培養(yǎng)基的制備、貯存及質(zhì)量控制方法
發(fā)布時(shí)間：
2022-12-27
作者：
沉降菌測(cè)試用胰酪大豆胨瓊脂培養(yǎng)基平板
培養(yǎng)基的制備、貯存及質(zhì)量控制方法
培養(yǎng)基可按處方配制，也可使用按處方生產(chǎn)的符合規(guī)定的脫水培養(yǎng)基。
在制備培養(yǎng)基時(shí)，應(yīng)選擇質(zhì)量符合要求的脫水培養(yǎng)基或單獨(dú)配方組分進(jìn)行配制。脫水培養(yǎng)基應(yīng)附有處方和使用說(shuō)明，配制時(shí)應(yīng)按使用說(shuō)明上的要求操作以確保培養(yǎng)基的質(zhì)量符合要求，不得使用結(jié)塊或顏色發(fā)生改變的脫水培養(yǎng)基。脫水培養(yǎng)基或單獨(dú)配方組分應(yīng)在適當(dāng)?shù)臈l件下儲(chǔ)存，如低溫、干燥和避光，所有的容器應(yīng)密封，尤其是盛放脫水培養(yǎng)基的容器。商品化的成品培養(yǎng)基除了應(yīng)附有處方和使用說(shuō)明外，還應(yīng)注明有效期、貯存條件、適用性檢查試驗(yàn)的質(zhì)控菌和用途。
為保證培養(yǎng)基質(zhì)量的穩(wěn)定可靠，各脫水培養(yǎng)基或各配方組分應(yīng)準(zhǔn)確稱量，并要求有一定的精確度。配制培養(yǎng)基最常用的溶劑是純化水，特殊情況下，可能需要用去離子水和蒸餾水。應(yīng)記錄各稱量物的重量和水的使用量。配制培養(yǎng)基所用容器和配套器具應(yīng)潔凈，可用純化水沖洗玻璃器皿以消除清潔劑和外來(lái)物質(zhì)的殘留。對(duì)熱敏感的培養(yǎng)基如糖發(fā)酵培養(yǎng)基其分裝容器一般應(yīng)預(yù)先進(jìn)行滅菌，以保證培養(yǎng)基的無(wú)菌性。
脫水型培養(yǎng)基應(yīng)完全溶解于水中，再行分裝與滅菌。配制時(shí)若需要加熱助溶，應(yīng)注意不要過(guò)度加熱，以避免培養(yǎng)基顏色變深。如需要添加其它組分時(shí)，加入后應(yīng)充分混勻。
應(yīng)按照生產(chǎn)商提供或使用者驗(yàn)證的參數(shù)進(jìn)行培養(yǎng)基的滅菌。商品化的成品培養(yǎng)基必須附有所用滅菌方法的資料。培養(yǎng)基滅菌一般采用濕熱滅菌技術(shù)，特殊培養(yǎng)基可采用薄膜過(guò)濾除菌。
培養(yǎng)基若采用不適當(dāng)?shù)募訜岷蜏缇鷹l件，有可能引起顏色變化、透明度降低、瓊脂凝固力或 pH 的改變。因此，培養(yǎng)基應(yīng)采用驗(yàn)證的滅菌程序滅菌，培養(yǎng)基滅菌方法和條件，應(yīng)通過(guò)無(wú)菌性試驗(yàn)和促生長(zhǎng)試驗(yàn)進(jìn)行驗(yàn)證。此外，對(duì)高壓滅菌器的蒸汽循環(huán)系統(tǒng)也要加以驗(yàn)證，以保證在一定裝載方式下的正常熱分布。溫度緩慢上升的高壓滅菌器可能導(dǎo)致培養(yǎng)基的過(guò)熱，過(guò)度滅菌可能會(huì)破壞絕大多數(shù)的細(xì)菌和真菌培養(yǎng)基促生長(zhǎng)的質(zhì)量。滅菌器中培養(yǎng)基的容積和裝載方式也將影響加熱的速度。因此，應(yīng)根據(jù)滅菌培養(yǎng)基的特性，進(jìn)行全面的滅菌程序驗(yàn)證。,
應(yīng)確定每批培養(yǎng)基滅菌后的 pH（冷卻至室溫 25℃測(cè)定）。若培養(yǎng)基處方中列出 pH 的范圍，除非經(jīng)驗(yàn)證表明培養(yǎng)基的 pH 允許的變化范圍很寬，否則，pH的范圍不能超過(guò)規(guī)定的±0.2。
制成平板或分裝于試管的培養(yǎng)基應(yīng)進(jìn)行下列檢查：容器和蓋子不得破裂，裝量應(yīng)相同，盡量避免形成氣泡，固體培養(yǎng)基表面不得產(chǎn)生裂縫或漣漪，在冷藏溫度下不得形成結(jié)晶，不得污染微生物等。應(yīng)檢查和記錄批數(shù)量、有效期及培養(yǎng)基的無(wú)菌檢查。
培養(yǎng)基的貯存
自配的培養(yǎng)基應(yīng)標(biāo)記名稱、批號(hào)、配制日期等信息，并在已驗(yàn)證的條件下貯藏。商品化的成品培養(yǎng)基標(biāo)簽上應(yīng)標(biāo)有名稱、批號(hào)、生產(chǎn)日期、失效期及培養(yǎng)基的有關(guān)特性，生產(chǎn)商和使用者應(yīng)根據(jù)培養(yǎng)基使用說(shuō)明書上的要求進(jìn)行保存，所采用的保藏和運(yùn)輸條件應(yīng)使成品培養(yǎng)基限度的失去水分并提供機(jī)械保護(hù)。
培養(yǎng)基滅菌后若儲(chǔ)藏在高壓滅菌器中，質(zhì)量可能會(huì)受影響，一般不提倡這種存放法。瓊脂培養(yǎng)基不得在 0℃或 0℃以下存放，因?yàn)槔鋬隹赡芷茐哪z特性。
培養(yǎng)基應(yīng)避光保存，若要長(zhǎng)期保存，應(yīng)置于密閉容器中以防止水分流失。瓊脂平板現(xiàn)配現(xiàn)用，如置冰箱保存，一般不超過(guò)一周，且應(yīng)密閉包裝，若延長(zhǎng)保存，保存期需經(jīng)驗(yàn)證確定。
固體培養(yǎng)基滅菌后的再融化只允許一次，以避免因過(guò)度受熱造成培養(yǎng)基質(zhì)量下降或微生物污染。培養(yǎng)基的再融化一般采用的水浴加熱或流通蒸汽。若使用微波爐，應(yīng)避免培養(yǎng)基過(guò)度受熱及水分的蒸發(fā)，更要注意安全。融化的培養(yǎng)基應(yīng)置于 45～50℃的水浴中，不得超過(guò) 8 小時(shí)。傾注培養(yǎng)基時(shí)，應(yīng)擦干培養(yǎng)基容器外表面的水分，避免容器外壁的水滴進(jìn)入培養(yǎng)基中造成污染。
使用過(guò)的培養(yǎng)基（包括失效的培養(yǎng)基）應(yīng)按照國(guó)家污染廢物處理相關(guān)規(guī)定進(jìn)行處理。
培養(yǎng)基的質(zhì)量控制試驗(yàn)
實(shí)驗(yàn)室應(yīng)對(duì)試驗(yàn)用培養(yǎng)基建立質(zhì)量控制程序（也就是要有培養(yǎng)基質(zhì)控sop），以確保所用培養(yǎng)基質(zhì)量符合相關(guān)檢測(cè)的需要。
實(shí)驗(yàn)室配制或商品化的成品培養(yǎng)基的質(zhì)量依賴于其制備過(guò)程，采用不適宜方法制備的培養(yǎng)基將影響微生物的生長(zhǎng)或復(fù)蘇，從而影響試驗(yàn)結(jié)果的可靠性。
配制好的培養(yǎng)基應(yīng)定期進(jìn)行質(zhì)量控制試驗(yàn)。實(shí)驗(yàn)室配制的培養(yǎng)基的常規(guī)監(jiān)控項(xiàng)目是 pH，適用性檢查試驗(yàn)，定期的穩(wěn)定性檢查以確定有效期。培養(yǎng)基在有效期內(nèi)應(yīng)依據(jù)適用性檢查試驗(yàn)確定培養(yǎng)基質(zhì)量是否符合要求。有效期的長(zhǎng)短將取決于在一定存放條件下（包括容器特性及密封性）的培養(yǎng)基其組成成分的穩(wěn)定性。
在實(shí)驗(yàn)室中，若采用已驗(yàn)證的配制和滅菌程序制備培養(yǎng)基且過(guò)程受控，那么同一批脫水培養(yǎng)基的適用性檢查試驗(yàn)可只進(jìn)行一次，試驗(yàn)的菌種可根據(jù)培養(yǎng)基的用途從相關(guān)附錄中進(jìn)行選擇，也可增加從生產(chǎn)環(huán)境及產(chǎn)品中常見(jiàn)的污染菌株。培養(yǎng)基的質(zhì)量控制試驗(yàn)若不符合規(guī)定，應(yīng)尋找不合格的原因，以防止問(wèn)題重復(fù)出現(xiàn)。任何不符合要求的培養(yǎng)基均不能使用。
用于環(huán)境監(jiān)控的培養(yǎng)基須特別防護(hù)。用于關(guān)鍵區(qū)域監(jiān)控的培養(yǎng)基要雙層包裝和終端滅菌，如果不能采用終端滅菌的培養(yǎng)基，那么在使用前應(yīng)進(jìn)行100%的預(yù)培養(yǎng)以防止外來(lái)的污染物帶到環(huán)境中及避免出現(xiàn)假陽(yáng)性結(jié)果。
其實(shí)在我們微生物實(shí)驗(yàn)室，培養(yǎng)基的質(zhì)控一直都是很多實(shí)驗(yàn)室很困擾的一個(gè)問(wèn)題，好好研讀GB 4789.28-2013 《培養(yǎng)基和試劑的質(zhì)量要求》，相信對(duì)大家還是很有幫助的。
Preparation, storage and quality control of culture medium
The culture medium can be prepared by prescription, or the dehydration medium produced by prescription can be used.
When preparing the medium, the dehydrated medium with the required quality or individual formula components should be selected for preparation. Dehydrated medium shall be accompanied by a prescription and instructions, preparation should be according to the requirements of the instructions on the operation to ensure quality meets the requirements of culture medium, dehydrated medium agglomerate or color change shall not be used. Dehydrated media or individual formula components should be stored under appropriate conditions, such as low temperature, dry and light-proof. All containers should be sealed, especially those containing dehydrated media. In addition to the prescriptions and instructions for use, the commercialized finished media should also indicate the expiry date, storage conditions, quality control bacteria and USES for the applicability test.
In order to ensure the stability and reliability of medium quality, each dehydrated medium or each formula component should be accurately weighed, and a certain degree of accuracy is required. The most commonly used solvent for preparing media is purified water. In special cases, deionized water and distilled water may be needed. The weight of each weighing object and the amount of water used shall be recorded. The containers and accessories used in preparation of the media shall be clean, and the glassware shall be washed with purified water to eliminate the residue of detergent and foreign substances. Heat sensitive media, such as sugar fermentation media, should be sterilized in advance to ensure the sterility of the medium.
The dehydrated medium should be completely dissolved in water, and then separated and sterilized. If the medium needs to be heated to aid dissolution, care should be taken not to overheat it to avoid the color of the medium getting darker. When additional groups are needed, they should be thoroughly mixed.
Sterilization of the medium shall be carried out according to parameters provided by the manufacturer or verified by the user. Commercial finished media must be accompanied by information on sterilization methods used. The sterilization technology of culture medium is wet and hot. The special culture medium can be sterilized by membrane filtration.
Improper heating and sterilization of the medium may result in color changes, decreased transparency, agarose coagulability or pH changes. Therefore, the culture medium should be sterilized by a proven sterilization procedure, and the sterilization method and conditions of the culture medium should be verified by aseptic test and growth-promoting test. In addition, the steam circulation system of high pressure sterilizer should be verified to ensure the normal heat distribution under certain loading mode. The high pressure sterilizer with slow rising temperature may cause the medium to overheat, and the excessive sterilization may damage the growth quality of most bacteria and fungi medium. The volume and loading of medium in sterilizer will also affect the rate of heating. Therefore, according to the characteristics of sterilization medium, a comprehensive sterilization procedure should be carried out. ,
Be sure that the pH of each batch of medium sterilization after cooling to room temperature (25 ℃). If the pH range is listed in the medium prescription, the pH range should not exceed the prescribed plus or minus 0.2 unless empirical evidence indicates that the pH range of the medium is wide.
Made into tablet or partial shipments in in vitro culture medium should be checked for the following: container and lid may not burst, the load should be the same, as far as possible avoid air bubble formation, solid medium surface cracks or ripple is not allowed in the cold storage temperature shall not crystallize, shall not be contaminated microbe, etc. Aseptic examination of batch number, period of validity and culture medium shall be checked and recorded.
Storage of culture medium
The self-prepared culture medium shall be marked with name, batch number, preparation date and other information and stored under verified conditions. Commercialization of finished product medium should be marked on the label name, batch number, production date, expiry date, and the related properties of culture medium, manufacturers and users should according to the requirement of the culture medium on the instruction manual to preserve, the preservation and transportation should be adopted by the minimum loss of moisture and make product medium provides mechanical protection.
If the medium is stored in a high pressure sterilizer after sterilization, the quality may be affected, which is generally not recommended. AGAR medium shall not be below 0 ℃ and 0 ℃ to deposit, because frozen may destroy the gel properties.
The medium should be kept away from light, and if long-term preservation is required, it should be placed in a closed container to prevent water loss. Agar-agar plate had better match to use now, if buy freezer to save, do not exceed a week commonly, and should close packing, if extend save, the shelf life needs experience certificate to determine.
The remelting of the solid medium after sterilization is allowed only once to avoid medium quality degradation or microbial contamination due to excessive heat exposure. Remelting of the culture medium usually USES a water bath to heat or circulate steam. If use microwave oven, should avoid medium excessive heat and moisture evaporation, more should pay attention to safety. Melt medium should be put in 45 ~ 50 ℃ water bath, shall not exceed 8 hours. When pouring the culture medium, the water on the outer surface of the culture medium container should be dried to avoid contamination caused by water drops on the outer wall of the container entering the culture medium.
Used media (including inactive media) shall be treated in accordance with national regulations on the disposal of contaminated wastes.
Quality control test of culture medium
The laboratory shall establish quality control procedures (i.e., medium quality control SOP) for test media to ensure the quality of the medium used meets the requirements of relevant tests.
Laboratory preparation or commercialization of finished products the quality of the medium depends on its preparation, preparation of inappropriate methods medium will affect the growth of microorganisms or recovery, thus affecting the reliability of test results.
The prepared medium should be tested regularly for quality control. The routine monitoring items for laboratory prepared media are pH, suitability test, periodic stability check to determine the validity period. The quality of the medium shall be determined in accordance with the applicability test within the period of validity. The duration of the period of validity will depend on the stability of the composition of the media under certain storage conditions, including the characteristics of the container and the sealing property.
In the lab, if adopting the authenticated preparation and preparation of culture medium and sterilization program controlled process, so the applicability of the same dehydrated medium can check test only once, test strains can undertake choosing according to the purpose of culture medium from the relevant appendices, also can increase the pollution of production environment and the product of common strains. If the quality control test of the medium does not meet the requirements, the reasons for the unqualified should be found to prevent the recurrence of problems. No medium that does not meet the requirements shall be used.
Media for environmental monitoring shall be specially protected. Critical areas for monitoring of medium to double packing and terminal sterilization, best if you can't use terminal sterilization medium, so before use should be 100% of the culture in order to prevent foreign pollutants to the environment and avoid false positive results.
Actually in our microbiology laboratory, quality control of culture medium has been a lot of laboratory is a puzzling question, a good study of GB 4789.28 2013 reagent and medium quality requirements, believe that is very helpful to everybody.