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Culture Media Supplements

COBA SELECTIVE MEDIUM

A selective supplement for the isolation of Streptococcus species.

COLUMBIA BLOOD AGAR BASE

Code: CM0331

Formula

gm/litre

Special peptone

23.0

Starch

1.0

Sodium chloride

5.0

Agar

10.0

pH 7.3 ± 0.2

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

STREPTOCOCCUS SELECTIVE SUPPLEMENT (COBA)

Code: SR0126

Vial contents (each vial is sufficient for 500 ml of medium)

per vial
per litre

Colistin sulphate

5.0 mg

10.0 mg

Oxolinic acid

2.5 mg

5.0 mg


Directions
Reconstitute one vial as directed, aseptically add the contents to 500ml of sterile Columbia Blood Agar Base containing 5% Defibrinated Horse Blood SR0050 cooled to approximately 50°C. Mix gently and pour into sterile Petri dishes.

Description
Streptococcus Selective Supplement is based on the formulation of Petts (COBA Medium)1 and is recommended for the selective isolation of
streptococci of medical and veterinary importance.
COBA Medium possesses advantages over other media described for selective isolation of streptococci.
Agents previously recommended for inhibition of Gram-positive organisms can be shown to have severe effects on streptococci even at subminimal
inhibitory concentrations. The antibiotics gentamicin2,3,4,5,6, amikacin7, fucidic acid8, neomycin9 and cotrimoxazole10 have all been shown to have
adverse effects as have the long established inhibitors crystal violet and sodium azide. Both colistin and oxolinic acid are thermostable and can, if necessary,
be stored without refrigeration.
Streptococci are commonly isolated from the upper respiratory tract. They are also often isolated from burns and other sites where frequently there is an
abundance of competing organisms. In order to isolate streptococci, especially when present in small numbers, it is necessary to inhibit the competing flora
without any adverse effect by the selective agents upon the Streptococcus species. The selective agents colistin sulphate (10 mg/ml) and oxolinic acid
(5 mg/ml) have been found to have no inhibitory effect on Streptococcus species although amongst Group D organisms Enterococcus faecalis colonies are somewhat smaller. The combination of these two selective agents results in total inhibition of Gram-negative organisms and almost all non-streptococcal Gram-positive organisms. A very few staphylococci and coryneform organisms may grow with reduced colony size. The haemolytic reactions on media containing blood are clearly defined, and the colonial size and growth recovery of streptococcal groups A, B, C, D and G and S. pneumoniae are comparable to that on a nonselective medium. The selective agents can also be used with Islam’s Medium (GBS Agar CM0755) for the isolation of Group B streptococci without loss of pigmentation occurring.

Technique
1. Prepare the medium from Columbia Blood Agar Base, Streptocccus Selective Supplement and Defibrinated Horse Blood SR0050,
according to the directions.
2. Inoculate the plates in the normal way and incubate at 35°C overnight in an atmosphere enriched with 5% carbon dioxide or anaerobically.*
3. Confirm that the colonies are streptococci by microscopy, biochemical or serological tests. The Oxoid Streptococcus Grouping Kit DR0585 or Dryspot DR0400 recommended for this purpose.
* Improved haemolytic reactions are achieved by anaerobic incubation. Gram-positive anaerobic cocci (Peptostreptococcus and Peptococcus species) would be selectively isolated under these conditions1.

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

Positive controls:

Expected results

Streptococcus pyogenes ATCC® 19615

Good growth; gas production.

Negative control:

Staphylococcus aureus ATCC® 25923

Inhibited

* This organism is available as a Culti-Loop®

References
1. Petts D. N. (1984) J. Clin. Microbiol. 19. 4-7.
2. Gray B. M., Pass M.A. and Dillon M. C. Jr. (1979) J. Clin. Microbiol. 9. 466-470.
3. Milatovic D. (1981) J. Clin. Path. 34. 556-588.
4. Nichols T. and Freeman R. (1980) J. Clin. Path. 33. 770-773.
5. Sondag J. E., Morgens R. K., Hoppe J. E. and Marr J. J. (1977) J. Clin. Microbiol. 5. 397-400.
6. Waitkins S. A. (1982) Med. Lab. Sci. 39. 185-188.
7. Petts D. N., MSc Thesis, University of Surrey.
8. Lowbury E. J. L., Kidson A. J. and Lilly H. A. (1964) J. Clin. Pathol. 17. 231-235.
9. Wren M. W. D. (1980) J. Clin. Pathol. 33. 61-65.
10. Dykstra M. A., McLoughlin J. C. and Bartlett R. C. (1979) J. Clin. Microbiol.9. 236-238.

 
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