a localized drop in pH around the colonies.  The colonies take red color due to the presence of neutral red. Mannitol-negative bacteria form colorless and translucent colonies. The medium is selective due to the of presence bile salt, sodium deoxycholate, and crystal violet, which inhibit gram-positive and a number of gram-negative bacteria. Sodium Chloride keeps the osmotic balance of the medium while sodium pyruvate and magnesium sulfate stimulate bacterial growth. Agar is the solidifying agent. Peptone is an enzymatic digest of animal protein and the principal source of organic nitrogen for growing bacteria. Lab-Lemco powder( beef extract) and yeast extract are water-soluble ingredients of nutrient agar that contribute to vitamins, carbohydrates, nitrogen, and salts.

The incorporation of antibiotic supplements makes it highly selective for Yersinia. Y. enterocolitica typical colony will form dark red colonies resembling the bull’s eye, which are normally surrounded by a transparent border. Yersinia colony size, smoothness, and the ratio of the border to center diameter may vary among different serotypes.

Composition of Yersinia Selective Agar

Ingredients  Gms / Litre

• Peptone, special: 20.0
• Yeast extract: 2.0
• Mannitol: 20.0
• Sodium pyruvate: 2.0
• Sodium chloride: 1.0
• Magnesium sulfate: 0.01
• Sodium deoxycholate: 0.5
• Neutral red: 0.03
• Crystal violet:  0.001
• Agar: 12.5
  Final pH ( at 25°C) 7.4±0.2

 Preparation of Yersinia Selective Agar

1. Suspend 29.02 grams in  500 ml of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Aseptically add reconstituted contents of 1 vial of Yersinia Selective Supplement.
6. Mix properly it.
7. Pour medium into each plate and leave plates on the sterile surface until the agar has solidified.
8. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Yersinia Selective Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples- feces,  food, and dairy samples. or growth of bacteria)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Escherichia coli ATCC 25922 and  Yersinia enterocolitica ATCC 27729)
• Culture Media

Procedure of Yersinia Selective Agar

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at  22-32°C for 24-48 hours.
6. Examine colonial characteristics.

Result -Interpretation of  Yersinia Selective Agar

• Control strains i.e. Escherichia coli ATCC 25922 (negative control): No growth
• Yersinia enterocolitica ATCC 27729 (positive control): Growth
• Enterococcus faecalis, Staphylococcus aureus
  Proteus mirabilis and Pseudomonas aeruginosa: No Growth

Colony Morphology of Yersinia Selective Agar

1. Yersinia enterocolitica: Good-luxuriant growth with translucent with a dark pink center and bile precipitate.
2. Other Gran positive and negative bacteria: Generally show no growth

Uses of Yersinia Selective Agar

• It is a selective culture medium for the isolation of Yersinia enterocolitica from clinical specimens (feces) as well as non-clinical samples ( food and dairy samples).
• This medium may also be used for the isolation of Yersinia species other than Y. enterocolitica, e.g., for Y. pseudotuberculosis, Y. frederiksenii, Y. kristensenii, and Y. intermedia.

Keynotes on Yersinia Selective Agar

• Serratia liquefaciens, Citrobacter freundi, and Enterobacter agglomerans may resemble Yersinia enterocolitica which can be further identified by biochemical assays.
• Yersinia Selective Supplement composition ( each vial for 500 ml medium) is as follows-

1. Cefsulodin:7.5mg
2. Irgasan: 2.0mg
3. Novobiocin:1.25mg

• Yersinia enterocolitica is widely found in lakes and reservoirs,
• Y.  enterocolitica is the etiological agent of epizootic outbreaks of diarrhea, lymphadenopathy, pneumonia, and spontaneous abortions that occur in various animals.
• This organism is biochemically more active at room temperature than at 37°C.

Further Readings on Yersinia Selective Agar

1. Foods, American Public Health Association, Washington, D.C.
2. https://himedialabs.com/TD/M843.pdf
3. https://labmal.com/product/yersinia-selective-supplement-1-x-10-vials/
4. https://www.thermofisher.com/document-connect/document-connect.html?
5. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
6. https://assets.thermofisher.com/TFSAssets%2FMBD%2Fcertificate%2FCertificates-of-Analysis%2FCOA_CM0653B_3555463_.pdf
7. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, American Public Health Association, Washington, D.C.
8. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.