Download TRYPTONE-SOY AGAR (blood agar base)

TRYPTONE-SOY AGAR (blood agar base)
INTENDED USE
Tryptone-Soy Agar, used as a base to be supplemented with blood, is prepared with selected starting
materials which do not turn brown. It was specially designed to detect beta-hemolytic reactions of
streptococci in Lancefield groups A and B, and to favor the growth of particularly fastidious aerobic
and anaerobic bacteria. It is also used in the CAMP test.
PRINCIPLES
- The combination of casein digest and papaic digest of soybean meal leads to optimal growth due
to synergy between the protein supply of casein and the carbohydrate supply of soybeans.
- Sodium chloride maintains osmotic balance.
- Sterile defibrinated sheep blood used to enrich the medium demonstrates the hemolysis
characteristics of different bacteria.
PREPARATION
-
Suspend 40.0 g of dehydrated medium (BK028) 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.
INSTRUCTIONS FOR USE
- Melt the medium (if prepared in advance).
- Cool and maintain at 44-47°C.
- Aseptically add the supplements required for the growth of the bacteria to be detected (sterile
defibrinated sheep or horse blood, growth accelerators, selective agents).
- Pour into sterile Petri dishes.
- Let solidify on a cold surface.
- Dry in an incubator with the covers partially removed.
- Inoculate.
- Incubate at 37°C for 24 to 48 hours aerobically or in CO2-enriched atmosphere for Brucella and
Neisseria.
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RESULTS
Group A hemolytic streptococci are small gray colonies, translucent or opaque, surrounded by a zone
of beta type hemolysis. Other bacteria may exhibit the same type of hemolysis : Listeria, hemolytic
staphylococci, Escherichia coli and Pseudomonas. It is thus necessary to carry out a Gram stain to
confirm the results. Streptococci exhibiting the following characteristics : Bacitracin-negative, CAMPpositive, beta-hemolytic, are considered to be presumptively belonging to group B. The CAMP test
can be carried out with the medium supplemented with 5% sheep blood as follows :
ƒ
ƒ
Inoculate a 10 hour culture of beta-hemolytic Staphylococcus aureus ATCC® 33862 as a
single median streak.
Perpendicular to the initial streak, streak a culture of the streptococcus to identify,
approaching the first as closely as possible (2-3 mm) without touching it.
Group B streptococci produce a heat-resistant extracellular substance (CAMP factor) which leads to a
triangle of total hemolysis in the zone of incomplete hemolysis of the staphylococcus, at the junction
of the two cultures. This procedure should be done with known collection strains in parallel with the
unknown strains to be identified. Group B streptococci generally present smaller hemolytic zones than
those observed with Streptococcus pyogenes. Pneumococci appear as flat, smooth, grayish and
sometimes mucous colonies, surrounded by a narrow greenish zone of alpha type hemolysis.
Staphylococci form opaque golden yellow or white colonies, with or without beta type hemolysis.
Listeria form small beta hemolysis zones. Other non-pathogenic bacteria may also grow on this nonselective medium.
TYPICAL COMPOSITION of the base medium
(can be adjusted to obtain optimal performance)
For 1 liter of medium :
- Tryptone .........................................................................................15.0 g
- Papaic digest of soybean meal ........................................................5.0 g
- Sodium chloride ...............................................................................5.0 g
- Bacteriological agar .......................................................................15.0 g
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2.
QUALITY CONTROL
- Dehydrated medium : cream-white 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 (qualitative method of inoculation) :
Microorganisms
Streptococcus pneumoniae
Streptococcus pyogenes
Listeria monocytogenes
Listeria monocytogenes
Bacillus cereus
(1)
Enterococcus faecalis
(1)
Legionella pneumophilia
(1)
Growth
ATCC® 6303
ATCC 19615
CIP 79.31
CIP 59.53
ATCC 11778
CCM 2541
ATCC 35152
good, score 2
good, score 2
good, score 2
good, score 2
good, score 2
good
inhibited
Type of
hemolysis
alpha
beta
beta
beta
N/A
72 hours of incubation at 36°C (qualitative method of inoculation). Hemolysis non-applicable.
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STORAGE / SHELF LIFE
Dehydrated base 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.
- Complete media in plates (with sheep blood) : 1 month at 2-8°C.
PACKAGING
Code
Dehydrated medium :
- 500 g bottle
BK028HA
PHOTO SUPPORT :
Product reference : BK028HA
Media used for : Observation of haemolytic reactions and the growth of fastidious microorganisms.
Group D Streptococci (β-hemolysis)
Tryptone Soy agar (Blood agar base)
Ref : BK028HA
Incubation : 24 hours / 37°C
Characteristics : β-hemolysis : clear, distinct zones in the blood.
α-hemolysis : incomplete clearing often with a greenish tint.
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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
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BIBLIOGRAPHY
FACKLAM, R.R., and CAREY, R.B.. 1985. Streptococci and Aerococci in LENNETTE, E.H., BALOWS, A., HAUSLER, W.J.,
and SHADOMY, H.J. (ed). Manual of Clinical Microbiology 4 th Ed., ASM Washington DC, 154-175.
MacFADDIN, J.F.. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria. Williams & Wilklins,
Baltimore, volume 1: 794-806.
XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. 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 T 90-461. Juillet 2001 et NF T 90-461/A1. Juin 2005. Qualité de l’eau. Microbiologie. Contrôle qualité des milieux de
culture.
ISO 21871. Janvier 2006. Microbiologie des aliments. Méthode horizontale pour le dénombrement de Bacillus cereus
présumés en petit nombre. Technique du nombre le plus probable et méthode de recherche.
NF T 90-431. Septembre 2003 et NF T 90-431/A1. Avril 2006. Qualité de l’eau. Recherche et dénombrement de Legionella
spp et de Legionella pneumophila. Méthode par ensemencement direct et après concentration par filtration sur membrane
ou centrifugation.
*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 2009-02-17.
They are susceptible to modification at any time, without warning.
Code document : BK028/A/2003-01 : 7.
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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com