Media Preparation 培養(yǎng)基制備

Culture media are the basis for most microbiological tests. Safeguarding the quality of the media is therefore critical to the success of the microbiology laboratory. Media preparation, proper storage, and quality control testing can ensure a consistent supply of high-quality media.

培養(yǎng)基是微生物實(shí)驗(yàn)的基礎(chǔ)。保證培養(yǎng)基的質(zhì)量因而成為微生物實(shí)驗(yàn)室成功的關(guān)鍵。培養(yǎng)基的制備、適宜的存儲(chǔ)條件和質(zhì)量控制試驗(yàn)是提供優(yōu)質(zhì)培養(yǎng)基的保證。

It is important to choose the correct media or components in making media based on the use of accepted sources or references for formulas.The manufacturer's formula and instructions for preparation routinely accompany dehydrated media and ready-made media. Because different media types may have different preparation requirements (e.g., heating, additives, and pH adjustment), it is important to follow these instructions to ensure preparation of acceptable media quality. A certificate of analysis describing expiration dating and recommended storage conditions accompanies ready-made media, as well as the quality control organisms used in growth-promotion and selectivity testing of that media.

在制備培養(yǎng)基過程中使用已接受的來源或配方標(biāo)準(zhǔn)，選擇正確的培養(yǎng)基或成分是非常重要的。一般生產(chǎn)商在供應(yīng)干粉培養(yǎng)基和成品培養(yǎng)基時(shí)，其配方和使用說明都會(huì)隨貨發(fā)送。由于不同的培養(yǎng)基類型可能有不同的配制要求（例如：加熱、添加劑，和pH值調(diào)節(jié)），重要的一點(diǎn)是需遵守其提供的使用說明以保證配制出的培養(yǎng)基質(zhì)量。如果是成品培養(yǎng)基，隨貨應(yīng)有指明有效期和存儲(chǔ)條件的分析報(bào)告，以及促生長(zhǎng)試驗(yàn)、選擇性試驗(yàn)所用的微生物。

Water is the universal diluent for microbiological media.Purified Water is most often used for media preparation, but in certain cases the use of deionized or distilled water may be appropriate. Water of lesser quality should not be used for microbiological media preparation.The volume of the water used should be recorded.

水是配制培養(yǎng)基常用的溶劑。一般使用純化水，但在某些情況下，也可以使用去離子水或蒸餾水。更低品質(zhì)的水不應(yīng)用于微生物培養(yǎng)基制備。水的用量應(yīng)記錄。

Consistent preparation of media requires accurate weighing of dehydrated media or media constituents. A calibrated balance with the appropriate weight range for the ingredients should be used (See Weighing on an Analytical Balance <1251)>.Clean weighing containers and tools (such as spatulas) should be used to prevent foreign substances from entering the formulation. The weight of the components should be recorded.

要保證培養(yǎng)基配制的一致性，需要對(duì)培養(yǎng)基干粉或培養(yǎng)基組分進(jìn)行準(zhǔn)確稱量。應(yīng)使用經(jīng)過校準(zhǔn)的天平，其稱量范圍應(yīng)與所稱的重量相符（見1251分析天平稱量）。應(yīng)對(duì)稱量容器和工具（例如稱量勺）進(jìn)行清潔以保證無異物進(jìn)入所配培養(yǎng)基。各成分的重量應(yīng)進(jìn)行記錄。

Dehydrated media should be thoroughly dissolved in water before dispensing and sterilization. If heating is necessary to help dissolve the media, care should be taken not to overheat media, because all culture media, to a greater or lesser extent,are heat-sensitive. Equipment used in the preparation of media should be appropriate to allow for controlled heating, constant agitation, and mixing of the media. Darkening of media (Maillard-type reaction or nonenzymatic browning) is a general indication of overheating.When adding required supplements to media, adequate mixing of the medium after adding the supplement should be performed.

培養(yǎng)基干粉應(yīng)在水中完全溶解，然后進(jìn)行滅菌。如果需要加熱溶解，要注意不能過度加熱，因?yàn)樗械呐囵B(yǎng)基，或多或少，都是對(duì)熱敏感的。使用合適的設(shè)備配制培養(yǎng)基配制，以便控制加熱、持續(xù)攪拌和混合培養(yǎng)基。培養(yǎng)基變黑（美拉德類型反應(yīng)或非酶褐變）一般說明過熱。在向培養(yǎng)基中加入所需要的補(bǔ)充成分時(shí)，在加入后需要進(jìn)行充分?jǐn)嚢杌靹颉?/span>

Preparation of media in poorly cleaned glassware can allow inhibitory substances to enter the media.Inhibitory substances can come from detergent residue after cleaning glassware or from prior materials used in the glassware. Be sure that the cleaning process removes debris and foreign matter, and that the detergent is thoroughly rinsed out with Purified Water.See Cleaning Glass Apparatus <1051> for additional guidance.

在清潔不徹底的玻璃器皿中配制培養(yǎng)基會(huì)使得抑制性物質(zhì)帶入培養(yǎng)基。抑制性物質(zhì)可能來于玻璃器皿中的清潔劑殘留，或來自于玻璃器皿中上次所盛裝的物料。要保證清洗程序可以去除殘?jiān)屯鈦砦镔|(zhì)，并且清潔劑可以被純化水徹底沖洗掉。參見1051玻璃容器的清潔。

Sterilization of media should be performed within the parameters provided by the manufacturer or validated by the user.Commercially prepared media should provide documentation of the sterilization method used. Autoclaving by moist heat is the preferred sterilization technique, except in instances when boiling is required in order to avoid deterioration of heat-labile components of the media. Sterilization by filtration may also be appropriate for some formulations.

培養(yǎng)基滅菌應(yīng)在生產(chǎn)商提供的參數(shù)范圍內(nèi)，或用戶驗(yàn)證的參數(shù)范圍內(nèi)實(shí)施。商品化的預(yù)制培培養(yǎng)基應(yīng)隨貨有所用滅菌方法的資料。濕熱滅菌是較好的滅菌技術(shù)，除非需要煮沸以避免培養(yǎng)基中不耐熱成分被破壞。有些配方可能適用過濾滅菌。

The effects of the sterilization method and conditions on the media should be validated by sterility and growth-promotion testing of the media. In addition, if sterilized by moist heat, the autoclave cycle should be validated to ensure proper heat distribution for selected loads and volumes. Typically, manufacturers recommend using an autoclave cycle of 121° for 15 minutes using a validated autoclave. These conditions apply to time at temperature of the media. As container size and the load configuration of the autoclave will influence the rate of heating, longer cycles may be required for larger loads. However, the sterilization time will be dependent on the media volume and autoclave load. Sterilization cycles in which the autoclave is slow to come up to temperature may result in overheating of the media.Therefore, care must be taken to validate a sterilization cycle,balancing the need for sterile media against the tendency of the media to degrade under excessive heating.Storage of the media in the autoclave after the liquid cycle is completed is not recommended after cooling, as it may damage the media.Improper heating or sterilizing conditions—for commercially prepared or internally prepared media—may result in a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturer's recommended range, as well as reduced growth-promotion activity and/or selectivity.

滅菌方法和條件對(duì)培養(yǎng)基的影響應(yīng)經(jīng)過無菌驗(yàn)證和培養(yǎng)基促生長(zhǎng)試驗(yàn)確認(rèn)。另外，如果采用濕熱滅菌，滅菌周期應(yīng)進(jìn)行驗(yàn)證以保證在所選的負(fù)載和體積下的熱分布。生產(chǎn)商一般會(huì)推薦采用121℃15分鐘作為滅菌條件，培養(yǎng)基滅菌即采用此條件。由于容器尺寸和滅菌器的負(fù)載參數(shù)會(huì)影響加熱速度，因此較大的負(fù)載可能需要較長(zhǎng)的滅菌時(shí)間。當(dāng)然，滅菌時(shí)間還是取決于培養(yǎng)基體積和滅菌負(fù)載。升溫較慢的滅菌條件可能會(huì)導(dǎo)致培養(yǎng)基過熱，因此需要特別注意滅菌周期的驗(yàn)證，在培養(yǎng)基滅菌要求和培養(yǎng)基在過熱條件下分解中尋找平衡點(diǎn)。在滅菌結(jié)束冷卻后，不建議將培養(yǎng)基留在滅菌器中存儲(chǔ)，因?yàn)榭赡軙?huì)對(duì)培養(yǎng)基造成損壞。不適當(dāng)?shù)募訜峄驕缇鷹l件--對(duì)于商業(yè)制備的培養(yǎng)基或公司自制的培養(yǎng)基--可能會(huì)導(dǎo)致顏色變化差異、澄清度差、凝膠強(qiáng)度改變、或pH值超出生產(chǎn)商指定范圍、以及促生長(zhǎng)和/或具有選擇性下降。

The pH of each batch of medium should be confirmed after it has cooled to room temperature (20°–25°) by aseptically withdrawing a sample for testing. Refrigerated purchased media should be allowed to warm up to ambient room temperature if it is to be checked for pH confirmation. A flat pH probe is recommended for agar surfaces, and an immersion probe is recommended for liquids. See pH <791> for guidance with pH measurement and instrument calibration. The pH of media should be in a range of ±0.2 of the value indicated by the manufacturer, unless a wider range is acceptable by the validated method.

每個(gè)批次培養(yǎng)基在冷卻到室溫后（20~25℃），應(yīng)采用無菌方式取樣檢測(cè)pH值；冷藏的的培養(yǎng)基應(yīng)在升溫至室溫后檢測(cè)pH值。建議對(duì)培養(yǎng)基平面采用扁平的pH探頭，液體培養(yǎng)基則采用浸入式探頭。pH值測(cè)量和儀器校正指南見pH791.除非有驗(yàn)證過的方法可以接受一個(gè)更寬的范圍，否則培養(yǎng)基的pH值應(yīng)在生產(chǎn)商指示的pH值±0.2之內(nèi)。

•Excessive darkening or color change 太黑或顏色變化

•Crystal formation from possible freezing 可能因?yàn)槔鋬鲆鸬慕Y(jié)晶

•Excessive number of bubbles 過量氣泡

•Microbial contamination 微生物污染

•Status of redox indicators (if appropriate) 氧化還原指示劑狀態(tài)（適用時(shí)）

•Lot number and expiration date checked and recorded 檢查批號(hào)和有效期并記錄

•Sterility of the media 培養(yǎng)基滅菌

•Cleanliness of plates (lid should not stick to dish) 培養(yǎng)基用碟清潔（蓋不應(yīng)粘在碟上）

原文鏈接：https://mp.weixin.qq.com/s/Qsz_NNFKWUoIb_sh0NYm5Q

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