微生物菌種管理的相關(guān)流程
發(fā)布時間：
2022-12-27
作者：
微生物菌種管理的相關(guān)流程
1.1菌株的采集
實驗室用菌種一是到國家法定機構(gòu)采購，二是購買商業(yè)派生菌種，三是科研中菌種交流。不管是哪種來源，均統(tǒng)一采集。采集中嚴(yán)格按照規(guī)范執(zhí)行。要求包裝可靠，采集迅速，不泄漏不污染。保證菌種合格和環(huán)境安全。采集中要有菌種傳代標(biāo)識。選擇有資質(zhì)的標(biāo)準(zhǔn)菌株的合格供應(yīng)商，每批標(biāo)準(zhǔn)菌株必須附帶有供應(yīng)商的合格證或檢測報告或說明書，來證明所采購的標(biāo)準(zhǔn)菌株是合格的。
1.2標(biāo)準(zhǔn)菌株和驗收
實驗室收到標(biāo)準(zhǔn)菌株，首先應(yīng)進(jìn)行符合性感官檢查，記錄菌株號和標(biāo)準(zhǔn)菌株來源途徑信息，確保溯源性清楚。同時還應(yīng)記錄標(biāo)準(zhǔn)菌株名稱和數(shù)量、生產(chǎn)日期、接收日期和有無破損等情況。
1.3凍干標(biāo)準(zhǔn)菌株的復(fù)活
1.3.1開啟產(chǎn)品包裝：先用70%酒精棉擦拭外包裝，再打開使用。
1.3.2復(fù)活：選擇合適的培養(yǎng)基和培養(yǎng)條件（根據(jù)菌種使用說明書，見附錄）進(jìn)行復(fù)活。菌種的首次活化最好是在非選擇性瓊脂培養(yǎng)基上，除非特殊情況或特別推薦，一般不用液體培養(yǎng)基。凍干菌株的傳代次數(shù)不得超過5代，從標(biāo)準(zhǔn)菌株保藏中心購買的凍干標(biāo)準(zhǔn)菌株為第F0代。
注：培養(yǎng)基后面的數(shù)字編號為廣東環(huán)凱微生物有限公司的產(chǎn)品編號，如需采購，請聯(lián)系經(jīng)銷商或業(yè)務(wù)員。上表提供了菌種傳代短期保存的方法，供參考。
1.4工作菌株確認(rèn)方法及依據(jù)
用無菌接種環(huán)取上述培養(yǎng)物，在相應(yīng)的培養(yǎng)基平板（營養(yǎng)瓊脂、大豆胰蛋白胨瓊脂）上或相應(yīng)的細(xì)菌鑒別平板（如伊紅美藍(lán)、麥康凱、BP等上劃線分離單個菌落，置適宜條件下培養(yǎng)（若該類微生物為厭氧菌，則培養(yǎng)條件應(yīng)為厭氧條件）。以同樣方法取真菌和酵母菌至SDA（薩布羅培養(yǎng)基）平板上或玫瑰紅鈉培養(yǎng)基平板上，23～28℃下培養(yǎng)7d；培養(yǎng)后觀察是否具有典型的菌落狀態(tài)，然后挑取單一純菌落，進(jìn)行革蘭染色、鏡檢，觀察其染色特征及菌體形態(tài)以確定菌種。
1.5 污染處理
假如在該平板上發(fā)現(xiàn)有其他菌落生長，則說明操作有污染或菌種不純。要將
該污染培養(yǎng)物做滅菌處理，尋找原因，重新分離挑選純菌落。
1.6菌種保存
所有菌種均由實驗室專人(雙人雙鎖)保存于專用冰箱或其它保存方式。要建立菌種登記臺帳，詳細(xì)記錄菌種采集、保管、制備、使用、處置情況；每一種菌種一定要有檢測鑒定報告（具體的的鑒定方法見菌種質(zhì)量報告鑒定證書上的方法）；
具體菌種保存方法一般為：
將復(fù)蘇后肉湯與滅菌甘油按15%甘油比例（肉湯8.5mL+甘油1.5mL）混合，-30 ℃凍存，（可用2mL凍存管凍存多管），作為保藏儲備菌株F1代，也可使用商品化的菌種保存管。
1.7菌種的傳代
1.7.1標(biāo)準(zhǔn)菌株的復(fù)蘇：
① 菌種操作應(yīng)在無菌條件下進(jìn)行，防止雜菌污染。
② 每次使用和復(fù)蘇時只需將小珠在平板上滾動或置肉湯中培養(yǎng)。
1.7.2標(biāo)準(zhǔn)儲備菌株每轉(zhuǎn)接一次須進(jìn)行確認(rèn)。（確認(rèn)方法一般為他們各自獨有的形態(tài)，在鑒別培養(yǎng)基上的形態(tài)）
1.7.3工作菌株轉(zhuǎn)接的方法：
①、配制營養(yǎng)瓊脂培養(yǎng)基（溶血性弧菌加3%氯化鈉 ），121℃高壓滅菌15分鐘后分裝在試管內(nèi)，冷卻后備用。
②、在無菌條件下，用接種環(huán)挑取菌苔至新鮮試管上作“米”字形劃線接種，放置在36℃的培養(yǎng)箱內(nèi)培養(yǎng)24小時。
1.7.4工作菌株的使用
1.7.4.1內(nèi)部質(zhì)量控制：一個月一次陽性對照、培養(yǎng)基每批驗收；
1.7.4.2外部質(zhì)量控制：能力驗證、實驗室比對；
1.7.5工作菌株的期間核查
1.7.5.1期間核查的頻率：每半年對使用標(biāo)準(zhǔn)菌株進(jìn)行一次期間核查。
1.7.5.2工作菌株期間核查的方法及依據(jù)：同工作菌株的確認(rèn)一樣。
1.7.5.3建立標(biāo)準(zhǔn)菌株期間核查記錄。
1.8菌種的標(biāo)識和使用期限：
1.8.1標(biāo)識：菌種代數(shù)的計算為干粉菌種為第0代，轉(zhuǎn)接一次加一代。具體標(biāo)識為：例單增李斯特氏菌第0代，標(biāo)識為DZ-0，第一代標(biāo)識為DZ-01第一代有12支，則DZ-01-01、……、DZ-01-12；并標(biāo)明日期：如2016.02.13這樣填寫。
1.9 菌種的保藏
1.9.1將試管內(nèi)菌種放入冰箱中2～8℃冷藏保存。
1.9.2將傳代并經(jīng)過培養(yǎng)后的菌種放入冰箱中2～8℃保存。每支保存菌種需標(biāo)明菌名、標(biāo)準(zhǔn)編號、傳次、傳代日期。
1.10菌種的銷毀
1.10.1菌種使用后或超過貯存期的應(yīng)進(jìn)行銷毀。
1.10.2需銷毀的菌種，應(yīng)用高壓蒸汽（121℃）滅菌30分鐘。
1.10.3滅菌后，再進(jìn)行清洗和處理。
1.10.4銷毀的菌種應(yīng)做好記錄。銷毀時由化驗負(fù)責(zé)人監(jiān)督銷毀，保管人員負(fù)責(zé)銷毀。
參考資料：http://m.ricoportland.com/products_list/pmcId=25.html
The process of microbial strain management
1.1 collection of strains
Laboratory fungi are purchased from national legal institutions, commercial derived strains are purchased, and the exchange of strains in scientific research is conducted. No matter which source, all unified collection. The collection is carried out in strict accordance with the specifications. Require reliable packaging, rapid collection, no leakage and no pollution. To ensure qualified strains and environmental safety. In the collection, the identification of bacterial species should be carried out. Select qualified standard strains of qualified suppliers, each batch of standard strain must be attached with the supplier's certificate or test report or the instruction, to prove the purchase standard strains is qualified.
1.2 standard strains and acceptance
When the standard strains are received in the laboratory, the first step is to carry out the compliance sensory examination, record the number of strains and the source information of the standard strains, and ensure that the traceability is clear. At the same time, the name and quantity of the standard strain, the date of production, the date of receiving and whether there is any damage should be recorded.
1.3 revival of freeze-dried standard strains
1.3.1 open the product packaging: first wipe the outer packaging with 70% alcohol cotton, and then open it for use.
1.3.2 resurrection: select the appropriate medium and culture conditions (according to the instructions of the species, see appendix) for the resurrection. The first activation of the bacteria is preferably in a non-selective agar-agar medium, which is generally not used in liquid medium unless it is particularly recommended or under special circumstances. The number of generations of freeze-dried strains shall not exceed 5 generations. The freeze-dried standard strains purchased from the reserve center of standard strains shall be the F0 generation.
Note: the number behind the medium is the product number of guangdong huankai microorganism co., LTD. If you need to purchase, please contact the dealer or salesman. The above table provides a method for the short-term preservation of germ propagation for reference.
1.4 methods and basis for identification of working strains
With aseptic inoculation loops in these cultures, in the corresponding media tablet (nutrient AGAR, soybean tryptone AGAR) or the corresponding bacteria identification plate (such as eosin methylene blue and macconkey, BP streak on a single colony, place under the condition of appropriate training (if the microbes for anaerobic bacteria, the culture conditions for anaerobic conditions). In the same way in fungi and yeasts to SDA (broad culture medium) tablet or rosy sodium medium plate, 23 ~ 28 ℃ under training 7 d; After culture, it was observed whether there was a typical colony state, and then a single pure colony was selected for gram staining and microscopic examination.
1.5 pollution treatment
If other colonies are found on the plate, the operation is contaminated or the species is not pure. will
The contaminated culture was sterilized, the reasons were found, and the pure colonies were re-isolated and selected.
1.6 species preservation
All strains are kept by laboratory specializers (double lock) in special refrigerators or other storage methods. To establish a seed registry account and record in detail the collection, storage, preparation, use and disposal of strains; Each strain must have a test and identification report (the specific identification method is shown in the identification certificate of germ quality report);
Specific conservation methods of strains are generally:
The broth and sterilization after recovery glycerin 15% glycerol ratio (8.5 mL) 8.5 mL + glycerin broth mixture, and 30 ℃ cryopreserved, tube (or 2 mL cryopreserved cryopreserved multiple tube), the preservation reserve strain F1 generation, also can use commercial strains preservation pipe.
1.7 generation of strains
1.7.1 recovery of standard strains:
The germplasm operation should be carried out under sterile conditions to prevent contamination of miscellaneous bacteria.
In each use and resuscitation, tiny beads are rolled on a plate or incubated in a broth.
1.7.2 standard reserve strains shall be confirmed every time they are transferred. (the confirmation method is usually their own unique form, in the differentiation medium form)
1.7.3 methods of transfer of working strains:
(1), the preparation of nutrient AGAR medium (hemolytic vibrio and 3% sodium chloride), 121 ℃ high pressure sterilization packaging in vitro after 15 minutes, cooling backup.
(2), under aseptic conditions, using pick bacteria moss on the vaccination to fresh in vitro on "m" glyph crossed, placed in the incubator of the 36 ℃ for 24 hours.
1.7.4 use of working strains
1.7.4.1 internal quality control: once a month, the positive control and each batch of culture medium were checked and accepted;
1.7.4.2 external quality control: capability verification, laboratory comparison;
1.7.5 verification of working strains during the period
1.7.5.1 frequency of verification during the period: periodic verification of the use of standard strains is conducted every six months.
1.7.5.2 methods and basis of verification during the period of working strains: same as the identification of working strains.
1.7.5.3 establish inspection records of standard strains during the period.
1.8 identification and duration of use:
1.8.1 identification: the calculation of bacterial species algebra is that the dry powder species are the 0th generation, which is transferred to the first generation. The specific identification is as follows: for example, generation 0 of listeria monocytogenes was identified as dz-0, generation 1 was identified as dz-01, generation 1 had 12 branches, then dz-01-01, , DZ - 01-12; Date: such as 2016.02.13.
1.9 storage of strains
1.9.1 strains in vitro to be included in the refrigerator 2 ~ 8 ℃ cold storage.
1.9.2 will extend and after training were placed in the refrigerator for 2 ~ 8 ℃. Each conserved strain shall be marked with the name, standard number, transmission, and date of transmission.
1.10 destruction of strains
1.10.1 bacterial species shall be destroyed after use or beyond the storage period.
1.10.2 to destroy bacteria, the application of high pressure steam (121 ℃) sterilization for 30 minutes.
1.10.3 after sterilization, cleaning and treatment shall be carried out.
1.10.4 the destroyed strains shall be recorded. Destruction shall be supervised and destroyed by the person in charge of laboratory technology, and the custodian shall be responsible for destruction.