标准菌珠的管理

Procedure for Maintaining and Handling Reference Microorganism

标准菌株的管理与控制

1. Aim 目的

To maintain and handle stock cultures of reference strains for quality control and performance testing purposes. This procedure will minimize the opportunity for cross contamination, mutation and alteration of typical characteristic of the reference strains.

规范用于微生物实验质量控制和操作评估的标准菌株管理与使用程序，尽量减少交叉污染确保实验

结果可靠与实验室安全。

2. Scope 范围

The working instruction applies to maintaining and handling of reference microorganism in the microbiological laboratory

本工作指引适用于微生物实验室所有标准菌株的管理与使用。

3. Reference 参考

1. LI 00.703 Performance Test of Culture Media.

2. Microbank Advance Microorganism Storage System Package Insert

4. Contents 内容

4.1 Procedure 程序

1. Resuscitate the reference microorganism strains by:

标准菌株的复壮

a. Ensure that the appropriate liquid medium containing 0.5 – 1.0 ml (e.g. Tryptone

Soya Broth or Buffer Peptone Water) is at room temperature

将复壮所用的0.5-1.0ml液体培养基 (如TS或BPW)至于室温。

b. Remove the loop from the refrigerator and warm it to room temperature.

将标准菌株从冰箱取出至于室温。

c. Remove the sheath from the loop aseptically.

无菌操作去处菌株的包装物。

d. Put the liquid medium in the strain container or Cut off the loop shaft from the

handle using a pair of sterilized scissors into the liquid medium.

无菌操作将液体培养基倒入菌株容器；或者无菌操作剪开菌株包装物，将菌株倒入液

体培养基。

e. Incubate the tube in a 37°C incubator long enough just for the film to dissolve

completely out of the loop.

将试管在37°C下放置一段时间后，使菌种完全溶解到培养集中。

f. Shake the tube gently to suspend the microorganism.

轻轻摇动试管形成悬浮液。

g. Inoculate several drops of the suspended microorganism using a Pasteur pipette

onto the appropriate medium (e.g. Nutrient agar slant) and streak in the usual

method.

从菌种悬浮液中接种几环到培养基平板(如营养琼脂)划线培养。

h. Incubate the plates in an appropriate atmosphere and temperature for the optimal

growth.

将接种过的平板置于合适的温度和条件下培养复壮。

2. Take a few colonies from one of the slant and streak onto the test media as per LI for each

microorganism.

从培养过的平板或斜面上挑取几个菌落，按照相应的LI接种到选择性培养基上。

3. When the cultures are plated out, checked for correct morphology. Gram stained for cell

morphology and purity and run through the whole biochemical or confirmation tests to

check for purity of culture strains. Record all observation on the reference culture check worksheet.

将挑选出的菌落按照相应的LI进行菌种确认。进行革兰氏染色或者镜检，并纯化后按照相应的LI进行生化试验确认。记录下所有的现象。

4. Reference strain check as in step 3 should be done yearly or when ever a new lot of

reference strain is purchased.

在每年复壮或者买入时都要进行菌种确认试验。

5. If test on culture purity is satisfactory, preserve the reference stock culture using the

Microbank Frozen Beads system as follow:

菌种确认达到要求后，使用Microbank Frozen Beads标准储备菌株。

a. Take a generous inoculum (18 – 24 hours) from the reference stock and transfer to

the vial containing frozen beads and broth aseptically. Close vial tightly and invert 4

– 5 times to emulsify the organism. DO NOT VORTEX.

取一定量的标准菌株培养物（18-24小时），无菌操作接种到装有小珠的小瓶内并加

入肉汤（BPW）。关紧小瓶并颠倒4-5次乳化培养物。不要剧烈摇晃，避免液体撒

出。

b. The excess cryopreservative should be well aspirated leaving the inoculated beads

free of liquid if possible. Close the vial finger tight.

将多余的液体吸出。关紧小瓶。

c. Label with permanent marker the vials as reference stock cultures with date of

preparation.

在小瓶上贴上标签，标明菌种名称和准备日期。

d. The vials are frozen (approximately -20°C) for storage in the freezer for a year.

小瓶在冷冻条件下（大约-20°C）可储存一年。

e. To prepare the working cultures from the Microbank: A single bead is aseptically

removed from the vial and rolled across the surface of a non- selective agar plate or

slant or may be placed into appropriate liquid medium. After removal, beads should

never be returned to the vial for any reason. The vial is returned to the freezer

immediately.

从Microbank中准备工作菌株：从小瓶中无菌操作取出一个小珠，接种到非选择性培

养基平板或斜面，或者放入液体培养集中。小珠从小瓶中取出后决不可以再放回。立

即将小瓶放回冷冻。

f. The inoculated plate, slant or broth is incubated at 37°C for 24 hours – 48 hours.

Plate/ slant or broth should be taken out from the incubator once growth (e.g. turbid)

is observed