CHARCOAL AGAR

Code: CM0119

A medium for the cultivation and isolation of Bordetella pertussis and Haemophilus influenzae

百日咳博德特氏菌和流感嗜血杆菌培养分离培养基

Typical Formula*	gm/litre
`Lab-Lemco’ powder	10.0
Peptone	10.0
Starch	10.0
Charcoal bacteriological	4.0
Sodium chloride	5.0
Nicotinic acid	0.001
Agar	12.0
pH 7.4 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 51g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C, add 10% of defibrinated blood and mix gently. The medium is made selective for the isolation of Bordetella pertussis and Bordetella parapertussis by the addition of Bordetella Selective Supplement SR0082.

To one vial add 2ml of sterile distilled water and dissolve the contents completely. Add this solution to 500ml of sterile, molten Charcoal Agar, cooled to 50°C, together with 10% v/v defibrinated horse blood SR0050. Mix well before pouring into sterile Petri dishes.
For Haemophilus influenzae, omit the selective agents and convert to `chocolate’ agar.

Transport Medium for Bordetella pertussis
The vial contents may be added to 500ml of half-strength Charcoal Agar + 10% v/v defibrinated horse blood SR0050 for use as a transport medium for B. pertussis.

Description
Charcoal Agar was developed by Oxoid to provide a non-blood containing medium for the cultivation of Bordetella pertussis and Haemophilus influenzae. Proom¹ showed that nicotinic acid was an essential growth factor for the bordetellae. Ensminger et al.² used a charcoal medium for the growth of Bordetella pertussis in vaccine production and found that the medium could replace Bordet-Genou. Mishulow et al.³ used charcoal agar for Bordetella pertussis cultivation.

Haemophilus influenzae is cultivated on the medium containing 10% `chocolated’ blood but no antibiotics. The inoculated plate is incubated for 2 to 3 days at 37°C. The colonies are usually small, transparent and droplet-like, but some transformation to the `rough’ type colony may occur. Species differentiation is performed by examination of the need for X and V growth factors, on Blood Agar Base CM0055.
The greatest problem in the isolation of Bordetella species from naso-pharyngeal secretions is the suppression of unwanted flora during the long incubation period on very nutritious media.

Fleming’s first in vitro demonstration of penicillin was to show that it could help isolate Bordetella pertussis on media⁴. Lacey⁵ confirmed this but found that the penicillin-resistant flora still caused problems. He supplemented penicillin with 2µg/ml 4,4’ diamidino-diphenylamine dihydrochloride (M & B 938) thereby increasing the selectivity of this medium.

Broome et al.⁶ found methicillin to be superior to penicillin in suppressing unwanted naso-pharyngeal flora but the earlier publication of Sutcliffe and Abbott⁷ where cephalexin (40µg/ml) was shown to be superior to penicillin, has proved to be the most significant advance.

The benefits of cephalexin as a selective agent for Bordetella pertussis have been confirmed ^8,9,10,11. The ability to recover stressed cells and the much longer shelf life (6-8 weeks) are added benefits to its superiority at suppressing unwanted naso- pharyngeal growth.

Regan and Lowe⁸ showed that half-strength Oxoid Charcoal Agar, supplemented with 40µg/ml cephalexin SR0082 v/v lysed, defibrinated horse blood was an excellent enrichment and transport medium.

The efficacy of this transport medium has been confirmed by other workers¹².

方向
将 51 克悬浮在 1 升蒸馏水中。煮沸以完全溶解。通过在 121°C 高压灭菌 15 分钟进行灭菌。冷却至 50°C，加入 10% 的去纤维化血液并轻轻混合。通过添加博德特氏菌选择性补充剂 SR0082，该培养基可选择性地分离百日咳博德特氏菌和副百日咳博德特氏菌。

向一个小瓶中加入 2ml 无菌蒸馏水并完全溶解内容物。将此溶液与 10% v/v 去纤维化马血 SR0050 一起添加到 500 毫升无菌、熔融的木炭琼脂中，冷却至 50°C。在倒入无菌培养皿之前充分混合。
对于流感嗜血杆菌，省略选择性试剂并转化为“巧克力”琼脂。

百日咳博德特氏菌运输培养基
可将小瓶内容物添加到 500ml 半浓度木炭琼脂 + 10% v/v 去纤维化马血 SR0050 中，用作百日咳杆菌的转运培养基。

描述
木炭琼脂由 Oxoid 开发，为百日咳博德特氏菌和流感嗜血杆菌的培养提供不含血的培养基。 Proom1 表明烟酸是博德氏菌的必需生长因子。 Ensminger 等人 2 在疫苗生产中使用木炭培养基培养百日咳博德特氏菌，发现该培养基可以替代 Bordet-Genou。 Mishulow 等人 3 使用木炭琼脂培养百日咳博德特氏菌。

流感嗜血杆菌在含有 10%“巧克力”血但不含抗生素的培养基上培养。接种板在 37°C 下孵育 2 至 3 天。菌落通常很小、透明且呈液滴状，但可能会发生一些向“粗糙”型菌落的转变。通过在血琼脂培养基 CM0055 上检查对 X 和 V 生长因子的需求来进行物种分化。
从鼻咽分泌物中分离博德特氏菌的最大问题是在非常有营养的培养基上的长潜伏期内抑制不需要的菌群。

Fleming 对青霉素的首次体外演示是为了证明它可以帮助在培养基上分离百日咳博德特氏菌4。 Lacey5 证实了这一点，但发现耐青霉素的菌群仍然会引起问题。他在青霉素中添加了 2µg/ml 4,4′ 二脒基-二苯胺二盐酸盐 (M & B 938)，从而提高了该培养基的选择性。

Broome 等人 6 发现甲氧西林在抑制不需要的鼻咽菌群方面优于青霉素，但早先发表的 Sutcliffe 和 Abbott7 表明头孢氨苄 (40µg/ml) 优于青霉素，已被证明是最重要的进步.

头孢氨苄作为百日咳博德特氏菌的选择剂的益处已得到证实 8,9,10,11。恢复受压细胞的能力和更长的保质期（6-8 周）使其在抑制不必要的鼻咽生长方面的优势更加明显。

Regan 和 Lowe8 表明，半强度 Oxoid Charcoal Agar，辅以 40µg/ml 头孢氨苄 SR0082 v/v 裂解、去纤维化的马血，是一种极好的富集和转运培养基。

这种运输介质的功效已得到其他工人的证实12。

Technique
The following technique for the laboratory diagnosis of Pertussis is recommended¹¹.
1. Collect pernasal swabs in the early stage of the illness and place in tubes of half-strength Charcoal Agar supplemented with 10% v/v lysed, defibrinated horse blood and 40mg/ml cephalexin.
2. Generously inoculate the swabs on to thick layers of Charcoal Agar containing 10% v/v defibrinated horse blood and 40µg/ml cephalexin (SR0082).
A non-selective medium in which the cephalexin is omitted may be used in addition.
3. Perform direct fluorescent antibody (DFA) tests on the secretions, using Bordetella pertussis and Bordetella parapertussis-conjugated antisera, to help make an earlier diagnosis.
4. Replace the swabs in the original transport medium and hold at room temperature. If the culture plates become overgrown with commensal flora or fungi, use the swabs to inoculate fresh plates of medium.
5. Incubate the plates at 35°C in a moist atmosphere (60-70% humidity) for up to six days. Examine the plates after 40 hours incubation and twice-daily thereafter.
6. Look for small, shiny, greyish-white, round convex colonies. Suspicious colonies should be Gram stained, using a two- minute safranin counterstain. Some pleomorphic cells may be seen, caused by the cephalexin in the selective medium.
7. Confirm the identification with DFA tests on the suspicious colonies.

Precautions
Stuart’s transport medium or similar formulation media should not be used for Bordetella-containing specimens¹³.
Two pernasal swabs should be taken from each patient, one through each nostril¹⁴.
Make sure the charcoal remains in suspension when dispensing the medium by gently swirling the flask.
Lysed horse blood is used in the transport medium but whole blood is used in the isolation medium.
Most naso-pharyngeal flora are inhibited by cephalexin but Pseudomonas aeruginosa and some fungi may grow through. Amphotericin B can be added (12mg/ml) as an antifungal agent to prevent the growth of filamentous fungi. However, this level of amphotericin B can be inhibitory to Bordetella pertussis and should not be used routinely.

技术
推荐以下用于百日咳实验室诊断的技术11。
1. 在疾病早期收集鼻拭子，置于半强度木炭琼脂管中，添加 10% v/v 裂解、去纤维的马血和 40mg/ml 头孢氨苄。
2. 将拭子大量接种到含有 10% v/v 去纤维化马血和 40µg/ml 头孢氨苄 (SR0082) 的厚层木炭琼脂上。
另外，也可以使用省略了头孢氨苄的非选择性培养基。
3. 使用百日咳博德特氏菌和副百日咳博德特氏菌结合抗血清对分泌物进行直接荧光抗体 (DFA) 测试，以帮助做出早期诊断。
4. 将拭子更换到原来的运输介质中并保持在室温下。如果培养板长满了共生菌群或真菌，请使用棉签接种新鲜的培养基板。
5. 将板在 35°C 的潮湿环境（60-70% 湿度）中孵育长达 6 天。孵育 40 小时后检查板，之后每天两次。
6. 寻找小的、有光泽的、灰白色的圆形凸状菌落。可疑菌落应使用两分钟的番红复染剂进行革兰氏染色。可以看到一些多形性细胞，这是由选择性培养基中的头孢氨苄引起的。
7. 用 DFA 测试对可疑菌落进行确认。

防范措施
Stuart 的运输培养基或类似的配方培养基不应用于含有博德特氏菌的标本 13。
应从每位患者身上采集两支经鼻拭子，一个通过每个鼻孔 14。
轻轻旋转烧瓶，确保在分配培养基时木炭保持悬浮状态。
裂解的马血用于运输介质，但全血用于隔离介质。
大多数鼻咽菌群被头孢氨苄抑制，但铜绿假单胞菌和一些真菌可能会通过。可以添加两性霉素 B (12mg/ml) 作为抗真菌剂，以防止丝状真菌的生长。然而，这种水平的两性霉素 B 可以抑制百日咳博德特氏菌，不应常规使用。

METRONIDAZOLE SUSCEPTIBILITY TEST FOR

Helicobacter pylori
Charcoal agar supplemented with a concentrate of essential growth factors has been reported to be a reliable testing medium for determining metronidazole resistance in Helicobacter pylori¹⁵.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C.

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

甲硝唑药敏试验

幽门螺杆菌
据报道，添加了必需生长因子浓缩物的木炭琼脂是确定幽门螺杆菌对甲硝唑耐药性的可靠测试培养基15。

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。
将准备好的培养基板储存在 2-8°C。

外貌
脱水介质：黑色、自由流动的粉末
制备培养基：黑色凝胶

With Cephalexin:
Positive controls:	Expected results
Bordetella pertussis ATCC® 8467	Good growth; grey coloured colonies
Bordetella parapertussis NCTC 10521	Good growth; grey coloured colonies
Negative controls:
Staphylococcus aureus ATCC® 25923*	inhibited
Klebsiella pneumoniae ATCC® 13883*	inhibited
Without Antibiotics:
Positive control:
Haemophilus influenzae ATCC® 35056*	Good growth; grey coloured colonies
Negative control:
Uninoculated medium	No change

* This organism is available as a Culti-Loop®

References 参考文献
1. Proom H. (1955) J. Gen. Microbiol. 12 (1). 63-75.
2. Ensminger P.W., Culbertson C.G. and Powell H.M. (1953) J. Infect. Dis. 93 (3). 266-268.
3. Mishulow Lucy, Sharpe L.S. and Choen Lillian L. (1953) Amer. J. Pub. Health 43 (11). 1466-1472.
4. Fleming A. (1932) J. Path. Bact. 35. 831-842.
5. Lacey B.W. (1954) J. Hyg. 59. 273-303.
6. Broome C.V., Fraser D.W. and English J.W. (1979) In Internat. Symp. on Pertussis DHEW J. Washington DC pp 19-29.
7. Sutcliffe E.M. and Abbott J.D. (179) BMJ ii. 732-733.
8. Regan J. and Lowe F. (177) J. Clin. Microbiol. 6. 303-309.
9. Stauffer L.R., Brown D.R. and Sandstrom R.E. (1983) J. Clin. Microbiol. 17. 60-62.
10. Giligan P.H. and Fisher M.C. (1984) J. Clin. Microbiol. 20. 891-893.
11. Young S.A., Anderson G.L. and Mitchell P.D. (1987) Clin. Microbiol. Newsletter 9. 176-179.
12. Hoppe J.E., Worz S. and Botzenhart K. (1986) Eur. J. Clin. Micro. 5. 671-673.
13. Gastrin L., Kallings O. and Marcetic A. (1968) Acta. Path. Microbiol. Scand. 74. 371–375.
14. Regan J. (1980) Clin. Microbiol. Newsletter 2. 1-3.
15. Henriksen T.H., Brorson O, Schoyen R. et al. (1997) J. Clin. Microbiol. 35. 1424-1426.