Oxoid现货 CM0739B CM1183B OXOID无血弯曲杆菌选择性琼脂基础培养基

Oxoid现货 CM0739B CM1183B OXOID无血弯曲杆菌选择性琼脂基础培养基

Dehydrated Culture Media

CAMPYLOBACTER BLOOD-FREE SELECTIVE MEDIUM (MODIFIED CCDA- PRESTON)

A medium, which when prepared from Campylobacter Blood-Free Selective Agar Base CM0739 and CCDA Selective Supplement SR0155, can be used for the isolation of Campylobacter jejuni, Campylobacter coli and Campylobacter laridis.

弯曲杆菌无血选择性培养基(改良 CCDA-普雷斯顿)

培养基由无血弯曲杆菌选择性琼脂培养基 CM0739 和 CCDA 选择性补充剂 SR0155 制备而成,可用于分离空肠弯曲杆菌、大肠杆菌和海里弯曲杆菌。

CAMPYLOBACTER BLOOD-FREE SELECTIVE AGAR BASE

Code: CM0739

Typical Formula*

gm/litre

Nutrient Broth No.2

25.0

Bacteriological charcoal

4.0

Casein hydrolysate

3.0

Sodium desoxycholate

1.0

Ferrous sulphate

0.25

Sodium pyruvate

0.25

Agar

12.0

pH 7.4 ± 0.2 @ 25°C
* Adjusted as required to meet performance standards

CCDA SELECTIVE SUPPLEMENT

Code: SR0155
An improved selective supplement for Campylobacter Blood-Free Selective Agar Base.

Vial contents (each vial is sufficient for 500ml of medium)
per vial
per litre
Cefoperazone

16mg

32mg
Amphotericin B

5mg

10mg

Directions
Suspend 22.75g of Campylobacter Blood-Free Selective Agar Base in 500ml of distilled water and bring to the boil to dissolve. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C. Aseptically add 1 vial of CCDA Selective Supplement SR0155 reconstituted as directed. Mix well and pour into sterile Petri dishes.

方向
将 22.75g 无血弯曲杆菌选择性琼脂基质悬浮在 500ml 蒸馏水中并煮沸溶解。 通过在 121°C 高压灭菌 15 分钟进行灭菌。 冷却至 50°C。 无菌添加 1 瓶 CCDA 选择性补充剂 SR0155,按照指示重新配制。 充分混合并倒入无菌培养皿中。

Description
Modified CCDA Medium is based on the original formulation described by Bolton et al.1 which was developed to replace blood with charcoal, ferrous sulphate and sodium pyruvate. Improved selectivity was achieved when cephazolin in the original formulation was replaced by cefoperazone as the selective agent 2. More recent work has shown an increased isolation rate can be achieved if the plates are incubated at 37°C rather than 42°C3.

Amphotericin B has been added to the formula to suppress the growth of yeast and fungal contaminants that may occur at 37°C.

Modified CCDA medium and Campy-BAP medium were equal in performance in a rapid colony-lift procedure for detection of thermophilic campylobacters in which membranes are blotted on agar cultures and then subjected to immunoassay 5.

In a study of healthy puppies and kittens for carriage of Campylobacter species6, modified CCDA medium was found to be a suitable medium and more productive for Campylobacter upsaliensis in this application than CAT medium. Modified CCDA medium has been confirmed as suitable for isolation of Campylobacter spp. from non-clinical samples following enrichment in Exeter broth7.

The use of Campylobacter Blood-Free Medium is specified by the U.K. Ministry of Agriculture, Fisheries and Food (MAFF) in a validated method for isolation of Campylobacter from foods4.

描述
改良的 CCDA 培养基基于 Bolton 等人 1 描述的原始配方,该配方旨在用木炭、硫酸亚铁和丙酮酸钠代替血液。当原始配方中的头孢唑啉被头孢哌酮代替作为选择性试剂 2 时,选择性得到了提高。最近的研究表明,如果将板在 37°C 而不是 42°C 下孵育,则可以提高分离率3。

配方中添加了两性霉素 B,以抑制在 37°C 时可能发生的酵母和真菌污染物的生长。

改良的 CCDA 培养基和 Campy-BAP 培养基在用于检测嗜热弯曲杆菌的快速菌落提升程序中的性能相同,其中膜被印迹在琼脂培养物上,然后进行免疫测定 5。

在一项关于携带弯曲杆菌属的健康幼犬和小猫的研究中,发现改良的 CCDA 培养基是一种合适的培养基,并且在该应用中比 CAT 培养基更能生产上山弯曲杆菌。改良的 CCDA 培养基已被证实适用于分离弯曲杆菌属。来自埃克塞特肉汤富集后的非临床样品7。

英国农业、渔业和食品部 (MAFF) 指定使用无血弯曲杆菌培养基,这是一种经过验证的从食品中分离弯曲杆菌的方法4。

Technique
1. Prepare Campylobacter Blood-Free Selective Agar as described in the directions.
2. Emulsify approximately 0.5g of the specimen in 5ml of sterile 0.1% peptone water to form an approximate 1:10 dilution.
3. Inoculate onto the selective medium with cotton tipped swabs so that single isolated colonies are formed.
4. Incubate the plates in an atmosphere consisting of approximately 5-6% oxygen, 10% carbon dioxide and 84-85% nitrogen for 48 hours at 37°C.

This can best be achieved by using the Oxoid Gas Generating Kit for campylobacter (BR0056) in conjunction with the Oxoid Anaerobic Jar (HP0011) and an active catalyst (BR0042). For jars of smaller capacity AnaeroJar(2.5 litres) use the Oxoid Gas Generating Kit for Campylobacters (BR0060). Alternatively use CampyGen CN0025 or CN0035 which does not require the use of a catalyst or the addition of water.

The colonial morphology of campylobacters can be used as a guideline for identification to species level. Campylobacter jejuni strains produce grey, moist flat spreading colonies. Some strains may have a green hue or a dry appearance, with or without a metallic sheen. Campylobacter coli strains tend to be creamy-grey in colour, moist, slightly raised and often produce discrete colonies.

Colonies tend to swarm when initially isolated from clinical specimens.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the selective supplement in the dark at 2-8°C and use before the expiry date on the label.
The prepared medium may be stored for up to 2 weeks at 2-8°C .

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

技术
1. 按照说明制备弯曲杆菌无血选择性琼脂。
2. 将大约 0.5g 样品在 5ml 无菌 0.1% 蛋白胨水中乳化,形成大约 1:10 的稀释液。
3. 用棉签接种到选择性培养基上,形成单个分离的菌落。
4. 将板在由大约 5-6% 氧气、10% 二氧化碳和 84-85% 氮气组成的气氛中在 37°C 下孵育 48 小时。

这可以通过将用于弯曲杆菌 (BR0056) 的氧化型气体生成套件与氧化型厌氧罐 (HP0011) 和活性催化剂 (BR0042) 结合使用来最好地实现。对于容量较小的 AnaeroJar(2.5 升)罐,请使用适用于弯曲杆菌的氧化物气体生成套件 (BR0060)。或者使用不需要使用催化剂或添加水的 CampyGen CN0025 或 CN0035。

弯曲杆菌的菌落形态可作为物种水平鉴定的指南。空肠弯曲杆菌菌株会产生灰色、潮湿、扁平的蔓延菌落。一些菌株可能具有绿色或干燥的外观,带有或不带有金属光泽。弯曲杆菌菌株往往呈乳灰色、潮湿、略微隆起,并且经常产生离散的菌落。

当最初从临床标本中分离出来时,菌落往往会成群结队。

储存条件和保质期
将脱水培养基储存在 10-30°C 并在标签上的有效期之前使用。
将选择性补充剂储存在 2-8°C 的黑暗中,并在标签上的有效期之前使用。
制备的培养基可在 2-8°C 下储存长达 2 周。

外貌
脱水介质:黑色,自由流动的粉末
制备培养基:黑色凝胶

Quality control.
Incubation at 37°C for 48 hours.

Positive control: Expected results
Campylobacter jejuni ATCC® 33291 * Good growth; grey coloured colonies
Negative control:
Escherichia coli ATCC® 25922 * No growth
* This organism is available as a Culti-Loop®

References
1. Bolton, F.J., Hutchinson, D.N. and Coates, D. (1984) J. Clin. Microbiol. 19, 169-171.
2. Hutchinson, D.N. and Bolton, F.J. (1984) J. Clin. Path. 34, 956-957
3. Bolton, F.J., Hutchinson, D.N. and Parker, G. (1988) Eur. J. Clin. Microbiol. Infect. Dis. 7. 155-160.
4. MAFF Validated Methods for the Analysis of Foodstuffs: Method for the detection of thermotolerant Campylobacter in Foods (v30) J. Assoc. Publ. Analysts (1993) 29. 253-262.
5. Rice, B.E., Chinta Lamichhane, Joseph, S.W. and Rollins, D.M. (1996) Clin. Diag. Lab. Immunol. 3, 669-677.
6. Hald, B. and Madsen, M. (1997) J. Clin. Microbiol. 35, 3351-3352.
7. Humphrey, T.J., Martin, K.W. and Mason, M.J. (1997) PHLS Microbiology Digest 13 (2), 86-88.

CEFOPERAZONE, AMPHOTERICIN B, TEICOPLANIN SUPPLEMENT (CAT)

Code: SR0174

A selective supplement for the isolation of thermophilic Campylobacter spp. and improved recovery of Campylobacter upsaliensis from faeces.

Vial contents(each vial is sufficient to supplement 500 ml of medium).

per vial

per litre

Cefoperazone

8.0mg

16.0mg

Teicoplanin

4.0mg

8.0mg

Amphotericin B

10.0mg

20.0mg

Directions
Prepare 500ml of sterile Blood-Free Campylobacter Agar Base as directed. Cool to 50°C and aseptically add one vial of SR0174 reconstituted as directed.
Mix well and pour the resulting CAT medium into sterile Petri dishes. Incubate cultures at 37°C for 48-72 hours in a microaerobic atmosphere.

Description
Because of the sensitivity of Campylobacter upsaliensis to a wide range of antibiotics, isolation of the organism from faeces using selective media has hitherto been difficult. The recommended isolation method uses a membrane filter culture technique on non-selective agar. This does not give good recovery from faeces containing less than 105 CFU/g5, and is a technically demanding method which is relatively slow to perform.
CAT Supplement SR0174 is based on the formulation described by Aspinall et al.7. When added to Blood-Free Campylobacter Agar Base which contains charcoal, it gives good isolation of thermophilic Campylobacter spp. The isolation of Campylobacter upsaliensis on a selective medium is possible because CAT Supplement contains reduced levels of cefoperazone compared to other campylobacter supplements. This inhibits most Enterobacteriaceae, but not enterococci. Teicoplanin is included to inhibit enterococci. Amphotericin B is added as an antifungal agent.
Further work confirmed the effectiveness of CAT medium as an alternative to membrane filtration culture for selective isolation of thermophilic campylobacters including Campylobacter upsaliensis8.

Atabay, Corry and On9 isolated a previously unknown catalase-negative, urease-positive Campylobacter from cattle faeces using CAT medium. This organism could not be cultured on blood-free Campylobacter medium (CCDA).

A study in which the productivity of CAT medium, blood-free media and semi-solid medium were compared, showed that CAT medium, used in parallel with membrane filtration on non-selective blood agar, is likely to be the most productive method for recovery of the greatest number of Campylobacter and Arcobacter species10.

Storage conditions and Shelf life
CAT Supplement SR0174 should be stored at 2-8°C in the dark. When stored as directed, the reagents remain stable until the stated expiry date shown on the packaging.

Quality control

Positive controls: Expected results
Campylobacter upsaliensis ATCC® 43954 * Good growth; pale colonies
Campylobacter jejuni ATCC® 33291 * Good growth; grey coloured colonies
Negative control:
Enterococcus faecalis ATCC® 33186 Inhibited
* This organism is available as a Culti-Loop®

References
1. Atabay, I., Corry, J.E.L., Post, D. 8th International Campylobacter Workshop 1995.
2. Sandstedt, K., Ursing, J., Walder, M. (1983). Curr. Microbiol. 8: 209-213.
3. Sandstedt, K. and Ursing, J. (1991). Sys. Appl. Microbiol. 14: 39-45.
4. Patton, C.M., Shaffer, N., Edmonds, P. et al. (1989). J. Clin. Microbiol. 27: 66-73.
5. Goosens, H., Vlaes, L., Butzler, J.P. et al. (1991). Lancet. 337: 1486-7.
6. Bolton, F.J., Hutchinson, D.N., Parker, G. (1987). J. Clin. Pathol. 40: 702-3.
7. Aspinall, S.T., Wareing, D.R.A., Hayward, P.G. and Hutchinson, D.N. (1993). J. Clin. Pathol. 46: 829-831.
8. Aspinall, S.T., Wareing, D.R.A., Hayward, P.G. and Hutchinson, D.N. (1996). J. Appl. Bact. 80: 667-672.
9. Atabay, H.I., Corry, J.E.L. and On, S.L.W. (1997). Lett. Appl. Microbiol. 24: 59-64.
10. Atabay, H.I., Corry, J.E.L. and Post, D.E. (1996). Campylobacters, Helicobacters and Related Organisms. Newell, D.G., Ketley, J.M. and Feldman, R. A. (eds) Part 1-5. Plenum Press, New York.

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