Oxoid CM0961B BAIRD-PARKER AGAR BASE MODIFIE 500G培养基说明书

Oxoid CM0961B BAIRD-PARKER AGAR BASE MODIFIE 500G培养基说明书

Dehydrated Culture Media

BAIRD-PARKER AGAR BASE (RPF)

Code: CM0961

a new improved base medium for use with RPF Supplement (SR0122). This conforms to ISO 6888 Part 21 for the enumeration of coagulase-positive staphylococci.

一种新的改良基础培养基,可与 RPF 补充剂 (SR0122) 一起使用。 这符合 ISO 6888 第 21 部分关于凝固酶阳性葡萄球菌计数的规定。

Typical Formula*

gm/litre

Pancreatic digest of casein

10.0

Meat extract

5.0

Sodium pyruvate

10.0

Yeast extract

1.0

Glycine

12.0

Lithium chloride

5.0

Agar

20.0

pH 7.2 ± 0.2 @ 25ºC
* Adjusted as required to meet performance standards

RPF SUPPLEMENT

Code: SR0122

Typical Formula

SR0122A
(1 vial per 100ml
medium)

per litre
Bovine fibrinogen

375mg

3.75g

Rabbit plasma

2.5ml

25.0ml

Trypsin inhibitor

2.5mg

25.0mg

Potassium tellurite

2.5mg

25.0mg

Directions
Suspend 6.3g of Baird-Parker Agar Base (RPF) in 90ml of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121ºC for 15 minutes. Cool to 48ºC and add 1 vial of SR0122A, reconstituted as directed. Mix well and pour plates.

方向
将 6.3g Baird-Parker Agar Base (RPF) 悬浮在 90ml 蒸馏水中。 煮沸以完全溶解。 通过在 121ºC 高压灭菌 15 分钟进行灭菌。 冷却至 48ºC 并加入 1 瓶 SR0122A,按照指示重新配制。 充分混合并倒入盘子。

N.B.: Baird-Parker Agar Base (RPF) should only be used with RPF Supplement SR0122

Description
Staphylococcus aureus is a Gram-positive coccus capable of producing enterotoxin which can induce food poisoning. The organisms may be present in small numbers in many foods, and, if allowed to multiply unchecked, may produce highly heat resistant enterotoxins. The ability of Staphylococcus aureus to produce lecithinase and lipase has been recognised for many years, and the detection of these enzymes in egg yolk media has become a widely used procedure for the identification of this organism. Its ability to produce coagulase using a similar basal formulation enables confirmatory diagnosis with the incorporation of rabbit plasma into the base medium.

Rabbit Plasma Fibrinogen (RPF) Agar is based on the formulation described by Beckers et al2. This medium is a modification of Baird-Parker Medium and is recommended for the selective isolation, enumeration and confirmation of Staphylococcus aureus from food and other specimens3. The RPF Agar formulation retains the Baird-Parker Agar Base which has been specifically formulated to resuscitate injured cells4. This medium differs from Baird-Parker Medium in that the egg yolk emulsion has been replaced by fibrinogen, rabbit plasma and trypsin inhibitor. The fibrinogen was added to enhance the coagulase reaction in the RPF Agar5. The addition of rabbit plasma was found to be more specific for the coagulase activity when compared to other sources of plasma2. Trypsin inhibitor was added to prevent fibrinolysis.

The RPF Supplement (SR0122) has been modified in one respect from the original formulation, in that the potassium tellurite content has been reduced four-fold, from 0.01% to 0.0025% w/v. This reduction was necessary as it was discovered in the Oxoid laboratory that some strains of Staphylococcus aureus were sensitive to potassium tellurite when used at 0.01% w/v in RPF Agar6. This modification of RPF Agar was found to give comparable growth and selectivity to that achieved on Baird-Parker Medium (CM0275 or CM1127 and SR0054). The improved productivity of RPF Agar has also been confirmed by other laboratories7,8. The reduction in potassium tellurite concentration in RPF Agar results in Staphylococcus aureus strains forming white, grey or black colonies, which are surrounded by an opaque halo of precipitation, i.e. the coagulase reaction.

描述
金黄色葡萄球菌是一种革兰氏阳性球菌,能产生肠毒素,可诱发食物中毒。许多食物中可能存在少量微生物,如果任其繁殖,可能会产生高度耐热的肠毒素。金黄色葡萄球菌产生卵磷脂酶和脂肪酶的能力已被认可多年,在蛋黄培养基中检测这些酶已成为鉴定该生物体的广泛使用的程序。它使用类似的基础配方产生凝固酶的能力能够通过将兔血浆掺入基础培养基中进行确认诊断。

兔血浆纤维蛋白原 (RPF) 琼脂基于 Beckers 等人描述的配方2。该培养基是 Baird-Parker 培养基的改良版,推荐用于从食物和其他标本中选择性分离、计数和确认金黄色葡萄球菌3。 RPF 琼脂配方保留了 Baird-Parker 琼脂基质,该基质专为复苏受损细胞而配制4。该培养基与 Baird-Parker 培养基的不同之处在于蛋黄乳液已被纤维蛋白原、兔血浆和胰蛋白酶抑制剂取代。添加纤维蛋白原以增强 RPF Agar5 中的凝固酶反应。与其他血浆来源相比,发现添加兔血浆对凝固酶活性更具特异性2。添加胰蛋白酶抑制剂以防止纤维蛋白溶解。

RPF 补充剂 (SR0122) 在一个方面对原始配方进行了修改,其中亚碲酸钾含量降低了四倍,从 0.01% 降至 0.0025% w/v。这种减少是必要的,因为在 Oxoid 实验室中发现,当在 RPF Agar6 中以 0.01% w/v 使用时,一些金黄色葡萄球菌菌株对亚碲酸钾敏感。发现这种对 RPF 琼脂进行修饰的生长和选择性与 Baird-Parker 培养基(CM0275 或 CM1127 和 SR0054)相当。 RPF 琼脂的生产力提高也得到了其他实验室7,8 的证实。 RPF琼脂中亚碲酸钾浓度的降低导致金黄色葡萄球菌菌株形成白色、灰色或黑色菌落,这些菌落被不透明的沉淀晕圈包围,即凝固酶反应。

Technique
Surface Inoculation Method

  1. Prepare the RPF Agar plates as directed.
  2. Process the food sample in a stomacher or Waring blender using the recommended sample size and diluent.
  3. Separate plates are inoculated with 0.1ml of the prepared samples and the subsequent decimal dilutions of them.
  4. Incubate at 35°C or 37°C and examine after 24 and 48 hours incubation.
  5. Count all the colonies that have an opaque halo of precipitation around them. Do not limit the count to black colonies.
  6. Report as number of coagulase positive staphylococcus isolated per gram of food.

Pour Plate Method

  1. Prepare the RPF Agar as directed and hold at 48°C.
  2. Process the food sample in a stomacher or Waring blender using the recommended sample size and diluent.
  3. Add 1ml of the prepared sample (initial suspension and subsequent decimal dilution) into each sterile Petri dish.
  4. Add aseptically 20ml of sterile RPF Agar and prepare pour plates.
  5. Incubate at 35°C or 37°C and examine after 24 to 48 hours.
  6. Count all the colonies that have an opaque halo of precipitation around them.
  7. Report as number of coagulase positive staphylococcus isolated per gram of food.

Storage Conditions and Shelf Life
Store the dehydrated medium at 10-30°C and RPF Supplement below 0°C. Use before the expiry date on the label.
Prepared plates of medium are best used freshly prepared1,2.

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

Quality Control

Quality control of the medium includes testing in accordance with ISO 111339.

Positive control: Expected results
Staphylococcus aureus ATCC® 25923*
WDCM00034
1-3 mm grey/black colonies coagulase zones
Negative control:
Staphylococcus saprophyticus ATCC® 15305*
WDCM00159
No growth pinpoint – 2 mm grey/black colonies, no zone
Escherichia coli ATCC® 25922*
WDCM00013
No growth
* This organism is available as a Culti-Loop®

Precautions
Colonies of some contaminating organisms growing in close proximity to the coagulase positive colonies may partially digest the coagulase halo reaction.

References  参考文献

1. International Organization for Standardization ISO 6888-2 (1999) Microbiology of food and animal feeding stuffs – Enumeration of coagulase positive staphylococci (Staphylococcus aureus and other species) – Part 2 Technique using rabbit plasma fibrinogen agar.
2. Beckers H. J., van Leusden F. M., Hogeboom W. M. and Delfgon-van Asch E. H. M. (1980) (English summary) De Ware(n)-Chemicals 10. 125-130.
3. Beckers H. J., van Leusden F. M., Bindshedler O. and Guerraz D. (1984) Can. J. Microbiol. 30. 470-474.
4. Baird-Parker A. C. (1962) J. Appl. Bacteriol. 25. 12-19.
5. Hauschild A. H. W., Park C. E. and Hilsheimer R. (1979) Can. J. Microbiol. 25. 1052-1057.
6. Sawhney D. (1986) J. Appl. Bact. 61. 149-155.
7. Beckers H. J. (1985) Personal Communication.
8. van Schothorst M. (1985) Personal Communication.
9. International Organization for Standardization ISO 11133:2014 Microbiology of food, animal feed and water – Preparation, production, storage and performance testing of culture media

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