easy staph

TECHNICAL DATA SHEET
EASY STAPH ® AGAR
ENUMERATION OF COAGULASE-POSITIVE STAPHYLOCOCCI
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INTENDED USE
®
EASY Staph is an alternative method of enumeration of coagulase-positive Staphylococcus in food and environmental
products. This method allows an enumeration of pathogenic staphylococci in 22 hours without confirmation instead of
48 hours for the normalized method NF EN ISO 6888-2.
The medium may be used for surface inoculation, pour plate inoculation or Spiral method.
®
EASY Staph is officially certified by AFNOR Certification for the enumeration of coagulase-positive Staphylococcus in
dairy products, meat products and seafood, under the reference number BKR 23/10-12/15, of which the validity runs
until 3 December 2019.
BKR 23/10 – 12/15
ALTERNATIVE ANALYSIS METHOD
FOR AGRIBUSINESS
Certified by AFNOR Certification http://nf-validation.afnor.org/en
In the context of NF VALIDATION, test portions weighing more than 25 g have not been tested.
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PRINCIPLES
The base medium is specifically formulated to ensure a growth of Staphylococcus aureus in 22 hours.
The optimization of selective system improves the inhibition of secondary flora usually observed on Baird-Parker agar.
®
The EASY Staph lyophilisate supplement corresponds to an optimized Rabbit Plasma Fibrinogen. The composition is
adjusted to obtain a development of halo of fibrin in 22 hours.
Rabbit plasma was chosen for its excellent specificity towards staphylococcal coagulase and by its aptitude to rapidly
produce clot formation by forming staphylothrombin from prothrombin. The rabbit plasma is reinforced with bovine
fibrinogen.
Staphylothrombin acts by cutting the A and B fibrinopeptides of fibrinogen, thereby initiating the polymerization process
that results in the appearance of fibrin halos surrounding the colonies.
Soybean trypsin inhibitor prevents fibrinolysis.
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TYPICAL COMPOSITION
Composition may be adjusted to obtain optimal performances.
For 1 L of complete medium :
- Peptones ................................................................................................................... 19.4 g
- Activators................................................................................................................... 21.7 g
- Buffer system .............................................................................................................. 1.4 g
- Selective system ......................................................................................................... 4.5 g
- Bacteriological agar ................................................................................................... 14.4 g
- Bovine fibrinogen ........................................................................................................ 5.3 g
- Rabbit plasma, EDTA................................................................................................ 25 mL
- Trypsin inhibitor ......................................................................................................... 25 mg
- Potassium tellurite ..................................................................................................... 25 mg
pH of ready-to-use medium at 25 °C : 7.3 ± 0.2.
Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – 60002 Beauvais Cedex – France
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Page 1 of 5
For 68.2 g of dehydrated base BK216
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For a vial of supplement BS090
(qsp 100 mL)
- Peptones ......................................................................... 21.6 g
- Activators ........................................................................ 24.1 g
- Buffer system .................................................................... 1.5 g
- Selective system ............................................................... 5.0 g
- Bacteriological agar ......................................................... 16.0 g
- Bovine fibrinogen ...................................... 0.53 g
- Rabbit plasma, EDTA............................... 2.5 mL
- Trypsin inhibitor ........................................ 2.5 mg
- Potassium tellurite .................................... 2.5 mg
For 190 ml of ready-to-melt base medium of BT012 Kit (BM185)
For a vial of supplement (BS086) of BT012 Kit
- Peptones ........................................................................... 4.3 g
- Activators .......................................................................... 4.8 g
- Buffer system .................................................................... 0.3 g
- Selective system ............................................................... 1.0 g
- Bacteriological agar ........................................................... 3.2 g
- Bovine fibrinogen ......................................1.06 g
- Rabbit plasma, EDTA ..................................5 mL
- Trypsin inhibitor ...........................................5 mg
- Potassium tellurite .......................................5 mg
PREPARATION
Preparation of dehydrated medium :
•
•
•
•
•
Suspend 68.2 g of dehydrated base medium (BK216) in 1 L of distilled or
deionized water.
Slowly bring to boiling, stirring with constant agitation until complete dissolution.
Dispense in flasks by adding 90 mL per flask or multiples of 90 mL.
Sterilize in an autoclave at 121 °C for 15 minutes.
Cool and maintain at 44-47°C.
 Reconstitution :
68.2 g/L
 Sterilization :
15 min at 121 °C
Use of ready-to-melt base medium :
•
•
Melt the base medium included in the kit (BT012 or BT013) or prepared in advance for the minimum amount of time
necessary in order to achieve total liquefaction.
Cool and maintain at 44-47°C.
Reconstitution of supplements :
•
•
•
Using aseptic techniques, add the required volume of sterile distilled water :
o
Qsp 100 mL of complete medium (BS090) : 10 mL of sterile water
o
Qsp 100 ml of complete medium from the kit BT012 (BS086) : 10 mL of sterile water
Using preheated water will accelerate the dissolution. Use water at room temperature or preheated without
exceeding 44 °C.
Turn end-over-end to dissolve. Avoid frothing the solution. Immediate dissolution is not always obtained. To
accelerate the process, a mechanical agitator (vortex) for tubes can be used to fully dissolve the lyophilisate. The
supplement should be completely dissolved before using.
Preparation of complete medium :
•
•
•
•
For 190 mL of base medium from the kit BT012 (BM185), add aseptically 10 mL of
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reconstituted supplement EASY Staph (BS086).
For 90 mL of prepared base medium from dehydrated or from the Kit BT013
®
(BM189), add aseptically 10 mL of reconstituted supplement EASY Staph
(BS090).
Mix well.
Use instantly for pour plate inoculation or pour in empty Petri dishes for surface
inoculation.
Complete medium
Add 10 mL of BS086 to
190 mL of BM185
or
Add 10 mL of BS090 to
90 mL of prepared
medium
NOTE : Do not keep the complete medium at 44-47°C.
Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – 60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
Page 2 of 5
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USE INSTRUCTIONS
Respect the good laboratory practices (follow the NF EN ISO 7218 standard).
Pour plate inoculation :
• Transfer 1 mL of the sample to analyze and its tenfold dilution series into sterile Ø
90 mm Petri dishes.
• Pour 10 to 15 mL of complete medium.
• Homogenize by swirling.
• Let solidify on a cool surface.
• Incubate at 37 ± 1°C for (24 ± 2) hours.
 Inoculation :
1 mL in pour plate
 Incubation :
24 h at 37°C
NOTE : Incubation may be prolonged until 72 hours, if this coinced with better laboratory organization.
Surface inoculation :
•
•
•
Transfer 0.1 mL of the suspension, and if necessary, any serial dilutions onto the
surface of prepared plate or pre-poured plate (BM187) previously brought to room
temperature.
Spread the inoculum on the surface with the aid of sterile triangle or « hochey
stick ».
Incubate at 37 ± 1 °C for (24 ± 2) hours.
 Inoculation :
0.1 mL in surface
 Incubation :
24 h at 37 ± 1 °C
NOTES :
- The method is also validated with the Spiral inoculation technology. Inoculation may be realized with 50 µL or 100 µL
in exponential mode.
- The enumeration limit can be reduced by a factor of 10, in inoculating 1 mL of suspension on the surface of 3 Petri
dishes (Ø 90 mm).
- Incubation may be prolonged until 72 hours for spiral inocualtion method or 48 hours for manual spreading, if this
coinced with better laboratory organization.
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READING
Coagulase-positive Staphylococcus are characterized by the formation of white, grey or black colonies surrounded by
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an opaque halo of fibrin that is clear-cut, stable and well visible on EASY Staph agar.
Enumerate colonies following the NF EN ISO 7218 standard.
See ANNEX 1 : PHOTO SUPPORT.
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QUALITY CONTROL
Dehydrated base medium : cream-white powder, free-flowing and homogeneous.
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EASY Staph Supplement : white to pinkish lyophilisate, giving a clear amber to slightly opalescent solution.
Complete prepared agar : amber agar.
Typical culture response after 24 hours of incubation at 37 °C :
Microorganisms
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus saprophyticus
Escherichia coli
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WDCM 00034
WDCM 00032
WDCM 00159
WDCM 00013
Growth
(Productivity Ratio : PR)
Characteristics
PR ≥ 50 %
PR ≥ 50 %
Slowed, score 0-1
Inhibited, score 0
Colonies with opaque halo
Colonies with opaque halo
Colonies without opaque halo
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STORAGE
Dehydrated base medium : 2-30 °C.
®
EASY Staph supplements : 2-8 °C.
Kit : 2-8 °C.
Pre-poured medium in Petri dishes : 2-8°C.
The expiration dates are indicated on the labels.
Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – 60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
Page 3 of 5
Prepared base medium in vials (*) : 6 months at 2-8 °C.
Reconstituted supplement (*) : 8 days at 2-8 °C. Before use, preheat between 25 to 37°C. The white precipitate
observed at 2-8 °C will disappear after heating.
Prepared complete medium in Petri dishes : 1 month at 2-8 °C, shielded from light.
Prepared complete medium in vials (*) : Use immediately.
(*) Benchmark value determined under standard laboratory conditions following manufacturer’s instructions.
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PRESENTATION
Dehydrated base medium
Vial of 500 g ......................................................................................................................................................... BK216HA
®
EASY Staph Supplement
8 vials qsf 100 mL ................................................................................................................................................ BS09008
Kit (6 * 200 mL)
6 vials of 190 mL of base medium and 6 lyophilisate supplements ..................................................................... BT01208
Kit (6 * 100 mL)
6 vials of 90 mL of base medium and 6 lyophilisate supplements ....................................................................... BT01308
Pre-poured medium in Petri dishes (Ø 90 mm)
20 plates .............................................................................................................................................................. BM18708
Pre-poured medium in Petri dishes (Ø 90 mm)
120 plates ............................................................................................................................................................ BM19008
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BIBLIOGRAPHY
Loeb, L.. 1903. The influence of certain bacteria on the coagulation of blood. Journal of Medical Research, 10 : 407419.
Duthie, E.S.. 1954. Evidence of two forms of staphylococcal coagulase. Journal of General Microbiology, 10 : 427-436.
Baird-Parker, A.C.. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci.
Journal of Applied Bacteriology, 25 : 12-19.
Hauschild, A.H., Park, C.E. and Hilsheimer, R.. 1979. A modified pork plasma agar for the enumeration of
Staphylococcus aureus in foods. Canadian Journal of Microbiology, 25 : 1052-1057.
Beckers, H.J., van Leusden, F.M., Bindschedler, O. and Guerraz, D.. 1984. Evaluation of a pour-plate system with a
rabbit plasma-bovine fibrinogen agar for the enumeration of Staphylococcus aureus in food. Canadian Journal of
Microbiology, 30 : 470-474.
NF EN ISO 6888-2. Octobre 1999. Microbiologie des aliments. Méthode horizontale pour le dénombrement des
staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 2 : Technique utilisant le milieu
gélosé au plasma de lapin et au fibrinogène.
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OTHER INFORMATIONS
Document code :
Creation date
:
EASY STAPH_EN_V1
01-2016
Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – 60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
Page 4 of 5
ANNEX 1 : PHOTO SUPPORT
EASY Staph® Agar
Enumeration of coagulase-positive Staphylococcus.
Reading :
Growth obtained after 24 hours of incubation at 37 °C.
Staphylococcus aureus
Characteristic colony :
Surrounded by an opaque
halo of fibrin
Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – 60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
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