哥伦比亚血琼脂

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CBA培养基产品基本信息

培养基名称: 哥伦比亚血琼脂
英文名称:
Columbia Blood Agar
 
培养基类型: 营养培养基
产品目录号:
M216-02,M800-01
 
产品规格: --
保存条件:
避光,密封,阴凉干燥处保存。配制好的培养基应该在2天内使用,不能长期放置。
 
产品性状: 红色至深红色凝胶,不透明。
注意事项:
避免摄入、呼入、皮肤接触。配制时在通风橱中进行,戴口罩、手套、护目镜。
 
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产品描述:


哥伦比亚血琼脂(Columbia Blood Agar)培养基是以哥伦比亚琼脂或胰蛋白胨大豆琼脂(TSA培养基)为培养基基础,加5%脱纤羊血配制而成的。哥伦比亚血琼脂是一种高度营养型的固体培养基,可以用来培养和回收各种苛养和非苛养型微生物,是应用非常广泛的培养基。

哥伦比亚血琼脂高度营养性来自于配方中的特殊混合蛋白胨,酵母提取物则提供B族维生素,刺激微生物的生长,尤其是细菌的生长。羊血提供X因子(亚铁血红素),是很多细菌生长所必须的,但是缺少V因子(烟酰胺腺嘌呤二核苷酸,NAD)。因此,既需要X因子又需要V因子的流感嗜血杆菌不能在哥伦比亚血琼脂培养基生长。该培养基能够支持微生物快速茁壮的生长,还能提供高分辨率的溶血反应,更具特征性的菌落形态特征和更高的色素生成量。




哥伦比亚血琼脂培养基的用途


临床实验室里常用哥伦比亚血琼脂鉴定溶血类型。但需要注意的是,哥伦比亚学琼脂有相对较高的含糖量,造成链球菌的Beta溶血反应偏绿色,可能误读成Alpha。


哥伦比亚血琼脂培养基本身不具有选择性,加入不同的抑制剂,如抗生素及其组合,就成了具有选择性和鉴别性的培养基,用于各种微生物的分离。




哥伦比亚血琼脂培养基的配方与配制方法



哥伦比亚血琼脂培养基配方:

哥伦比亚血琼脂(ISO) 43g
脱纤维羊血          50ml

配制方法:

1. 称取43g哥伦比亚血琼脂(ISO),加1000mL去离子水重悬。

2. 边加热边搅动,煮沸1min,使其完全溶解。

3. 121°C高温蒸汽灭菌15min。

4. 待冷却至50°C左右时,加入50ml脱纤维羊血,混匀后倒平板。配制好的培养基应2-8°C避光保存。



哥伦比亚血琼脂的使用——实验方法


Elek Test
Elek-Test 示意图

即白喉棒状杆菌毒力实验,48h,即可见毒素-抗毒素沉淀线。

实验流程:
1. Mix a tube of melted nutrient agarwith 2 ml of sterile horse serum.
2. Rotate the tube to mix the serum and agar. Do not shake the tube.
3. Pour the mixture into a sterile petri dish.
4. Using lightly flamed forceps, lay the strip of anti-toxin impregnated filter paper across the centre of the petri dish allowing it to sink beneath the agar surface.
5. Allow the agar to set, then lift one corner of the lid and let the plate dry for 30-45 min in the incubator.
6. When dry inoculate with a toxinogenic strain of C. diphtheriae by streaking a single line of inoculum across the plate and paper strip at right angles to the strip.
7. Repeat this about 1 inch away from the C. diphtheriae inoculum with a test strain.
8. Incubate the plate for 24 hrs and observe the results.

实验结果:
After 24 hours of incubation at 37°C, plate is examined with transmitted light for the presence of fine precipitin lines at 45 degree angle to the streaks.
Positive Test: Precipitin lines form at zone of equivalence, test organism is toxigenic.



Nagler Test(Nagler Reaction)

是产气荚膜梭菌诊断实验。
100ml哥伦比亚学琼脂中加入5mlFildes提取物,10ml卵黄乳液,100-125mg/LNeomycin_1.html' target='_blank'>新霉素,产气荚膜梭菌抗毒素。


产气荚膜梭菌鉴定Nagleer-reaction示意图

实验流程:
1. Label and dry a egg yolk media plate and mark the plate into two halves.
2. Inoculate 60 µl of Clostridium perfringens type A antitoxin in half of the plate, spread over the surface of agar using a spreader and allow to absorb and dry.
3. Mark the side of the plate in which the antitoxin is inoculated.
4. Streak the test organism in a straight line from the toxin free agar half of the plate to toxin containing side. Repeat the same procedure with control strains on the same plate.
5. Incubate anaerobically at 35-37°C for 24-48 hrs.
6. Examine the plate for an opalescent halo around the inoculum and inhibition by antitoxin.

结果与分析:


产气荚膜梭菌Naglers-reaction结果

CAMP Test

初步鉴定无乳链球菌,将无乳链球菌与其它Beta溶血B群链球菌区别开来。初步鉴定单增李斯特菌。

A. Standard CAMP test
1. Using an inoculating loop, streak a beta-lysin-producing Staphylococcus aureus (ATCC25923) in a straight line across the center of a sheep blood agar plate.
2. Streak test organism in a straight line perpendicular to the S. aureus leaving 1cm space between the two streaks. (Multiple organisms can be tested on a single plate if they are 3 to 4mm apart).
3. Incubate the plate at 37 degree Celsius in ambient air for 18-24 hours.

B. CAMP Spot test
1. Remove test kit from the freezer and allow it to thaw.
2. Using a pipette, place one drop of CAMP spot test reagent next to a characteristic colony grown for 18-24 hours on blood agar plate.
3. Incubate the plate aerobically or in 5-10% CO2 at 37°C for 20-30 minutes.
4. Observe, using transmitted light, for an arc or circle of enhanced hemolysis next to the colony. If reaction is negative, re-incubate for an additional 30 minutes.

Reversed CAMP Test
是高度灵敏性和专一性的产气荚膜梭菌检测实验。与Nagler Test作用相同,可相互替换。

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