菌种管理规定菌种管理规范(中英文版)
密级Security of Classification：公开
文件编号Document No.：453453453004
版本/变更Version/Change: A/0
菌种管理规定/规范
Standard Operating Procedure of
M-6000 Electromagnetic Dynamic Mechanical Test System
人员信息
1.0目的Purpose：
为了规范菌种复活、传代、保藏、销毁的操作，特制订本标准操作规程。
In order to regulate the operation of bacterial species resurrection, passage, preservation and destruction, this STANDARD operating procedure is hereby formulated.
2.0范围Scope：
适用于检定菌复活、传代、保藏、销毁标准操作要求。
It is applicable to the standard operating requirements for the verification of bacterial resurrection, passage, preservation and destruction.
3.0术语和定义Terms and Definitions
无。
None.
4.0职责Responsibility
4.1检测员Inspector：
负责按本标准操作规程要求进行操作使用。
Be responsible for the operation and use according to the requirements of this standard.
4.2技术负责人Technical Director：
负责指导并监督检测员对本标准的执行。
Be responsible for guiding and supervising the implementation of this standard by inspectors.
5.0程序Process
5.1阳性菌取用过程中的防护Protection during the use of positive bacteria：
为避免菌种对人员的侵害，阳性菌的接收和取用，应佩戴一次性手套和口罩进行操作。
阳性试验应在阳性间生物安全柜内进行，操作时人员应佩戴一次性无菌手套和口罩，操作完
成后，应将所有与菌种相关的试验废弃物装在密封容器中，进行灭菌然后才能处理。
In order to avoid the invasion of bacteria on the personnel, the reception and use of positive bacteria, should wear disposable gloves and masks for operation. The positive test should be carried out in the biosafety cabinet of the positive room. During operation, personnel should wear disposable sterile gloves and masks. After completion of operation, all test wastes related to bacteria species should be packed in a sealed container for sterilization before disposal.
5.2冷冻菌种的管理Management of refrigerant strains：
5.2.1菌种复活Strains of resurrection
取符合要求的冻干菌种，清洁装有冻干菌种（G0）的容器（用75%乙醇）后，用适宜方法将装有菌种的容器打开。用无菌吸管（或经灭菌处理的移液管）吸取1-2ml适
量的培养基（金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、枯草芽孢杆菌接种到胰
酪大豆胨液体培养基，生孢梭菌接种到硫乙醇酸盐流体培养基中）滴加到容器中，轻
轻旋转容器使冻干菌种和液体培养基充分混匀并完全溶解，再将容器内的菌液用转移
至5ml胰酪大豆胨液体培养基（生孢梭菌用硫乙醇酸盐流体培养基）中复壮，根据菌
种类型采用适宜的培养条件（金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、枯草芽
孢杆菌、生孢梭菌培养温度30-35℃，18-24h；白色念珠菌培养温度20-25℃，2-3d；
黑曲霉培养温度20-25℃，5-7d）下培养（G1），观察培养基是否生长或浑浊。浑浊说明菌种复活生长；如不生长或浑浊，用121℃，30min高温高压灭菌。
After cleaning the container containing freeze-dried fungus (G0) with 75% ethanol, open the
container containing fungus (G0) with appropriate method.With the aseptic suction pipe (or
the sterilization process of pipette) from 1 to 2 ml of adequate medium (staphylococcus
aureus, e. coli, pseudomonas aeruginosa, bacillus subtilis, vaccination to pancreatic dairy
soy peptone liquid medium, clostridium raw spore inoculation to sulfur glycolic acid salt fluid medium) drops to the container, gently rotating container make the lyophilized strains and
liquid medium fully blending and completely dissolved, then the bacteria within the container liquid with a transfer to 5 ml pancreatic dairy soy peptone liquid medium (born spore
clostridium fluid medium) with sulfur glycolic acid salt of rejuvenation,Appropriate culture
conditions (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus
subtilis, and Clostridium genitalium) were adopted according to the strain types. The culture
temperature was 30-35℃for 18-24h. The culture temperature of Candida albicans was 20-
25℃, 2-3D. Aspergillus Niger was cultured at a temperature of 20-25℃for 5-7 days (G1),
and the growth or turbidity of the medium was observed. Turbidity indicates that the strain is
resurrecting and growing. If there is no growth or turbidity, it is sterilized at 121℃for
30min under high temperature and pressure.
5.2.2复壮菌种转接Rejuvenation strain transfer
使用无菌接种环沾取复壮后的菌种转接到相应的斜面培养基上或液体培养基中进行培养、使用、传代。金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、枯草芽孢杆菌接种到胰酪大豆胨琼脂培养基斜面30-35℃，培养不超过3d，生孢梭菌接种到硫乙醇酸盐流体培养基中30-35℃，培养不超过3d；白色念珠菌接种到沙氏葡萄糖琼脂培养斜面温度20-25℃，培养不超过5d；黑曲霉培养温度20-25℃，培养不超过5d。
Sterile inoculation rings were used to dip the rejuvenated strains into the corresponding inclined medium or liquid medium for culture, use and passage. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis were inoculated to tryptone AGAR medium inclined plane at 30-35℃for culture not more than 3d, and Clostridium biosporum was inoculated to thioglycolate fluid medium at 30-35℃for culture not more than 3D. Candida albicans was inoculated to the inclined surface temperature of 20-25℃for cultivation with glucose AGAR for no more than 5 days. Aspergillus Niger was cultured at 20-25℃for no more than 5 days.
5.2.3菌种传代Strains of batches
●试验用菌种每月传代一次，在阳性实验室内使用传代用菌种进行传代。工作用菌种
不可以再传代。
Test with bacteria to extend once a month, in the positive strains to extend the batches
using in the laboratory. Working strains may not be re-passed on.
●先点燃酒精灯，在火焰旁的上部空间操作。灼烧接种环至变红。现在是否在使用红
外接种环灭菌器
Lit alcohol lamp, first in the fire at the side of the upper space operations. Burn the inoculation ring until it turns red. Is the infrared inoculation ring sterilizer now in use.
●再将原有的菌种斜面培养基（简称菌种管）与待接种的新鲜斜面培养基（简称接种
管），持在左手拇指、食指及中指之间，要注意能清楚的观察到斜面，菌种管在前，接种管在后。应斜持试管呈45°角，使试管内斜面向上，两试管口平齐，注意不要持成水平，以免管底凝集水浸湿培养基表面。
Then original strains cant medium (hereinafter referred to as strain tube) and cant stay inoculation of fresh culture medium (hereinafter referred to as inoculation tube), hold in left hand between thumb and forefinger and middle finger, cant note can be observed clearly, strains in the former, vaccination tubes in the back. Hold the test tube at an Angle of 45°, so that the inclined plane in the test tube is upward and the two test tube mouths are flat. Be careful not to hold the test tube to the level, so as to avoid the bottom of the tube agglutination water wetting the medium surface.
●首先，以右手在火焰旁转动两管的塞子，以便接种时易于拔取。再以右手持接种环
的柄端，垂直或稍斜的把接种环在火焰上烧灼。顶端的环必须烧红，以彻底灭菌。
环以上凡接种时可进入试管的部分，也应通过火焰灼烧。灼烧时，应把环置于酒精灯外焰上，因外焰的温度比内焰高，容易烧红。
First, two tube plug rotates at his right hand in a fire, so that it is easy to pull out vaccination. Holding the handle end of the inoculation ring in the right hand, burn the inoculation ring on the flame vertically or slightly obliquely. The top ring must be burned red to sterilize thoroughly. The part above the ring that can be inoculated into the test tube should also be burned by flame. When burning, the ring should be placed on the external flame of the alcohol lamp, because the temperature of the external flame is higher than the internal flame, easy to burn red.
●使用右手的小指和手掌之间以及无名指与小指之间，在火焰旁分别拔出两支试管的
塞子，持住。再将试管口在火焰上通过，以杀灭管口可能沾污的细菌，但勿烧烫。
Use right hand little finger and ring finger and little finger between palm and between, pull
out the two test tubes, respectively, in a fire plug, hold on. Pass the mouth of the tube over the flame to kill bacteria that may contaminate the mouth of the tube, but do not burn it.
●现将烧灼过的接种环插入菌种管内，先接触培养基，如有熔印则表明接种环未冷却，
能烫死细菌。待冷却后，挑取少量菌种，插入接种管中划线接种。划线时一般先由下而上划一直线后，再由上而下划曲线。
Will now burning inoculated strains of ring insert tube of contact medium first, if there is any melting suggests vaccination ring not cooling, can be hot dead bacteria. After cooling, select a small number of strains and insert them into the inoculation tube for lining inoculation. When the line is drawn, a straight line is drawn first from bottom to top, and then a curve is drawn from top to bottom.
●当接种完毕，接种环灼烧灭菌。
When finished, inoculated ring burning sterilization.
5.2.4传代菌种保藏Preservation of subculture
●将传代后的斜面菌种放入冰箱2-8℃保存冷藏。
Cant strains after will extend the store in a refrigerator for 2-8 ℃refrigerated.
●制备4支斜面菌种，每支保存菌种需标明菌名，代次，传代日期，用途（3支工作
菌株、1支传代菌株）。
Cant preparation of four species, each save bacteria strains to indicate the name, generation time, extend the date, use 1 batches (3 job strain, strain).
5.2.5代数确定Algebra to determine
工作菌株的传代不得超过5代（从菌种保藏机构获得的标准菌株为0代）。任何形式的转种（在培养基中培养萌发）均被认为传代1次。使用时，记录应以当前复苏的菌株代数进行记录。
The passage of working strains shall not exceed 5 generations (the standard strains obtained from the species preservation mechanism shall be 0 generations). Any form of
transplanting (germination in culture medium) is considered to be passed once. When used, records shall be recorded in the current resuscitated strain algebra.
5.3商业菌株的管理Management of commercial strains
5.3.1商业菌种的保藏Preservation of commercial strains
新购的符合要求的商业菌种应贮存在-18℃以下的环境里，有效期为6个月。
Newly purchased qualified commercial strains shall be stored under -18℃for 6 months.
5.3.2菌种的复溶及稀释Recombination and dilution of strains
a.打开胶囊，用注射器按产品的规格取相应的复溶液。
Open the capsule and take the corresponding compound solution according to the
product specifications with a syringe.
b.将复溶液注入到装有菌种的瓶中。
Inject the complex solution into a bottle containing the strain.
c.将复溶的菌液放在旋涡混合仪中混匀1min，使其充分溶解，如取样间隔超过2min，
取样前还需要振3s,混匀后用移液器取1ml菌液，加入到9ml的0.9%无菌氯化
钠溶液中逐级稀释。
Put the redissolved bacterial solution in the vortex mixer for 1min to make it fully
dissolved. If the sampling interval exceeds 2min, vibrate 3s before sampling.
5.3.3菌种计数Species count
将稀释后的菌液取出1ml接种到相应的培养基（金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、枯草芽孢杆菌接种到胰酪大豆胨琼脂培养基，生孢梭菌接种到硫乙醇酸盐流体培养基中在30-35℃，培养18-24h；白色念珠菌培养温度20-25℃，2-3d；
黑曲霉培养温度20-25℃，5-7d）中进行培养计数。
The diluted bacterial liquid was taken out and inoculated into the corresponding medium (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis were inoculated into the soy peptone AGAR medium of trypanosporin, and Clostridium
biosporum was inoculated into the thioglycolate fluid medium at 30-35℃for 18-24h; The culture temperature of Candida albicans was 20-25℃, 2-3D. The culture temperature of Aspergillus Niger was 20-25℃, 5-7 days).
5.4菌种销毁Bacterial species destruction
销毁菌种应用高压蒸汽（121℃）灭菌30min。
The bacteria were destroyed and sterilized by high-pressure steam (121℃) for 30min.
6.0相关文件Related Documents：
无。
None.
7.0相关记录Related Records：
无。
None.
8.0修订说明Revision Explanation: