成品預(yù)裝培養(yǎng)基平皿實(shí)用技術(shù)手冊(cè) ：培養(yǎng)基平皿生產(chǎn)與質(zhì)量控制
發(fā)布時(shí)間：
2022-12-27
作者：
一次性平板TSA
成品預(yù)裝培養(yǎng)基平皿實(shí)用技術(shù)手冊(cè) ：培養(yǎng)基平皿生產(chǎn)與質(zhì)量控制
培養(yǎng)基平皿生產(chǎn)與質(zhì)量控制
上一章節(jié)介紹了成品預(yù)裝培養(yǎng)基平皿的質(zhì)量要求，那么我們?nèi)绾文艿玫椒项A(yù)期質(zhì)量要求的成品預(yù)裝培養(yǎng)基平皿呢？
首先了解一下簡(jiǎn)化的培養(yǎng)基平皿生產(chǎn)工藝。
1
配制：在制備培養(yǎng)基時(shí)，準(zhǔn)確稱量質(zhì)量符合要求的脫水培養(yǎng)基或按單獨(dú)配方組分進(jìn)行配制，待脫水培養(yǎng)基或按單獨(dú)配方組分完全溶解后定容、調(diào)節(jié)PH值，再進(jìn)行升溫滅菌。應(yīng)記錄各稱量物的重量和水的使用量。
2
滅菌：培養(yǎng)基配制結(jié)束后，選擇經(jīng)過驗(yàn)證的滅菌程序，立即快速升溫至滅菌溫度進(jìn)行滅菌，滅菌結(jié)束后必須快速冷卻到可分裝溫度。在工序中，溫度對(duì)培養(yǎng)基的質(zhì)量影響非常敏感，所以要根據(jù)滅菌培養(yǎng)基的特性，進(jìn)行全面的滅菌程序驗(yàn)證，以保證在一定裝載方式下的正常熱分布。
3
分裝：將經(jīng)滅菌、冷卻后的培養(yǎng)基分裝到無菌空皿中。在該工序中應(yīng)避免產(chǎn)生氣泡，裝量均一、穩(wěn)定，固體培養(yǎng)基表面不得產(chǎn)生裂縫或漣漪、不破損。分裝后冷卻過程應(yīng)有層流保護(hù)，不得污染微生物。
4
包裝：分裝好的培養(yǎng)皿要及時(shí)包裝，用于環(huán)境監(jiān)控的培養(yǎng)基須特別防護(hù)，需雙層包裝，若是用于A級(jí)區(qū)環(huán)境監(jiān)測(cè)的培養(yǎng)基平皿，建議三層無菌包裝。
5
終端滅菌：將包裝后培養(yǎng)基平皿進(jìn)行滅菌，確保其無菌性。特別是用于環(huán)境監(jiān)測(cè)的培養(yǎng)基平皿，《中國(guó)藥典（2015版）》9203中明確規(guī)定“用于環(huán)境監(jiān)控的培養(yǎng)基須特別防護(hù)，最好要雙層包裝和終端滅菌”。
6
檢驗(yàn)放行：培養(yǎng)基平皿生產(chǎn)結(jié)束后，在使用前必須對(duì)每一批次取樣檢驗(yàn)。檢驗(yàn)項(xiàng)目包括外觀、裝量、PH值、無菌性、微生物促生長(zhǎng)試驗(yàn)，確保在潔凈區(qū)使用的培養(yǎng)基平皿是符合預(yù)期質(zhì)量要求的產(chǎn)品。
培養(yǎng)基平皿生產(chǎn)工藝中工序并不多，但是在很多微生物實(shí)驗(yàn)室自制用于環(huán)境監(jiān)測(cè)的培養(yǎng)皿的過程中，比較容易忽略一些工序細(xì)節(jié)，例如使用單層包裝、非最終滅菌等。在使用前采取100%預(yù)培養(yǎng)雖然避免了“假陽性”，但同時(shí)還帶來了“假陰性”和培養(yǎng)基平皿因包裝保護(hù)不當(dāng)而帶來的交叉污染。
正因?yàn)榕囵B(yǎng)基生產(chǎn)工序不多，一些院校微生物實(shí)驗(yàn)室和部分制藥企業(yè)微生物實(shí)驗(yàn)室所使用的培養(yǎng)基平皿還是根據(jù)培養(yǎng)基各組分自配自用，也有部分是使用按處方生產(chǎn)的符合規(guī)定的脫水培養(yǎng)基進(jìn)行自行配制使用。但是根據(jù)《中華人民共和國(guó)藥典》通則9203“藥品微生物實(shí)驗(yàn)室質(zhì)量管理指導(dǎo)原則”中培養(yǎng)基制備的要求，手工配制培養(yǎng)基平皿難以滿足潔凈區(qū)環(huán)境監(jiān)測(cè)的需要，所以在法規(guī)嚴(yán)格要求及質(zhì)量意識(shí)普遍提高的時(shí)代背景下，用于潔凈區(qū)環(huán)境監(jiān)測(cè)的培養(yǎng)基平皿基本上是采用商品化的成品預(yù)裝培養(yǎng)基平皿。
每個(gè)產(chǎn)品的生產(chǎn)工藝都具有唯一性，成品預(yù)裝培養(yǎng)基平皿也如此，但是培養(yǎng)基的質(zhì)量控制方面，有著共同的關(guān)鍵因素，如人員、培養(yǎng)基、菌種、驗(yàn)證、設(shè)備、文件等。
人員
人員是生產(chǎn)活動(dòng)中最關(guān)鍵的要素，既要管理物料、操控設(shè)備，又要制訂規(guī)程、治理環(huán)境，生產(chǎn)的每道工序都是靠人來把控。
人員應(yīng)依據(jù)所在崗位和職責(zé)接受相應(yīng)的培訓(xùn)，如在上崗前接受勝任工作所必需的設(shè)備操作、微生物檢驗(yàn)技術(shù)等方面的培訓(xùn)，包括無菌操作、培養(yǎng)基制備、消毒、滅菌、平皿分裝、菌落計(jì)數(shù)、菌種的轉(zhuǎn)種、傳代和保藏、微生物檢查方法等，經(jīng)考核合格后方可上崗。
培養(yǎng)基
培養(yǎng)基作為成品培養(yǎng)平皿的原料，是微生物試驗(yàn)的基礎(chǔ)，直接影響微生物試驗(yàn)結(jié)果。適宜的貯藏條件和質(zhì)量控制試驗(yàn)是提供優(yōu)質(zhì)培養(yǎng)基的保證。所以在投產(chǎn)前必須要檢驗(yàn)脫水培養(yǎng)基質(zhì)量，如PH值、微生物促生長(zhǎng)試驗(yàn)等，這也是確保培養(yǎng)基平皿符合質(zhì)量要求的首要條件。
菌種
根據(jù)《中國(guó)藥典》要求，實(shí)驗(yàn)室應(yīng)該有標(biāo)準(zhǔn)化的菌種處理和保藏的程序，盡可能減少菌種污染和生長(zhǎng)特性的改變。規(guī)范操作程序制備的菌株是微生物試驗(yàn)結(jié)果一致性的重要保證。
標(biāo)準(zhǔn)菌株的復(fù)蘇、復(fù)壯或培養(yǎng)物的制備應(yīng)按供應(yīng)商提供的說明或按已驗(yàn)證的方法進(jìn)行。從國(guó)內(nèi)或國(guó)外菌種保藏機(jī)構(gòu)獲得的標(biāo)準(zhǔn)菌株經(jīng)過復(fù)活并在適宜的培養(yǎng)基中生長(zhǎng)后，即為標(biāo)準(zhǔn)貯備菌株。標(biāo)準(zhǔn)貯備菌株應(yīng)進(jìn)行純度和特性確認(rèn)。標(biāo)準(zhǔn)貯備菌株保存時(shí)，可將培養(yǎng)物等份懸浮于抗冷凍的培養(yǎng)基中，并分裝于小瓶中，建議采用低溫冷凍干燥、液氮貯存、超低溫冷凍（低于-30℃）等方法保存。低于-70℃或低溫冷凍干燥方法可以延長(zhǎng)菌種保存時(shí)間。標(biāo)準(zhǔn)貯備菌株可用于制備每月或每周1次轉(zhuǎn)種的工作菌株。冷凍菌種一旦解凍轉(zhuǎn)種制備工作菌株后，不得重新冷凍和再次使用。
工作菌株的傳代次數(shù)應(yīng)嚴(yán)格控制，不得超過5代（從菌種保藏機(jī)構(gòu)獲得的標(biāo)準(zhǔn)菌株為第0代），以防止過度的傳代增加菌種變異的風(fēng)險(xiǎn)。1代是指將活的培養(yǎng)物接種到微生物生長(zhǎng)的新鮮培養(yǎng)基中培養(yǎng)，任何形式的轉(zhuǎn)種均被認(rèn)為是傳代1次。必要時(shí)，實(shí)驗(yàn)室應(yīng)對(duì)工作菌株的特性和純度進(jìn)行確認(rèn)。
工作菌株不可替代標(biāo)準(zhǔn)菌株，標(biāo)準(zhǔn)菌株的商業(yè)衍生物僅可用作工作菌株。標(biāo)準(zhǔn)菌株如果經(jīng)過確認(rèn)試驗(yàn)證明已經(jīng)老化、退化、變異、污染等或該菌株已無使用需要時(shí)，應(yīng)及時(shí)滅菌銷毀。
實(shí)驗(yàn)室必須建立和保存其所有菌種的進(jìn)出、收集、貯藏、確認(rèn)試驗(yàn)以及銷毀的記錄，應(yīng)有菌種管理的程序文件（從標(biāo)準(zhǔn)菌株到工作菌株），該程序包括：標(biāo)準(zhǔn)菌種的申購記錄；從標(biāo)準(zhǔn)菌株到工作菌株操作及記錄；菌種必須定期轉(zhuǎn)種傳代，并做純度、特性等實(shí)驗(yàn)室所需關(guān)鍵指標(biāo)的確認(rèn)，并記錄；每支菌種都應(yīng)注明其名稱、標(biāo)準(zhǔn)號(hào)、接種日期、傳代數(shù)；菌種生長(zhǎng)的培養(yǎng)基和培養(yǎng)條件；菌種保藏的位置和條件；其他需要的程序。
驗(yàn)證
無論是自行制備還是商品化生產(chǎn)成品預(yù)裝培養(yǎng)基平皿，首先都必須要有滅菌程序的驗(yàn)證，即培養(yǎng)基應(yīng)采用通過驗(yàn)證的滅菌程序進(jìn)行滅菌，培養(yǎng)基滅菌方法和條件，應(yīng)通過無菌性試驗(yàn)和促生長(zhǎng)試驗(yàn)進(jìn)行驗(yàn)證。其次對(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。第三，瓊脂平板最好現(xiàn)配現(xiàn)用，如置冰箱保存，一般不超過1周，且應(yīng)密閉包裝，若延長(zhǎng)保存期限，保存期需經(jīng)驗(yàn)證確定，即產(chǎn)品的穩(wěn)定性考察。第四，產(chǎn)品的工藝驗(yàn)證。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1次。如果培養(yǎng)基的制備過程未經(jīng)驗(yàn)證，那么每一滅菌批培養(yǎng)基均要進(jìn)行適用性檢查試驗(yàn)。
設(shè)備
微生物實(shí)驗(yàn)室應(yīng)配備與檢驗(yàn)?zāi)芰凸ぷ髁肯噙m應(yīng)的儀器設(shè)備，其類型、測(cè)量范圍和準(zhǔn)確度等級(jí)應(yīng)滿足檢驗(yàn)所采用標(biāo)準(zhǔn)的要求。設(shè)備的安裝和布局應(yīng)便于操作，易于維護(hù)、清潔和校準(zhǔn)，并保持清潔和良好的工作狀態(tài)。用于試驗(yàn)的每臺(tái)儀器、設(shè)備應(yīng)該有唯一標(biāo)識(shí)。
儀器設(shè)備應(yīng)有合格證書，實(shí)驗(yàn)室在儀器設(shè)備完成相應(yīng)的檢定、校準(zhǔn)、驗(yàn)證、確認(rèn)其性能，并形成相應(yīng)的操作、維護(hù)和保養(yǎng)的標(biāo)準(zhǔn)操作規(guī)程后方可正式使用，儀器設(shè)備使用和日常監(jiān)控要有記錄。
文件
對(duì)實(shí)驗(yàn)室自行配制培養(yǎng)皿而言，文件應(yīng)當(dāng)充分表明試驗(yàn)是在實(shí)驗(yàn)室里按可控的檢查法進(jìn)行的，一般包括以下方面：人員培訓(xùn)與資格確認(rèn)；設(shè)備驗(yàn)收、驗(yàn)證、檢定（或校準(zhǔn)期間核查）和維修；設(shè)備使用中的運(yùn)行狀態(tài)（設(shè)備的關(guān)鍵參數(shù)）；培養(yǎng)基制備、貯藏和質(zhì)量控制；菌種管理；檢驗(yàn)規(guī)程中的關(guān)鍵步驟；數(shù)據(jù)記錄與結(jié)果計(jì)算的確認(rèn)；數(shù)據(jù)偏離的調(diào)査。
由于培養(yǎng)基平皿作為醫(yī)療器械類產(chǎn)品，其生產(chǎn)企業(yè)必須嚴(yán)格按照《醫(yī)療器械生產(chǎn)質(zhì)量管理規(guī)范》來制訂相關(guān)指導(dǎo)文件指導(dǎo)生產(chǎn)，如工藝規(guī)程、質(zhì)量標(biāo)準(zhǔn)、各操作規(guī)程等。
總之，成品預(yù)裝培養(yǎng)基平皿的質(zhì)量是生產(chǎn)出來的，所以在生產(chǎn)過程中要嚴(yán)格控制生產(chǎn)工序中每一個(gè)環(huán)節(jié)，著眼于整個(gè)質(zhì)量體系，比如人員的規(guī)范操作、物料的質(zhì)量控制、嚴(yán)格規(guī)范的生產(chǎn)過程、科學(xué)的質(zhì)量控制等。
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Practical technical manual for finished preloaded culture medium plate (serial no.5) : production and quality control of culture medium plate
Culture plate production and quality control
In the previous chapter, the quality requirements of the finished prepared medium plate were introduced. How can we get the finished prepared medium plate that meets the expected quality requirements?
First of all, I want to know the simplified medium plate production process.
1
Make up: in the preparation of culture medium, accurate weighing dehydrated medium quality to meet the requirements or preparation according to separate formula components, or be dehydrated medium according to the formula composition alone completely dissolved after the capacity, adjust PH value, and then to heat sterilization. The weight of each weighing object and the amount of water used shall be recorded.
2
Sterilization: after the preparation of culture medium, select the sterilization procedure that has been verified, and immediately heat up rapidly to the sterilization temperature for sterilization. After sterilization, the sterilization must be quickly cooled to the separable temperature. In the process, the temperature impact on the quality of medium is very sensitive, so be according to the characteristics of sterilization medium, thorough sterilization process validation, to ensure the normal heat distribution under certain loading way.
3
Separation: the sterilized and cooled medium is separated into a sterile empty dish. Bubbles should be avoided in this process, and the loading quantity should be uniform and stable. Cracks or ripples on the surface of solid medium should not be produced or damaged. The cooling process after installation should be protected by laminar flow and should not contaminate microorganism.
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4
Packing: packing good dishes are to be packed in A timely manner, medium must be special protection for environmental monitoring, need to double packing, if it is used for A level area environmental monitoring of medium plate, suggested that three layers of aseptic packaging.
5
Terminal sterilization: sterilization is carried out on the flat plate of the packaged medium to ensure its sterility. Especially for environmental monitoring of medium plate, "the Chinese pharmacopoeia (2015 edition) 2015 clearly stipulated in the" medium must be special protection for environmental monitoring, best to double packing and terminal sterilization ".
6
Inspection and release: after the end of the production of petri dishes, each batch of samples must be inspected before use. The inspection items include appearance, loading, PH value, asepsis, and microbial growth promotion test to ensure that the culture medium plate used in the clean area meets the expected quality requirements.
Process in medium plate production process is not much, but in many microbiology laboratory used for environmental monitoring in the process of a dish, it's easy to ignore some process details, such as using single packing, the final sterilization. Although 100% preculture before use avoids "false positive", it also brings cross contamination caused by improper packaging protection of "false negative" and culture plate.
Because of media production process is not much, some colleges microbiology laboratory and part of the pharmaceutical enterprise microbiology laboratory used by medium plate or according to the culture medium components from the match for private use, also has a part is used according to the prescription of dehydrated medium production preparation used on its own. But according to the general principles of the "pharmacopoeia of the People's Republic of China" 9203 "pharmaceutical microbiology laboratory quality management guidelines" in the culture medium preparation requirements, manual preparation medium plate is difficult to meet the demand of clean area environment monitoring and so on laws and regulations strictly, and general improvement in the quality consciousness of era background, the culture medium for clean area environment monitoring AGAR is basically the commercialization of the products with culture medium plate.
Each product the production process has uniqueness, finished with medium plate, but the medium quality control, common key factors, such as personnel, media, strains, validation, equipment, documents, etc.
personnel
Personnel is the most important factor in production activities. We should not only manage materials and control equipment, but also formulate rules and control environment.
Personnel should be based on the position and responsibility to accept the corresponding training, such as necessary to accept the job before mount guard operation of the device, microbial inspection technology and other aspects of training, including aseptic operation, medium preparation, disinfection and sterilization, AGAR, colony counting, the switched to, extend the cultivation and preservation of species, microbiological examination method and so on, after the inspection qualified rear can mount guard.
medium
As the raw material of finished product culture plate, culture medium is the basis of microorganism test, which directly affects the result of microorganism test. Suitable storage condition and quality control test are the guarantee of providing high quality medium. Therefore, the quality of dehydrated medium must be tested before it is put into production, such as PH value, microorganism growth promotion test, etc., which is also the primary condition to ensure that the medium plate meets the quality requirements.
Bacterial species
According to the requirements of the Chinese pharmacopoeia, the laboratory should have standardized procedures for the treatment and storage of strains, so as to minimize the change of bacterial contamination and growth characteristics. The strain prepared by standard operating procedure is an important guarantee for the consistency of microbial test results.
The preparation of the resuscitation, rejuvenation or culture of the standard strain shall be carried out according to the instructions provided by the supplier or in accordance with the proven method. The standard strains obtained from domestic or foreign mycelia preservation institutions are the standard reserve strains after being resurrected and growing in suitable medium. Standard strains should be identified for purity and characterization. Standard supply strains preserved, cultures can be equal parts suspended in resistance to freezing medium, and packing in small bottle, the proposal USES low temperature freeze drying, liquid nitrogen storage, ultra-low temperature freezing method such as save (less than 30 ℃). Lower than 70 ℃ or freeze drying method can prolong strains preserved in low temperature. The standard strains can be used to prepare the working strains which are transferred once a month or once a week. Once the refrigerated strains are thawed and transferred to prepare the working strains, they shall not be re-frozen and reused.
The number of generations of the working strains should be strictly controlled, no more than 5 generations (the standard strain obtained from the seed preservation mechanism is the 10th generation), so as to prevent the excessive propagation from increasing the risk of strain variation. Generation 1 refers to the inoculation of live culture materials into the fresh medium for microbial growth, and any type of transgenic species is considered to be transmitted once. If necessary, the laboratory should confirm the characteristics and purity of the working strains.
The working strains can not replace the standard strains, and the commercial derivatives of the standard strains can only be used as working strains. The standard strain shall be sterilized and destroyed in time if it has been confirmed that it has aged, degraded, mutated, contaminated, etc.
Laboratory must establish and maintain its all strains of pass in and out, collection, storage, validation test as well as the destruction of records, should have a diversity management program files (from the standard strains to job strain), the program include: the standard strains of purchase records; Operation and recording from standard strain to working strain; The bacteria must be transfected and transmitted on a regular basis, and the identification of key laboratory indicators such as purity and characteristics should be made and recorded. Each strain should have its name, standard number, date of inoculation, and algebra indicated. Culture medium and culture conditions for bacterial growth; The location and conditions of bacterial species preservation; Other required procedures.
validation
Preparation on their own, or whether it is commercialized products with medium plate, first is must have the sterilization process validation, the medium should be used through the validation of the sterilization procedures of sterilization, medium sterilization methods and conditions, should be validated aseptic test and the growth test. Secondly, the steam circulation system of high pressure sterilizer should be verified to ensure the normal heat distribution under certain loading mode. Third, AGAR plate is best matched with now, such as refrigerator, generally no more than 1 week, and should be sealed packaging, if extended shelf life, shelf life should be verified and determined, the stability of the product. Fourth, product process validation. In the laboratory, if the culture medium is prepared by a proven preparation and sterilization procedure and the process is controlled, the suitability test of the same batch of dehydrated media can be conducted only once. If the preparation of the culture medium is not verified, the applicability test of each sterilized batch medium shall be conducted.
equipment
The microbiological laboratory shall be equipped with instruments and equipment suitable for the inspection capability and workload, whose types, measuring ranges and accuracy levels shall meet the requirements of the standards used in the inspection. The installation and layout of the equipment should be easy to operate, easy to maintain, clean and calibrate, and keep clean and in good working condition. Each instrument and equipment used for testing shall be uniquely identified.
Instruments and equipment shall have the certificate of quality and laboratory in the instruments and equipment to complete the corresponding verification and calibration, verification, confirm its performance, and to form the corresponding behind the operation, maintenance, and maintenance of standard operating procedures can be used formally, the equipment use and daily monitoring records.
file
For the preparation of the laboratory's own petri dishes, the documents shall fully demonstrate that the tests are conducted in the laboratory under controlled inspection methods, generally including the following aspects: personnel training and qualification confirmation; Equipment acceptance, verification, verification (or verification during calibration) and maintenance; Operating status (key parameters of the equipment) in use; Preparation, storage and quality control of culture media; Germ management; Key steps in the inspection procedures; Confirmation of data records and results calculation; Survey of data deviation.
Because culture medium plate as a class of medical equipment products, the production enterprise must be in strict accordance with the "medical equipment production and the quality control standard to make the relevant guidance documents to guide the production, such as process planning, quality standards, the operation procedures, etc.
In a word, the quality of the finished product with culture medium plate is produced, so in the process of production to strictly control the production process each link, look at the entire quality system, such as personnel, material quality control, strict norms of standard operation of the production process, scientific quality control, etc.