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A selective supplement for the isolation of H. pylori from clinical specimens.

COLUMBIA BLOOD AGAR BASE

Code: CM0331

Directions
Add 39g to 1 litre of distilled water. Boil to dissolve and sterilise by autoclaving at 121°C for 15 minutes.

Code: SR0147

Directions
Reconstitute one vial  as directed, aseptically add the contents to 500 ml of Columbia Blood Agar Base cooled to approximately 50°C. Add 35 ml of Laked Horse Blood SR0048 and mix well before pouring into sterile Petri dishes.

Description
Helicobacter pylori is associated with a number of gastric conditions, chiefly gastritis and peptic ulcers1,2,3.
Helicobacter pylori Selective Supplement (Dent) was developed from Dent’s selective medium described for the isolation of H. pylori from gastric biopsies2. This is a modification of Skirrow’s medium4 in which polymixin B is replaced by cefsulodin and amphotericin B is added to inhibit Candida species.
When used routinely in the laboratory for 100 gastric biopsies, Dent’s medium achieved a higher isolation rate for H. pylori and lower contamination by other organisms when compared with Skirrow’s medium and chocolate blood agar2. The provision of a good selective medium for H. pylori will help establish the role of this organism in the aetiology of gastric disease.

Technique
1. Prepare the medium as directed. The plates can be stored at 4°C for three weeks but it is essential that they are kept moist. This can be achieved simply
by keeping the plates in a plastic bag.
2. Smear the specimen on to the medium.
Note - the recovery of H. pylori from gastric biopsies is improved by direct cultivation as soon as possible after collection. If transportation is necessary, then place the biopsy against the neck of a small, sterile glass bottle containing 0.1 ml of sterile saline2. The biopsy should adhere to the glass but be protected from dehydration by water vapour.
3. Incubate at 35°C for three to five days under microaerophilic conditions. Use Campylobacter Gas Generating Kit BR0056 or BR0060 with an active
catalyst BR0042. Alternatively use CampyGen CN025 or CN035. CampyGen does not require the addition of water or a catalyst.
4. Examine for the presence of discrete, translucent and non-coalescent colonies. Note that colonies of H. pylori do not resemble those of Campylobacter
species.
5. Confirm the identity of the isolates with the following tests:
Gram negative, curved or spiral bacillus. Growth at 35°C, no growth at 25°C, variable growth at 42°C. Urease positive, Catalase positive, Oxidase positive, Hippurate negative.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated Medium: Straw coloured, free-flowing powder.
Prepared medium: Opaque red-coloured gel.

Quality control

* This organism is available as a Culti-Loop®

References
1. Marshall B. K., Warren J. R., Blincow E. D., Phillips M., Goodwin C. S., Murray R., Blackbourne S. J. and Waters T. E. (1988) The Lancet, December 24/31, No 8626/8627.
2. Dent J. C. and McNulty C. A. M. (1988) Eur. J. Clin. Microbiol. Infec. Dis. 7. 555±568.
3. Buck G. E. (1988) Laboratory Management, 26, No.9.
4. Skirrow M. B. (1977) BMJ, 1. 9-11.