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 Insert4. 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. TryptoneSoya 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 thehandle 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 dissolvecompletely 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 pipetteonto the appropriate medium (e.g. Nutrient agar slant) and streak in the usualmethod.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 optimalgrowth.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 eachmicroorganism.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 cellmorphology and purity and run through the whole biochemical or confirmation tests tocheck 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 ofreference 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 theMicrobank 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 tothe 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 beadsfree 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 ofpreparation.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 asepticallyremoved from the vial and rolled across the surface of a non- selective agar plate orslant or may be placed into appropriate liquid medium. After removal, beads shouldnever be returned to the vial for any reason. The vial is returned to the freezerimmediately.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接种过的平板、斜面或者肉汤放在37°C 培养24-48小时。
一旦发现平板、斜面或者肉汤有微生物生长后立即从培养箱取出。
g. Label the plate, slant/broth with the cultures’ name, date of preparation and expirydate. Store in the refrigerator about 4°C.在平板、斜面或者肉汤上粘贴标签，注明菌种名称、准备日期和有效期。
放在冰箱大约4°C储存。
6. Issue a working culture plate/slant/broth as use frequency, usually as each week. Discardand use a new working culture if there is evidence of contamination. Working cultures can not more than 2 weeks in the refrigerator每次使用一支工作菌株，频率按照工作需要，通常为一个星期。
一旦发现菌株有污染现象后立即处理掉并使用新的工作菌株。