COLUMBIA AGAR (base)

Transcription

1 COLUMBIA AGAR (base) INTENDED USE Columbia Agar is a highly nutritive medium used for the growth and isolation of a large variety of microorganisms, particularly very fastidious bacteria : streptococci and pneumococci in animal samples. When blood, selective agents or growth accelerators are added, it becomes possible to prepare a wide variety of media adapted to specific uses. HISTORY Developed by Ellner in 1966, Columbia Agar enables luxuriant colonies, perfectly defined hemolytic zones and well characterized colonies and pigmentation to be obtained. PRINCIPLES - Peptones included in the composition of the medium favor the excellent growth of colonies. - Yeast extract is a source of vitamin B complex. - Starch is a detoxifying agent and also an energy source. - Defibrinated sheep blood, which can be added to the medium, favors the detection of hemolytic reactions and supplies X factor (heme) required for the growth of a large number of bacteria, but lacks V factor (nicotinamide adenine dinucleotide) due to the presence of an NADase, which destroys any NAD present. Haemophilus influenzae, which requires both X and V factors, does not grow on agar containing ordinary blood. The following media can be prepared with Columbia base : 1 - Blood agar By adding 5 or 10% sterile sheep blood after autoclaving and cooling, the medium is suitable for the growth of Streptococcus, Pneumococcus, Staphylococcus, Listeria and Erysipelothrix. It can be made selective by adding colistin and nalidixic acid to preclude the development of Gram-negative bacteria and Bacillus. 2 - Chocolate agar By adding 10% blood to sterile Columbia Agar and heating at 80 C until a chocolate color develops, an excellent medium is obtained for the growth of Haemophilus influenzae and Neisseria species. Sheep blood does not favor the growth of most Haemophilus strains. 3 - Base without enrichment Columbia Agar can be used to grow Brucella abortus, Yersinia pestis and Clostridium perfringens, as well as all enterobacteria. 1/5

2 PREPARATION - Suspend 42.5 g of dehydrated medium (BK019) in 1 liter of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Dispense in tubes or flasks. - Sterilize in an autoclave at 121 C for 15 minutes. NOTE : Incomplete agar melting during preparation will invariably lead to significant inconsistency in the gel strength of the solidified agar, after sterilization and cooling. INSTRUCTIONS FOR USE - Melt the medium (if prepared in advance). - Cool and maintain at C. - Add the supplements required for the growth of the bacteria to be detected (sterile defibrinated horse or sheep blood, growth accelerators, selective agents). - Homogenize well. - Pour into sterile Petri dishes. - Let solidify on a cold surface. - Dry the plates with the covers partially removed. - Inoculate. - Incubate at 37 C for 24 to 48 hours in the optimal culture conditions of the bacteria inoculated. TYPICAL COMPOSITION (base media) (can be adjusted to obtain optimal performance) For 1 liter of medium : - Polypeptone g - Pancreatic digest of heart g - Yeast extract g - Corn starch g - Sodium chloride g - Bacteriological agar g ph of the ready-to-use medium at 25 C : 7.3 ± /5

3 QUALITY CONTROL - Dehydrated medium : beige powder, free-flowing and homogeneous. - Prepared medium (with 5% defibrinated sheep blood) : opaque, red agar. - Typical culture response after 48 hours of incubation at 37 C with 5% sheep blood (qualitative method of inoculation) : Microorganisms Growth Type of hemolysis Streptococcus pyogenes ATCC Streptococcus pneumoniae ATCC 6303 Listeria monocytogenes ATCC Staphylococcus aureus ATCC Escherichia coli ATCC beta alpha beta STORAGE / SHELF LIFE Dehydrated medium : 2-30 C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Base media in vials : 6 months at 2-8 C. - Media in plates (with sheep blood) : 1 month at 2-8 C. PACKAGING Code Dehydrated base medium : g bottle BK019HA 3/5

4 PHOTO SUPPORT : Product reference: BK019HA + defibrinated sheep s blood Media used for: Growth of fastidious bacteria and hemolysis studies with added blood. Group D streptococci Clear zone surrounding the colony (β-haemolysis) Columbia blood Agar base Ref : BK019HA (with 10% added sheep s blood) Incubation 48 hours at 37 C (surface) Characteristic colonies with a clear zone or with an incomplete clear zone in the blood (clear zone : β-haemolysis ; incomplete clearing with a greenish tint : α-haemolysis) 4/5

5 BIBLIOGRAPHY NETER, E The effect of yeast concentrate on the growth and survival of Haemophilus influenza in infusion broth. Journal of Bacteriology, 54 : THAYER, J.D. and MARTIN, J.E Improved medium selective for cultivation of Neisseria gonorrhoeae and Neisseria meningitidis. Public Health Report, 81 : ELLNER, P.D., STOESSEL, C.J., DRAKEFORD, E. and VASI, F A new culture medium for medical bacteriology. American Journal of Clinical Pathology, 45 : NF V Décembre Conserves. Recherche des Clostridium thermophiles. XP CEN ISO/TS (V ). Janvier Microbiologie des aliments. Guide pour la préparation et la production des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture. NF U Décembre Méthodes d analyse en santé animale. Isolement et identification de Taylorella equigenitalis à partir de prélèvements génitaux d équidés. NF EN ISO (V ). Avril Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement des Campylobacter spp. Partie 1 : Méthode de recherche. Pharmacopée Européenne /2007: Contrôle microbiologique des produits non stériles : Recherche de microorganismes spécifiés. Solution et milieux de culture recommandés, United States Pharmacopeia Microbiological Tests / Microbiological Examination. Recommended Solutions and Culture Media, XP CEN ISO/TS Juin Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Campylobacter spp.. Partie 3 : Méthode semi-quantitative. *Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer s instructions. It is provided as a guide only and no warranty, implied or otherwise is associated with this information. The information provided on the package take precedence over the formulations or instructions described in this document. The information and specifications contained in this technical data sheet date from They are susceptible to modification at any time, without warning. Code document : BK019/A/ : 7. 5/5