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      培養基配制及適用性檢查標準操作規程

      發布時間:

      2022-11-16

      作者:

      買培養基的官網


      培養基配制及適用性檢查標準操作規程

      建立培養基配制及適用性檢查的標準操作規程,規范實驗人員的操作流程。
       
      范圍:適用于微生物檢驗人員對培養基的規范管理。
       
      依據:《藥品生產質量管理規范(2010年修訂)》,《中華人民共和國藥典》2015年版第四部
       
      職責:
      1.微生物檢驗員:負責實驗室所需求的培養基管理工作,使日常檢驗可以順利進行。
       
      2.微生物檢驗主管:負責對日常配制的規范操作進行監督。
       
      內容
      1 根據檢驗項目,工作量和工作進度的需求,提前一個月進行所需的培養基申購。申購時需指定培養基供應商,必要時進行供應商審計。
       
       
      2.1 培養基到貨后,微生物室檢驗員應進行驗收。驗收內容包括核對品名,數量,規格,生產廠商,應與申購單一致;檢查培養基包裝有無破損,包裝是否完整,產品是否在有效期內。
       
      2.2 驗收合格后,登記于《培養基領用臺賬》(附記錄文件編號:SMP-10-QC-009-02 (00))。
       
      3.1未開封的茶品貯存于陰涼室,處于低溫,干燥,和避光條件下。已開封的應蓋緊,貯存于陰涼庫。
       
      3.2滅菌后應進行預培養72h檢查無菌后,置4~8℃冷藏保存待用。滅菌后儲存期為7天。
       
      4.1培養基的配制和使用應填寫《培養基配制及使用記錄》(附記錄文件編號:SMP-10-QC-009-02(00))。
       
      4.2培養基配制批號原則:原材料批號-配制日期:比如2011年1月1日配制的培養基,干粉批號000000,配制批號為:000000-110101。配制好后需要貼上《培養基標簽》(附記錄文件編號:R-SMP-10-QC-009-03)。(注:同一天同一配置多次的,在原批號后加“-”加“數字”表示,如000000-110101-01)
       
      4.3.1 使用商品化脫水合成培養基時,應嚴格按照廠商提供的使用說明配制,如重量/體積,pH,滅菌條件和操作步驟等。實驗室使用各種基礎成分制備時,應按照配方準確配制,并記錄相關信息如:名稱和類型及試劑級別,每個成分物質含量,制造商,批號,pH值,體積/分裝體積,滅菌條件(滅菌方式,溫度及時間),配制日期,人員等,以便溯源。
       
      4.3.2 水,容器具要求:配制均采用純化水,特殊說明時采用去離子水和蒸餾水。培養基配制所用容器和配套器具應潔凈。對熱敏感的如糖發酵培養基其分裝容器應先進行預滅菌,以保證培養基的無菌性。
       
      4.3.3 稱量與分裝:快速稱量所需量的脫水合成培養基(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)。以免吸潮。先加入適量的水,充分混合。注意避免結塊,然后加水至所需的量。根據說明書要求選擇是否加熱。
       
      分裝體積不超過容器體積的2/3。配制斜面等含瓊脂的也需加熱煮沸至完全溶解后分裝。
       
      4.4 pH 值的測定和調整
       
      pH值采用pH計進行檢測,溫度25±2℃。取配后培養基10ml進行滅前pH測定。另取20ml于50ml三角瓶與同批配制培養基同時滅菌冷卻至25±2℃檢測滅后PH。滅菌前后pH應按下表進行控制。滅菌前可使用濃度約為40g/L(約1mol/L)的氫氧化鈉溶液或濃度約為36.5g/L(約1mol/L)的鹽酸溶液進行緩慢調整,避免反復調整而增加離子濃度。滅菌后不符合標準時視為不合格,不進行使用。
       
      4.5 培養基和試劑應采用濕熱滅菌法或過濾除菌法。濕熱滅菌在高壓滅菌鍋或蒸汽滅菌柜中進行,容器具若需濕熱滅菌時,滅菌條件為121℃滅菌30 min。培養基嚴格按說明書條件進行滅菌。培養基滅菌后應立即取出,不得存放于高壓滅菌器中。
       
      5.1計數培養基適用性檢查
       
      5.1.1菌種及菌液制備
       
      菌種:試驗用菌株的傳代次數不得超過5代(從菌種保藏中心獲得的干燥菌種為第0代),并采用適宜的菌種保藏技術進行保存,以保證試驗菌株的生物學特性。計數培養基適用性檢查和計數方法適用性試驗用菌株見表1。
       
      菌液制備:按表1規定程序培養各試驗菌株。取金黃色葡萄球菌,銅綠假單胞菌,枯草芽孢桿菌的新鮮培養物至胰酪大豆胨液體培養基中,35℃培養18~24小時,取上述培養物1ml加入9ml 0.9%無菌氯化鈉溶液中,制成10-1 的菌液,依法10倍稀釋至10-7,分取各菌懸液1ml注入平皿中,立即傾注胰酪大豆胨瓊脂培養基20ml,各菌懸液平行制備兩個平皿,平皿法培養計數,取小于100CFU/ml的菌液備用;取白色念珠菌的新鮮培養物至沙氏葡萄糖液體培養基中,25℃培養2~3天。取上述培養物1ml加入9ml 0.9%無菌氯化鈉溶液中,制成10-1的菌液,依法10倍稀釋至10-7,取菌懸液1ml注入平皿中,立即傾注沙氏葡萄糖瓊脂peiyangji25ml,各菌懸液平行制備兩個平皿,平皿法培養計數,取小于100CFU/ml的菌液備用;取黑曲霉的新鮮培養物接種至沙氏葡萄糖瓊脂斜面培養基,經25℃培養5~7天,加入3~5ml含0.05%(ml/ml)聚山梨酯80的0.9%無菌氯化鈉溶液,將孢子洗脫。然后吸出孢子懸液至無菌試管內,取上述培養物1ml加入9ml 0.9%無菌氯化鈉溶液中,制成10-1的菌液,依法10倍稀釋至10-7,分取各菌懸液1ml注入平皿中,立即傾注沙氏葡萄糖瓊脂培養基20ml,各菌懸液平行制備兩個平皿,平皿法培養計數,取小于100CFU/ml的菌液備用。
       
      菌液制備后若在室溫下放置,應在2 小時內使用;若保存在2?8℃,可在24小時內使用。黑曲霉孢子懸液可保存在2?8℃ ,在驗證過的貯存期內使用。
       
      5.1.2 陰性對照
       
      為確認試驗條件是否符合要求,應進行陰性對照試驗,陰性對照試驗應無菌生長。如陰性對照有菌生長,應進行偏差調查。
       
      微生物計數用的成品培養基,由脫水培養基或按處方配制的培養基均應進行適用性檢查。
       
      按表1 規定,接種不大于l00cfu的菌液至胰酪大豆胨液體培養基管或胰酪大豆胨瓊脂培養基平板或沙氏葡萄糖瓊脂平板。每一試驗菌株平行制備2管或2個平皿。同時,用相應的對照培養基替代被檢培養基進行上述試驗。
       
      被檢固體培養基上的菌落平均數與對照的菌落平均數的比值應在0.5?2 范圍內,且菌落形態大小應與對照的菌落一致;被檢液體培養基管與對照培養基管比較,試驗菌應生長良好。
       
      5.2控制菌檢查用培養基適用性檢查
       
      5.2.1 菌種及菌液制備
       
      菌種:試驗用菌株的傳代次數不得超過5 代(從菌種保藏中心獲得的干燥菌種為第0 代),并采用適宜的菌種保藏技術進行保存,以保證試驗菌株的生物學特性。
       
      菌液制備:按表1規定程序培養各試驗菌株。取大腸埃希菌,銅綠假單胞菌,金黃色葡萄球菌,乙型副傷寒沙門菌的新鮮培養物至胰酪大豆胨液體培養基中,35℃培養18~ 24小時,取上述培養物1ml加入9ml 0.9%無菌氯化鈉溶液中,制成10-1的菌液,依法10倍稀釋至10-7,分取各菌懸液1ml注入平皿中,立即傾注胰酪大豆胨瓊脂培養基20ml,各菌懸液平行制備兩個平皿,平皿法培養計數,取小于100CFU/ml的菌液備用;取白色念珠菌的新鮮培養物至沙氏葡萄糖液體培養基中,25℃培養2~3天。取上述培養物1ml加入9ml 0.9%無菌氯化鈉溶液中,制成10-1的菌液,依法10倍稀釋至10-7,取菌懸液1ml注入平皿中,立即傾注沙氏葡萄糖瓊脂培養基20ml,各菌懸液平行制備兩個平皿,平皿法培養計數,取小于100CFU/ml的菌液備用;
       
      菌液制備后若在室溫下放置,應在2小時內使用;若保存在2?8℃,可在24小時內使用。
       
      2.1.3 陰性對照
       
      為確認試驗條件是否符合要求,應進行陰性對照試驗,陰性對照試驗應無菌生長。如陰性對照有菌生長,應進行偏差調查。
       
      2.2 控制菌檢查用的成品培養基,由脫水培養基或按處方配制的均應進行適用性檢查。控制菌檢査用培養基的適用性檢查項目包括促生長能力,抑制能力及指示特性的檢查。各培養基的檢查項目及所用的菌株見表1。
       
      2.2.1 液體培養基促生長能力檢査分別接種不大于100cfu的試驗菌于被檢培養基和對照培養基中,在相應控制菌檢查規定的培養溫度及不大于規定的最短培養時間下培養,與對照培養基管比較,被檢培養基管試驗菌應生長良好。
       
      2.2.2 固體培養基促生能力檢査用涂布法分別接種不大于100cfu的試驗菌(表1 )于被檢培養基和對照平板上,在相應控制菌檢査規定的培養溫度及不大于規定的最短培養時間下培養,被檢培養基與對照培養基上生長的菌落大小,形態特征應一致。
       
      2.2.3 抑制能力檢査接種不少于l00cfu的試驗菌(表1)于被檢培養基和對照培養基中,在相應控制菌檢査規定的培養溫度及不小于規定的最長培養時間下培養,試驗菌應不得生長。
       
      2.2.4 培養基指示特性檢査用涂布法分別接種不大于100cfu的試驗菌(表1 )于被檢培養基和對照培養基平板上,在相應控制菌檢查規定的培養溫度及不大于規定的最短培養時間下培養,被檢培養基上試驗菌生長的菌落大小,形態特征,指示劑反應情況等應與對照培養基一致。
       
      6失效,檢測用過培養基(無論是否有菌生長)均按廢物進行高壓蒸汽滅菌處理。參照《實驗室廢棄物管理規程》(文件編號:SMP-10-QC-014(00))。
       
      Establish standard operating procedures for media preparation and applicability inspection, and standardize the operation process of laboratory staff.
      Scope: it is suitable for microbiological examination personnel to regulate the culture medium.
      Basis: "drug production quality management standard (revised 2010)", "People's Republic of China Pharmacopoeia" 2015 edition fourth.
      Duty:
      1. microbiological inspector: responsible for the management of the medium required by the laboratory, so that the daily inspection can be carried out smoothly.
      2. microbiological test supervisor: responsible for supervising the normal operation of daily preparation.
      content
      1 according to the requirements of the test items, workload and work schedule, one month ahead of time will be required for the purchase of the media. Supplier must be designated when purchasing, and supplier audit is necessary if necessary.
      2.1 after the culture medium arrives, the microbial room inspector should check. The acceptance includes checking the name, quantity, specification and manufacturer. It should be consistent with the purchase order; check whether the package is broken, the package is complete, and whether the product is in the period of validity.
      2.2 after acceptance, it is registered in the "training base ledger" (attached with the record number: SMP-10-QC-009-02 (00)).
      3.1 unopened tea is stored in a cool room to keep the medium at low temperature, dry and dark. The open seal should be covered tightly and stored in the cool storage.
      3.2 the medium with good sterilization should be pre incubated with 72h for asepsis and then stored at 4~8 C for storage. After sterilization, the storage period was 7 days.
      4.1 the preparation and use of the medium should fill in the record of the preparation and use of the medium (with the serial number: SMP-10-QC-009-02 (00)).
      4.2 culture medium preparation batch number principle: raw material batch number - preparation date: for example, the medium of preparation in January 1, 2011, dry powder batch number 000000, batch number is: 000000-110101. After making the preparation, you need to label the culture label (with the record number: R-SMP-10-QC-009-03). (Note: on the same day, the same medium is configured several times, with the addition of "-" and "digit" after the original batch number, such as 000000-110101-01).
      When 4.3.1 uses commercialized dehydration to synthesize the medium, it should be prepared in strict accordance with the instructions provided by the manufacturer, such as weight / volume, pH, sterilization conditions and operation steps. When the laboratory uses a variety of basic ingredients, it should be prepared accurately according to the formula, and records related information such as name and type and reagent level, the content of each component, manufacturer, batch number, pH value, medium volume / volume, sterilization condition (sterilization method, temperature and time), preparation date, personnel, etc. It traced to the source.
      4.3.2 water, vessel requirements: purified water is used for preparation, deionized water and distilled water are used for special instructions. The containers and matching utensils used in the preparation of culture medium should be clean. The heat sensitive medium, such as sugar fermentation medium, should be pre sterilized to ensure the aseptic nature of the medium.
      4.3.3 weighing and packing: a dehydrated synthetic medium for rapid weighing required (wearing a mask or operating in a ventilator when necessary in order to prevent inhalation of medium powder containing toxic substances). Lest the tide be sucked. First, add a proper amount of water and mix well. Be careful to avoid caking and then add water to the desired amount. Choose whether or not to be heated according to the instructions.
      The volume of the bulk is not more than 2/3 of the volume of the container. Preparation of agar media such as slants should also be heated to boil until completely dissolved and then packed.
      Determination and adjustment of 4.4 pH value
      The pH value was detected by pH, and the temperature was 25 + 2. After the preparation, the 10ml was determined by pH. 20ml was also taken from 50ml triangulation bottle and the same batch of culture medium at the same time, and then cooled to 25 + 2 degrees centigrade, then PH was detected. The pH should be controlled before and after sterilization. Before sterilization, a solution of sodium hydroxide, about 40g/L (about 1mol/L), or a solution of about 36.5g/L (about 1mol/L) of hydrochloric acid, can be used slowly to avoid repeated adjustment to increase the ion concentration. After sterilization, it does not conform to the standard and is deemed to be unqualified and not used.
      4.5 medium and reagents should be treated with wet heat sterilization or filtration sterilization. Damp heat sterilization is carried out in autoclave or steam sterilizing cabinet. If the container is to be sterilized by damp heat, the sterilization condition is 121 min 30. The culture medium is sterilized strictly according to the specification condition. After sterilization, the medium should be removed immediately and not stored in a high pressure sterilizer.
      5.1 count medium applicability examination
      Preparation of 5.1.1 strain and bacterial liquid
      Bacteria species: the number of passages of the tested strains should not exceed 5 generations (the zeroth generation of dry bacteria obtained from the Preservation Center of the bacteria), and the suitable bacteria preservation technology is used to preserve the biological characteristics of the tested strains. Enumeration medium, applicability test and enumeration method, applicability test strains are shown in Table 1.
      Preparation of bacterial liquid: according to the procedures specified in Table 1, all strains were cultured. The fresh culture of Pseudomonas aeruginosa, Pseudomonas aeruginosa and Bacillus subtilis were cultured in the medium of soy peptone liquid for 18~24 hours for 18~24 hours, and the culture substance 1ml was added to 9ml 0.9% aseptic Sodium Chloride Solution to produce 10-1 bacterial liquid, which was diluted to 10-7 by 10 times according to law, and the bacteria suspension was injected into the Petri dish by 1ml. 20ml was immediately poured into the soy peptone agar medium, and two plates were prepared in parallel to the suspension of the bacteria. The plate culture was counted and the bacterial liquid was less than 100CFU/ml; the fresh culture of Candida albicans was taken to the liquid culture medium of Salmonella glucose and cultured for 2~3 days at 25. The above culture medium 1ml was added to 9ml 0.9% aseptic Sodium Chloride Solution to make 10-1 bacteria liquid, diluted to 10-7 by 10 times according to law, and then injected the bacteria suspension 1ml into the plate and immediately poured the Salmonella glucose agar medium 25ml. The bacteria suspension was prepared in parallel to prepare two Petri dishes, the plate method culture was counted, and the bacterial liquid was less than 100CFU/ml, and the black koji was taken. The fresh culture of moldy was inoculated to the culture medium of Salmonella agar agar, and 0.9% aseptic Sodium Chloride Solution containing 0.05% (ml/ml) polysorbate 80 was added to the 0.05% (ml/ml) polysorbate 80 for 5~7 days, and the spores were eluted. Then the spore suspension was sucked into the aseptic test tube, and the culture substance 1ml was added to the 9ml 0.9% aseptic Sodium Chloride Solution to make 10-1 of the bacterial liquid, which was diluted to 10-7 by 10 times according to law. The bacteria suspension was injected into the Petri dish by 1ml injection, and immediately poured into the salser glucose agar medium 20ml, and the bacteria suspension was prepared in parallel to two Petri dishes, and the plate method was used for culture. Count, take the bacteria less than 100CFU/ml and spare.
      If the bacteria solution is placed at room temperature, it should be used within 2 hours. If it is stored at 2? 8 degrees, it can be used within 24 hours. Aspergillus niger spores suspension can be stored at 2? 8 C and used during the verified storage period.
      5.1.2 negative control
      In order to confirm whether the test conditions meet the requirements, negative control tests should be conducted, and negative control tests should be aseptic. If negative control has bacterial growth, deviation investigation should be conducted.
      The finished product culture medium for microbial counting should be checked for suitability by dehydrating medium or by prescription.
      According to table 1, the inoculation of bacterial liquid not more than l00cfu to soy peptone liquid culture medium tube or cheese soybean Peptone Agar Medium plate or Shashi glucose agar medium plate. 2 tubes or 2 Petri dishes were prepared in parallel to each test strain. At the same time, the corresponding control medium was used instead of the tested medium.
      The ratio of the average number of colonies on the tested solid medium and the average number of the colony on the control medium should be within the range of 0.5? 2, and the morphology of the colony should be the same as the colonies on the control medium; the tested liquid culture base tube should grow well compared with the control culture base tube.
      Examination of the applicability of culture medium for the examination of 5.2 controlled bacteria
      Preparation of 5.2.1 strain and bacterial liquid
      Bacteria species: the number of passages of the tested strains should not exceed 5 generations (the zeroth generation of dry bacteria obtained from the Preservation Center of the bacteria), and the suitable bacteria preservation technology is used to preserve the biological characteristics of the tested strains.
      Preparation of bacterial liquid: according to the procedures specified in Table 1, all strains were cultured. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella paratyphi B were cultured in the medium of soya peptone liquid for 18~ 24 hours for 18~ 24 hours, and 10-1 of the bacteria solution was prepared by adding the above culture to 9ml 0.9% aseptic Sodium Chloride Solution, which was diluted to 10-7 by 10 times according to law. In the liquid 1ml injection, 20ml was immediately poured into the soybean Peptone Agar Medium, and two Petri dishes were prepared in parallel with the suspension of the bacteria. The plate culture was counted to take the bacteria less than 100CFU/ml, and the fresh culture of Candida albicans was taken to the Salmonella glucose liquid medium and cultured for 2~3 days at 25. The above culture 1ml was added to 9ml 0.9% aseptic Sodium Chloride Solution to make 10-1 bacteria liquid, diluted to 10-7 by 10 times according to law, and injected the bacteria suspension 1ml into the Petri dish and immediately poured the salser glucose agar medium 20ml. The bacteria suspension was prepared in parallel with two Petri dishes, and the plate culture was counted, and the bacterial liquid was less than 100CFU/ml.
      If the bacteria solution is placed at room temperature, it should be used within 2 hours. If it is stored at 2? 8 degrees, it can be used within 24 hours.
      2.1.3 negative control
      In order to confirm whether the test conditions meet the requirements, negative control tests should be conducted, and negative control tests should be aseptic. If negative control has bacterial growth, deviation investigation should be conducted.
      2.2 culture medium applicability examination
       
      The finished product culture medium used for control bacteria inspection should be checked for suitability by dehydrating medium or prescription. The applicability of the culture medium for examination of control bacteria includes examination of growth promoting ability, inhibition ability and indication characteristics. Examination items of each medium and the strains used are shown in Table 1.
      The growth promoting ability of 2.2.1 liquid culture medium was examined in the tested medium and the control medium, which was not more than 100cfu, respectively. The culture temperature and the shortest culture time of the corresponding control bacteria were not greater than the shortest culture time. Compared with the control medium, the tested bacteria should grow well.
      The 2.2.2 solid culture medium was inoculated with the test bacteria not more than 100cfu (Table 1) on the tested culture medium and the control medium plate respectively. The culture temperature and the shortest culture time of the corresponding control bacteria were not greater than the shortest culture time. The colony size of the cultured culture medium and the control medium was found. The morphological characteristics should be consistent.
      The 2.2.3 inhibition ability was examined by the test bacteria inoculated with l00cfu (Table 1) in the tested medium and the control medium. The test bacteria should not grow at the culture temperature of the corresponding control bacteria and not less than the maximum culture time specified.
      The experimental bacteria (Table 1) that were not more than 100cfu were inoculated on the 2.2.4 culture medium (Table 1) on the tested culture medium and the control medium plate. The culture temperature and the shortest culture time of the corresponding control bacteria were not greater than the shortest culture time. The indicator reaction should be consistent with the control medium.
      6 failure, detection of used medium (whether or not bacteria growth) is treated by high pressure steam sterilization. Refer to "laboratory waste management regulations" (document number: SMP-10-QC-014 (00)).
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