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COLUMBIA BLOOD AGAR BASE

Code: CM0331

A multi-purpose medium suitable for the cultivation of fastidious organisms.

Typical Formula*

gm/litre

Special peptone

23.0

Starch

1.0

Sodium chloride

5.0

Agar

10.0

pH 7.3 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Add 39g to 1 litre of distilled water. Boil to dissolve the medium completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and add 5% sterile defibrinated blood.

Description
Traditionally, blood agar bases have been either casein hydrolysate or meat infusion media. The advantage of the first lies in the rapid production of large colonies, and of the second in clearly defined zones of haemolysis and good colonial differentiation.

Columbia Agar Base (CM0331) ( Ellner et al.1 ) combines the virtues of both these types of media to give an improved all-round performance.
This new base has shown versatility and superior performance in several applications.

Brucella
To prepare a selective medium add Brucella Selective Supplement SR0083 to 500ml of sterile, molten Columbia Blood Agar Base (CM0331), containing 5-10% v/v inactivated horse serum and 1% w/v dextrose2,3.

Campylobacter and Helicobacter
To prepare a selective medium add:
Campylobacter Selective Supplement (Skirrow) SR00694
or Campylobacter Selective Supplement (Butzler) SR00855,6
or Modified Butzler (ISO) Selective Supplement SR0214
or Campylobacter Selective Supplement (Blaser-Wang) SR00987,8,9
or Helicobacter pylori Supplement SR0147 to 500 ml of sterile, molten Columbia Agar Base (CM0331) containing Campylobacter Growth Supplement SR023210 and 5-7% v/v horse or sheep blood (SR0048, SR0050 or SR0051).
Egg Yolk Emulsion Agar made using Oxoid Columbia Agar Base (CM0331) and Egg Yolk Emulsion SR0047 has been shown to be a satisfactory isolation medium for Helicobacter pylori11.

Gram-positive cocci
see Staph/Strep Selective Supplement SR0070
see Modified CNA Selective Supplement SR0176
see Streptococcus Selective Supplement SR0126

Gardnerella
see Gardnerella vaginalis Selective Supplement SR0119

Other applications
Elek Test
Columbia Agar Base (CM0331) with added sterile serum provides an efficient Corynebacterium diphtheriae virulence test medium. After following the established technique, lines of toxin-antitoxin precipitation are clearly visible in 48 hours.

Nagler Test
The addition of 5ml Fildes Extract SR0046 and 10ml of Egg Yolk Emulsion SR0047 to 100ml of sterile, molten Columbia Blood Agar Base (CM0331) will provide a diagnostic medium for Clostridium perfringens, when used with Clostridium perfringens antitoxin (Nagler reaction) and neomycin (100-125m/ml)12.

Reversed CAMP Test for Clostridium perfringens
The reversed CAMP test13 is a highly sensitive and specific test for Clostridium perfringens which may be used as an alternative to the Nagler test.

Technique
Inoculate the culture suspected to be Clostridium perfringens in a straight line across a plate of Columbia sheep blood agar. Streak an overnight (or older) culture of Streptococcus agalactiae known to produce the CAMP factor at right angles to the first inoculation taking care that the lines do not touch. Incubate anaerobically at 35-37°C for 18-24 hours.

A positive reverse CAMP test is indicated by the formation of an ``arrowhead’’ of haemolysis between the lines of the Clostridium perfringens and Streptococcus agalactiae growth.

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 plates of medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive controls:

Expected results

Columbia Agar 
Staphylococcus aureus ATCC® 25923 *

Good growth; cream coloured colonies

Streptococcus pyogenes ATCC® 19615 *

Good growth; pale straw coloured colonies

Negative control:

 

Uninoculated medium.

No change

Brucella Medium 
Positive control: 
Brucella abortus ATCC® 4315Good growth
Negative control:  
Escherichia coli ATCC® 25922 *Inhibited
Campylobacter Media 
Positive control: 
Campylobacter jejuni ATCC® 33291 *Good growth; grey brown coloured colonies
Negative control:  
Escherichia coli ATCC® 25922 *Inhibited
Helicobacter pylori Medium. 
Positive control: 
Helicobacter pylori ATCC® 43526 Good growth; colourless colonies
Negative control:  
Candida albicans ATCC® 10231 *Inhibited or no growth
Staph./Strep. Medium 
Positive controls: 
Staphylococcus aureus ATCC® 25923 *

Good growth

Streptococcus pyogenes ATCC® 19615 *

Good growth; colourless/white colonies; ß-haemolysis

Negative control:  
Escherichia coli ATCC® 25922 *Inhibited
Streptococcus Selective Medium 
Positive control: 

Streptococcus pyogenes ATCC® 19615 *

Good growth; colourless/white colonies; ß-haemolysis

Negative control:  
Staphylococcus aureus ATCC® 25923 * No growth
Gardnerella Selective Medium 
Positive control: 
Gardnerella vaginalis ATCC® 14018 *Good growth; grey/white colonies
Negative control:  
Proteus mirabilis ATCC® 29906 *Inhibited
* This organism is available as a Culti-Loop®

Precautions
Brucella cultures are highly infective and must be handled under properly protected conditions. Incubate in 5-10% carbon dioxide atmosphere for 24-48 hours.
Campylobacter species are best grown at 42°C (except Campylobacter fetus subsp. fetus) in a micro-aerophilic atmosphere (Oxoid Campylobacter Gas Generating Kit BR0056 or BR0060 or CampyGen CN0025/CN0035).
Staph./Strep. supplemented plates should be incubated aerobically at 35°C for 18 hours. Incubation in carbon dioxide- enriched air will cause inhibition of staphylococcal growth14.
Streptococcus Selective Supplement SR0126 supplemented plates may be incubated aerobically or anaerobically at 35°C for 18 hours.
Prepared plates of both supplemented media should be used within 18 hours of preparation for maximum selectivity. Gardnerella supplemented plates should be incubated at 35°C for 48 hours in an atmosphere containing 7% carbon dioxide.
Carry out confirmatory tests on all colonies from horse blood medium and on beta-haemolytic colonies from human or rabbit blood medium.
Incubate plates of Clostridium E-Y Agar anaerobically at 35°C for 18 hours, look for lecithinase activity (pearly layer) and for proteolysis. Lecithinase activity is inhibited in the presence of specific anti-toxin.

References
1. Ellner P.D., Stoessel C.J., Drakeford E. and Vasi F. (1966) Tech. Bull. Reg. Med. Techn. 36. No. 3, reprinted in Amer. J. Clin. Path. (1966) 45. 502-504.
2. Farrel I.D. and Robinson L. (1972) J. Appl. Bact. 35. 625-630.
3. Hunter D. and Kearns M. (1977) Brit. Vet. J. 133. 486-489.
4. Skirrow M. B. (1977) B.M.J. (ii) 9-11.
5. DeKeyser P., Goussuin-Detrain M., Butzler J.P. and Sternon J. (1972) J. Infect. Dis. 125. 390-392.
6. Butzler J.P., De Keyser P., Detrain M. and Dehaen F. (1973) J. Pediat. 32. 493.
7. Blaser M.J., Hardesty H.L, Powers B. and Wang W. L. L. (1980) J. Clin. Microbiol. 11. 309-313.
8. Blaser M.J., Berkowitz I.D., La Force F.M., Dravens J., Reller L.B. and Wang W.L.L. (1979) Ann. Int. Med. 91. 179-185.
9. Blaser M.J., Cravens J., Powers B.U., La Force F.M. and Wang W.L.L. (1979) Amer. J. Med. 67. 715-718.
10. Hoffman P.S., George H.A., Krieg H.R. and Smibert R.M. (1979) Canad. J. Microbiol. 25. 8-16.
11. Westblom T.U., Madan E. and Midkiff B.R. (1991) J. Clin. Microbiol. 29. 819-821.
12. Lowbury E.J.L. and Lilly H.A. (1955) J. Path. Bact. 70. 105-108.
13. Hansen M.V. and Elliott L.P. (1980) J. Clin. Microbiol. 12. 617-619.
14. Morton C.E.G. and Holt H.A. (1989) Med. Lab. Sci. 46. 72-73.

 
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