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      【日水培養基】培養基的制備規程和使用規程

      發布時間:

      2022-12-27

      作者:


      培養基制備規程及使用規程
       
       
      1.目的
      規范本中心微生物實驗室培養基制備工作,保證培養基的質量。
       
      2.適用范圍
      本規程適用于使用商品化干粉培養基制備各種待用培養基。
      3.職責
      3.1培養基制備人員負責按本規程制備各類培養基,及時填寫成品培養基制備記錄,編制培養基目錄,粘貼標簽。
       
      3.2 科室負責人:負責指導、監督檢驗人員按規程制備培養基。
       
      4.制備程序
       
      4.1概述
      4.1.1制備培養基所用的化學藥品,均為化學純。有些試劑在使用前應先試用。
       
      4.1.2使用脫水培養基和其他含有有害物質(如膽鹽或其他選擇劑)的成分時,應遵循良好實驗室規范和生產廠商提供的使用說明。
       
      4.1.3使用商品化脫水合成培養基制備培養基時,應嚴格按照廠商提供的使用說明配制。如質量/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基時,應按配方準確配制,并記錄所使用成分的特性。
       
      4.1.5制備培養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。培養基中的含銅量過高時(>0.3mg/1000ml),細菌不易生長;含鐵量過高時(>0.14mg/1000ml),則妨礙細菌毒素的產生。
       
      4.2水
      4.2.1制備培養基應使用蒸餾水或相同質量的水,以排除測試條件下抑制或影響微生物生長的物質。電阻率在25℃時應≥300000Ω·cm。最好每周檢測一次。
       
      4.2.2制備培養基時避免采用離子交換器生產的去離子水,其微生物含量可能較高,這種水在過濾滅菌后仍可能帶有細菌生長的抑制因子。
       
      4.3稱量和復水
      4.3.1稱量所需量的脫水培養基(注意緩慢操作,必要時佩戴口罩或在通風柜中操作,以防吸入含有有毒物質的培養基粉末),先加入少量的水,充分混合(注意避免培養基結塊),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確性。
       
      4.4溶解和分裝
      脫水培養基加水后應適當加熱,并不停攪拌使其快速溶解,必要時,重新溶解。含瓊脂的培養基在加熱前應先浸泡幾分鐘。用各別成分制備的培養基應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  測定和調整PH
      4.5.1用PH計或精密PH紙測pH,必要時進行調整。
       
      4.5.2在實驗室用各別成分制備的培養基,除特殊說明外,培養基滅菌后冷卻至25℃時,PH的變化不應超過0.2個單位。
       
      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)的鹽酸溶液調整培養基的pH。
       
      4.6分裝
      將配制好的培養基根據使用要求分裝到適當的容器中,并及時密封好。
       
      4.7滅菌
      4.7.1分裝好的培養基應當天進行滅菌處理(2小時內滅菌最佳)。
       
      4.7.2實驗室采用濕熱滅菌對培養基進行滅菌處理,按照培養基使用說明采用合適的滅菌溫度和時間。
       
      4.8保存
      制就的培養基不宜保存過長,以少量勤做為宜。培養基存放于暗處和(或)4℃~12℃冰箱的密封袋或平皿筒中可延長貯存期限。
       
      4.9制備記錄
      培養基制備完畢后,應及時填寫“成品培養基制備記錄”和“成品培養基目錄清單”,并在外包裝上貼好相應的標簽,標明名稱,編號,制備日期和有效期等內容。
       
       
      培養基使用規程
      1. 目的
      規范本中心微生物實驗室培養基的使用和管理。
       
      2. 適用范圍
      本規程適用于培養基的儲存、融化和廢棄等。
       
      3. 職責
      3.1 檢驗人員:按照本規程規范使用培養基、定期檢查培養基是否失效。
       
      3.2 科室負責人:負責指導、監督檢驗人員規范使用培養基。
       
      4.使用程序
       
      4.1培養基的儲存
      4.1.1除特殊說明和標準規定,通常情況下基礎培養基(如營養瓊脂培養基)應在4℃冰箱中保存不超過3個月,或在室溫(20℃)下保存不超過一個月。
       
      4.1.2滅菌后的培養基應置適當條件下保存至規定的有效期,并對保存條件進行確證
       
      4.2瓊脂培養基的融化
      4.2.1將培養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。經過高壓的培養基應盡量減少重加熱時間,避免過度加熱。
       
      4.2.2培養基融化后放入47℃±2℃的恒溫水浴鍋或恒溫培養箱中保溫,直至使用。
       
      4.2.3融化后的培養基應盡快使用,放置時間一般不應超過4h,不可二次融化。
       
      4.3培養基的脫氣
      必要時,將培養基在使用前放到沸水浴或蒸汽浴中加熱15min,加熱時松開容器的蓋子;加熱后蓋緊,并迅速冷卻至使用溫度。
       
      4.4添加成分的加入
      對熱不穩定的添加成分應在培養基冷卻至47℃±2℃時再加入。滅菌的添加成分在加入之前,應先放置到室溫,避免冷的液體造成瓊脂凝結或形成片狀物。將加入添加成分的培養基緩慢充分混勻,盡快分裝到待用的容器中。
       
      4.5平板的制備和儲存
      4.5.1傾注融化的培養基到平皿中,使之在平皿中形成一個至少2mm厚的瓊脂層(直徑90mm的平皿通常要加入15mL瓊脂培養基)。將平皿蓋好皿蓋后放到水平平面使瓊脂冷卻凝固。
      4.5.2為了避免產生冷凝水,平板應冷卻后再裝入袋中。貯存前不要對培養基表面進行干燥處理。
       
      4.5.3凝固后的培養基應立即使用或放于暗處和(或)4℃~12℃冰箱中,儲存期限見“4.1培養基的儲存”。 并在外包裝上貼好相應的標簽,標明名稱,編號,制備日期和有效期等內容。
       
      4.5.4對于采用表面接種形式培養的固體培養基。應先對瓊脂表面進行干燥:揭開平皿蓋,將平板倒扣于烘箱/培養箱中(溫度設為25℃~50℃);或放在有對流風的無菌凈化臺中,直到培養基表面的水滴消失為止。注意不要過度干燥。商業化的平板瓊脂培養基應按照廠商提供的說明使用。
       
      4.6培養
      4.6.1培養時每垛最多堆放六個平板,平板間要留有空隙以保證空氣流通,使培養物的溫度盡快與培養箱溫度達到一致。
       
      4.6.2在培養過程中,培養基會損失水分。當水分損失的量大于培養基總量的15%時,就會影響微生物的生長。造成培養基水分損失的因素較多,如培養基成分,平皿中培養基總量和培養箱的類型等(如使用帶風扇的培養箱,培養箱中的濕度偏低,平板在培養箱中位置靠近培養箱內壁等),操作時應注意避免。
       
      4.7培養基的棄置
      培養基廢棄前,實驗室采用121℃、15分鐘蒸汽滅菌,或煮沸消毒30分鐘。
       
      食品論壇分享

      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
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