【日水培養(yǎng)基】培養(yǎng)基的制備規(guī)程和使用規(guī)程
發(fā)布時(shí)間：
2022-12-27
作者：
培養(yǎng)基制備規(guī)程及使用規(guī)程
1.目的
規(guī)范本中心微生物實(shí)驗(yàn)室培養(yǎng)基制備工作，保證培養(yǎng)基的質(zhì)量。
2.適用范圍
本規(guī)程適用于使用商品化干粉培養(yǎng)基制備各種待用培養(yǎng)基。
3.職責(zé)
3.1培養(yǎng)基制備人員負(fù)責(zé)按本規(guī)程制備各類培養(yǎng)基，及時(shí)填寫成品培養(yǎng)基制備記錄，編制培養(yǎng)基目錄，粘貼標(biāo)簽。
3.2 科室負(fù)責(zé)人：負(fù)責(zé)指導(dǎo)、監(jiān)督檢驗(yàn)人員按規(guī)程制備培養(yǎng)基。
4.制備程序
4.1概述
4.1.1制備培養(yǎng)基所用的化學(xué)藥品，均為化學(xué)純。有些試劑在使用前應(yīng)先試用。
4.1.2使用脫水培養(yǎng)基和其他含有有害物質(zhì)（如膽鹽或其他選擇劑）的成分時(shí)，應(yīng)遵循良好實(shí)驗(yàn)室規(guī)范和生產(chǎn)廠商提供的使用說(shuō)明。
4.1.3使用商品化脫水合成培養(yǎng)基制備培養(yǎng)基時(shí)，應(yīng)嚴(yán)格按照廠商提供的使用說(shuō)明配制。如質(zhì)量/體積、PH、制備條件、滅菌條件和操作步驟等。
4.1.4使用各別成分制備培養(yǎng)基時(shí)，應(yīng)按配方準(zhǔn)確配制，并記錄所使用成分的特性。
4.1.5制備培養(yǎng)基常用的容器為玻璃、不銹鋼鋁、鍋或搪瓷容器。不宜使用銅或鐵鍋。培養(yǎng)基中的含銅量過(guò)高時(shí)（＞0.3mg/1000ml），細(xì)菌不易生長(zhǎng)；含鐵量過(guò)高時(shí)（＞0.14mg/1000ml），則妨礙細(xì)菌毒素的產(chǎn)生。
4.2水
4.2.1制備培養(yǎng)基應(yīng)使用蒸餾水或相同質(zhì)量的水，以排除測(cè)試條件下抑制或影響微生物生長(zhǎng)的物質(zhì)。電阻率在25℃時(shí)應(yīng)≥300000Ω·cm。最好每周檢測(cè)一次。
4.2.2制備培養(yǎng)基時(shí)避免采用離子交換器生產(chǎn)的去離子水，其微生物含量可能較高，這種水在過(guò)濾滅菌后仍可能帶有細(xì)菌生長(zhǎng)的抑制因子。
4.3稱量和復(fù)水
4.3.1稱量所需量的脫水培養(yǎng)基（注意緩慢操作，必要時(shí)佩戴口罩或在通風(fēng)柜中操作，以防吸入含有有毒物質(zhì)的培養(yǎng)基粉末），先加入少量的水，充分混合（注意避免培養(yǎng)基結(jié)塊），然后再加水至所需的量。
4.3.2 必須選擇合適的天平和量筒，以確保稱量和移液的準(zhǔn)確性。
4.4溶解和分裝
脫水培養(yǎng)基加水后應(yīng)適當(dāng)加熱，并不停攪拌使其快速溶解，必要時(shí)，重新溶解。含瓊脂的培養(yǎng)基在加熱前應(yīng)先浸泡幾分鐘。用各別成分制備的培養(yǎng)基應(yīng)將不同成分分別加入適量的水中，并充分溶解，然后再加水至所需的量。
4.5 測(cè)定和調(diào)整PH
4.5.1用PH計(jì)或精密PH紙測(cè)pH，必要時(shí)進(jìn)行調(diào)整。
4.5.2在實(shí)驗(yàn)室用各別成分制備的培養(yǎng)基，除特殊說(shuō)明外，培養(yǎng)基滅菌后冷卻至25℃時(shí)，PH的變化不應(yīng)超過(guò)0.2個(gè)單位。
4.5.3一般使用濃度約為40g/L(約1mol/L)的氫氧化鈉溶液或濃度約為36.5g/L(約1mol/L)的鹽酸溶液調(diào)整培養(yǎng)基的pH。
4.6分裝
將配制好的培養(yǎng)基根據(jù)使用要求分裝到適當(dāng)?shù)娜萜髦?，并及時(shí)密封好。
4.7滅菌
4.7.1分裝好的培養(yǎng)基應(yīng)當(dāng)天進(jìn)行滅菌處理（2小時(shí)內(nèi)滅菌最佳）。
4.7.2實(shí)驗(yàn)室采用濕熱滅菌對(duì)培養(yǎng)基進(jìn)行滅菌處理，按照培養(yǎng)基使用說(shuō)明采用合適的滅菌溫度和時(shí)間。
4.8保存
制就的培養(yǎng)基不宜保存過(guò)長(zhǎng)，以少量勤做為宜。培養(yǎng)基存放于暗處和（或）4℃～12℃冰箱的密封袋或平皿筒中可延長(zhǎng)貯存期限。
4.9制備記錄
培養(yǎng)基制備完畢后，應(yīng)及時(shí)填寫“成品培養(yǎng)基制備記錄”和“成品培養(yǎng)基目錄清單”，并在外包裝上貼好相應(yīng)的標(biāo)簽，標(biāo)明名稱，編號(hào)，制備日期和有效期等內(nèi)容。
培養(yǎng)基使用規(guī)程
1. 目的
規(guī)范本中心微生物實(shí)驗(yàn)室培養(yǎng)基的使用和管理。
2. 適用范圍
本規(guī)程適用于培養(yǎng)基的儲(chǔ)存、融化和廢棄等。
3. 職責(zé)
3.1 檢驗(yàn)人員：按照本規(guī)程規(guī)范使用培養(yǎng)基、定期檢查培養(yǎng)基是否失效。
3.2 科室負(fù)責(zé)人：負(fù)責(zé)指導(dǎo)、監(jiān)督檢驗(yàn)人員規(guī)范使用培養(yǎng)基。
4.使用程序
4.1培養(yǎng)基的儲(chǔ)存
4.1.1除特殊說(shuō)明和標(biāo)準(zhǔn)規(guī)定，通常情況下基礎(chǔ)培養(yǎng)基（如營(yíng)養(yǎng)瓊脂培養(yǎng)基）應(yīng)在4℃冰箱中保存不超過(guò)3個(gè)月，或在室溫（20℃）下保存不超過(guò)一個(gè)月。
4.1.2滅菌后的培養(yǎng)基應(yīng)置適當(dāng)條件下保存至規(guī)定的有效期，并對(duì)保存條件進(jìn)行確證
4.2瓊脂培養(yǎng)基的融化
4.2.1將培養(yǎng)基放到沸水浴中或采用有相同效果的方法（如高壓鍋中的蒸汽，如100℃）使之融化。經(jīng)過(guò)高壓的培養(yǎng)基應(yīng)盡量減少重加熱時(shí)間，避免過(guò)度加熱。
4.2.2培養(yǎng)基融化后放入47℃±2℃的恒溫水浴鍋或恒溫培養(yǎng)箱中保溫，直至使用。
4.2.3融化后的培養(yǎng)基應(yīng)盡快使用，放置時(shí)間一般不應(yīng)超過(guò)4h，不可二次融化。
4.3培養(yǎng)基的脫氣
必要時(shí)，將培養(yǎng)基在使用前放到沸水浴或蒸汽浴中加熱15min，加熱時(shí)松開(kāi)容器的蓋子；加熱后蓋緊，并迅速冷卻至使用溫度。
4.4添加成分的加入
對(duì)熱不穩(wěn)定的添加成分應(yīng)在培養(yǎng)基冷卻至47℃±2℃時(shí)再加入。滅菌的添加成分在加入之前，應(yīng)先放置到室溫，避免冷的液體造成瓊脂凝結(jié)或形成片狀物。將加入添加成分的培養(yǎng)基緩慢充分混勻，盡快分裝到待用的容器中。
4.5平板的制備和儲(chǔ)存
4.5.1傾注融化的培養(yǎng)基到平皿中，使之在平皿中形成一個(gè)至少2mm厚的瓊脂層（直徑90mm的平皿通常要加入15mL瓊脂培養(yǎng)基）。將平皿蓋好皿蓋后放到水平平面使瓊脂冷卻凝固。
4.5.2為了避免產(chǎn)生冷凝水，平板應(yīng)冷卻后再裝入袋中。貯存前不要對(duì)培養(yǎng)基表面進(jìn)行干燥處理。
4.5.3凝固后的培養(yǎng)基應(yīng)立即使用或放于暗處和（或）4℃～12℃冰箱中，儲(chǔ)存期限見(jiàn)“4.1培養(yǎng)基的儲(chǔ)存”。 并在外包裝上貼好相應(yīng)的標(biāo)簽，標(biāo)明名稱，編號(hào)，制備日期和有效期等內(nèi)容。
4.5.4對(duì)于采用表面接種形式培養(yǎng)的固體培養(yǎng)基。應(yīng)先對(duì)瓊脂表面進(jìn)行干燥：揭開(kāi)平皿蓋，將平板倒扣于烘箱/培養(yǎng)箱中（溫度設(shè)為25℃～50℃）；或放在有對(duì)流風(fēng)的無(wú)菌凈化臺(tái)中，直到培養(yǎng)基表面的水滴消失為止。注意不要過(guò)度干燥。商業(yè)化的平板瓊脂培養(yǎng)基應(yīng)按照廠商提供的說(shuō)明使用。
4.6培養(yǎng)
4.6.1培養(yǎng)時(shí)每垛最多堆放六個(gè)平板，平板間要留有空隙以保證空氣流通，使培養(yǎng)物的溫度盡快與培養(yǎng)箱溫度達(dá)到一致。
4.6.2在培養(yǎng)過(guò)程中，培養(yǎng)基會(huì)損失水分。當(dāng)水分損失的量大于培養(yǎng)基總量的15%時(shí)，就會(huì)影響微生物的生長(zhǎng)。造成培養(yǎng)基水分損失的因素較多，如培養(yǎng)基成分，平皿中培養(yǎng)基總量和培養(yǎng)箱的類型等（如使用帶風(fēng)扇的培養(yǎng)箱，培養(yǎng)箱中的濕度偏低，平板在培養(yǎng)箱中位置靠近培養(yǎng)箱內(nèi)壁等），操作時(shí)應(yīng)注意避免。
4.7培養(yǎng)基的棄置
培養(yǎng)基廢棄前，實(shí)驗(yàn)室采用121℃、15分鐘蒸汽滅菌，或煮沸消毒30分鐘。
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Preparation and use of culture medium
Purpose 1.
To standardize the preparation of medium in microbiology laboratory of the center and ensure the quality of medium.
2. Scope of application
This procedure is applicable to the preparation of various media for use in commercial dry medium.
3. The responsibility
3.1 the media preparation personnel shall prepare all kinds of media according to this regulation, timely fill in the preparation records of the finished media, prepare the media catalogue and paste labels.
3.2 responsible person of the department: responsible for guiding and supervising the inspectors to prepare the media according to the regulations.
4. Preparation procedure
4.1 overview
4.1.1 all the chemicals used to prepare the culture medium are chemical pure. Some reagents should be tried before use.
4.1.2 when using dehydrated media and other ingredients containing harmful substances (such as bile salts or other selective agents), good laboratory specifications and instructions provided by the manufacturer shall be followed.
4.1.3 the preparation of commercial dehydrated synthetic media shall be in strict accordance with the instructions provided by the manufacturer. Such as quality/volume, PH, preparation conditions, sterilization conditions and operating procedures.
4.1.4 when preparing media with various ingredients, the media shall be prepared accurately according to the formula and the characteristics of the ingredients used shall be recorded.
4.1.5 the commonly used containers for preparing media are glass, stainless steel, aluminum, pot or enamel containers. Do not use copper or iron POTS. When the amount of copper in the culture medium is too high (> 0.3mg/1000ml), the bacteria will not grow easily. Excessive iron content (> 0.14mg/1000ml) hinders the production of bacterial toxins.
4.2 the water
4.2.1 distilled water or water of the same quality shall be used to prepare the culture medium to eliminate substances that inhibit or affect microbial growth under test conditions. Should be 300000 or higher resistivity at 25 ℃ Ω · cm. It's best to test it once a week.
4.2.2 when preparing the culture medium, deionized water produced by ion exchangers should be avoided, and its microbial content may be high. Such water may still contain inhibiting factors for bacterial growth after filtration and sterilization.
4.3 weigh and refill water
4.3.1 weighing the required amount of dehydrated medium (note that slow operation, wearing masks, or in the ventilation cabinet operation when necessary, to prevent inhalation culture medium containing toxic powder), add a small amount of water first, mix (avoid medium agglomeration), then add water to the required level.
4.3.2 suitable balance and measuring cylinder must be selected to ensure the accuracy of weighing and liquid transfer.
4.4 dissolve and disassemble
After adding water, the dehydrated medium should be heated properly, and it should be dissolved quickly without stopping stirring. If necessary, it should be redissolved. AGAR medium should be soaked for several minutes before heating. The medium prepared from each component should be added to the appropriate amount of water and dissolved thoroughly before adding water to the required amount.
4.5 determine and adjust PH
4.5.1 use a PH meter or precision PH paper to measure PH and adjust if necessary.
4.5.2 with culture medium of the preparation of individual components in the lab, except where noted, culture medium after sterilization cooling to 25 ℃, the change of PH should not exceed 0.2 units.
4.5.3 the pH of the medium is generally adjusted by using sodium hydroxide solution with a concentration of about 40g/L(about 1mol/L) or hydrochloric acid solution with a concentration of about 36.5g/L(about 1mol/L).
4.6 partial shipments
Divide the prepared medium into appropriate containers as required and seal it in time.
4.7 the sterilization
4.7.1 the prepared media shall be sterilized for a day (preferably within 2 hours).
4.7.2 wet and heat sterilization was used in the laboratory to sterilize the medium, and appropriate sterilization temperature and time were used according to the instructions of the medium.
4.8 save
The prepared medium should not be kept too long and should be used sparingly. Media stored in the dark and (or) 4 ℃ ~ 12 ℃ freezer bags or AGAR tube can prolong the storage period.
4.9 preparation records
Culture medium preparation has been completed, should be timely fill in the "finished product culture medium preparation record" and "product medium directory listing", and the corresponding labels pasted on the outer packing is good, indicate the name, number, date of preparation and validity, etc.
Protocol for use of culture medium
Purpose 1.
To standardize the use and management of medium in microbiology laboratory of the center.
2. Scope of application
This procedure is applicable to storage, melting and disuse of media.
3. The responsibility
3.1 inspectors: use the medium in accordance with the regulations and check whether the medium is invalid regularly.
3.2 responsible person of the department: responsible for guiding and supervising inspectors to standardize the use of media.
4. Application procedures
4.1 storage of culture medium
4.4.1 except for special specifications and standards, usually basal medium (such as nutrient AGAR culture medium) should be kept at 4 ℃ refrigerator in no more than three months, or at room temperature (20 ℃) to save no more than a month.
4.1.2 the sterilized medium shall be kept under appropriate conditions until the specified period of validity, and the preservation conditions shall be confirmed
4.2 melting of AGAR medium
2 the broth in a boiling water bath or using has the same effect (such as the steam in the pressure cooker, such as 100 ℃) to melt. After high pressure medium should reduce reheating time as far as possible, avoid excessive heating.
4.2.2 melted into the culture medium to 47 ℃ + 2 ℃ constant temperature water-bath water or constant temperature incubator in heat preservation, until use.
4.2.3 the melted media should be used as soon as possible, and generally should not be placed for more than 4h, and should not be melted again.
4.3 degassing of medium
When necessary, the culture medium shall be heated in a boiling water bath or steam bath for 15 minutes before use, and the lid of the container shall be loosened when heated. Cover tightly after heating and cool quickly to service temperature.
4.4 addition of ingredients
Add the ingredients to the thermal instability should be cool in the culture medium to 47 ℃ + 2 ℃ to join again. The sterilized ingredients should be placed at room temperature before being added to prevent the cold liquid from causing agar-agar condensation or sheet formation. Mix the added medium slowly and thoroughly and distribute it into the container to be used as soon as possible.
4.5 preparation and storage of tablets
4.5.1 pour the melted medium into the plate to form an AGAR layer at least 2mm thick (a 90mm diameter plate is usually added with 15mL AGAR medium). Cover the plate and put it on the horizontal plane to cool the AGAR.
4.5.2 in order to avoid condensing water, the plate should be cooled and then put into the bag. Do not dry the surface of the medium before storage.
4.5.3 after solidification of the medium should be used immediately or in the dark and (or) 4 ℃ ~ 12 ℃ refrigerator, storage life see "4.1 medium storage". The corresponding labels shall be affixed to the outer package, indicating the name, number, preparation date and expiry date.
4.5.4 solid medium cultured in the form of surface inoculation. Should be carried out on the AGAR surface drying: uncover plate cover, will buckle the tablet in the oven/incubator (temperature is 25 ℃ to 50 ℃); Or put it in a sterile purification stand with a flow of air until the water drops on the surface of the culture medium disappear. Be careful not to overdry. Commercial plate AGAR media shall be used in accordance with manufacturer's instructions.
4.6 develop
4.6.1 during the culture, a maximum of six plates shall be stacked in each stack, and there shall be space between the plates to ensure air circulation, so that the temperature of the culture material and the incubator can be reach