成品預裝培養(yǎng)基平皿實用技術手冊（連載三）： 成品預裝培養(yǎng)基平皿發(fā)展史
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2022-12-27
作者：
成品預裝培養(yǎng)基平皿實用技術手冊（連載三）： 成品預裝培養(yǎng)基平皿發(fā)展史
成品預裝培養(yǎng)基平皿發(fā)展史
成品預裝培養(yǎng)基平皿亦稱即用型培養(yǎng)基，是已經(jīng)滅菌分裝好的，無需再稱量滅菌分裝的、可以直接使用的培養(yǎng)基。
19世紀末，德國著名細菌學家科赫(Robert Koch，1843~1910)成功地制備了固體培養(yǎng)基，并發(fā)明了玻璃平皿，使細菌學得到重大的變革，極大地推動了細菌的分離、培養(yǎng)和鑒別技術，同時也促進了成品預裝培養(yǎng)基平皿的發(fā)展，滿足了微生物檢測的需要。
為了能直接觀察微生物的形態(tài)及生長情況，科學家希望能將微生物培養(yǎng)在固體表面上，就像微生物生長在橘子皮或土豆上一樣。羅伯特·科赫曾用煮沸消毒的土豆來培養(yǎng)細菌。此后，他試著用明膠作培養(yǎng)基的凝固劑。他將明膠加入液體培養(yǎng)基中進行融化，然后將混合均勻的液體緩慢地倒在一塊玻璃板的表面。當明膠冷卻凝固后，就在玻璃板表面形成一層固體培養(yǎng)基。為了防止空氣中雜菌的污染，科赫還用玻璃罩將玻璃板與周圍環(huán)境隔離開來。
但是，人們很快發(fā)現(xiàn)，明膠的凝固力非常弱。膠凝化溫度隨濃度及共存鹽類的種類、濃度、溶液的pH等因素變化而變化。溶解溫度與凝固溫度相差很小，明膠在20 ℃以上就變軟了，分離微生物的劃線操作非常困難。在溫度高于25 ℃時，明膠就液化了，而大多數(shù)細菌的培養(yǎng)溫度都不低于25 ℃。
羅伯特·科赫的同事Walter Hesse也為同樣的問題苦惱著。一次，Walter Hesse的妻子Fannie建議他試一試用瓊脂做凝固劑，因為Fannie用瓊脂做果凍做得不錯。Walter Hesse采納了妻子的建議，發(fā)現(xiàn)瓊脂比明膠更適合做培養(yǎng)基的凝固劑，這使得在固體培養(yǎng)基的技術改進上邁出一大步。
1887年，Richard Petri在Koch平皿技術上作了進一步的改進。Petri設計了一種圓形并帶有圍邊的雙盤，大盤作為皿蓋，而小盤當作皿底。制備固體培養(yǎng)基時，將融化的培養(yǎng)基倒入小盤內，然后再用大盤蓋在小盤上。這就是我們今天所使用的培養(yǎng)基平皿。
自此之后，出現(xiàn)了世界聞名的美國Difco和英國Oxoid等培養(yǎng)基生產(chǎn)專業(yè)公司。其中美國Difco至今已有一百多年歷史， 1917年即開始生產(chǎn)脫水干燥培養(yǎng)基；1979年開始商品化生產(chǎn)成品預裝培養(yǎng)基平皿。1994年該產(chǎn)品進入中國市場。
我國在20世紀50年代末開始研制干燥培養(yǎng)基，到目前已有100多個培養(yǎng)基廠家，干粉培養(yǎng)基品種已達300多種。近幾十年來，我國培養(yǎng)基事業(yè)有了飛速發(fā)展，在原材料的研究、開發(fā)及生產(chǎn)使用上都有了很大的進展，大大縮短了與發(fā)達國家的差距。
有資料顯示，最早在中國生產(chǎn)的培養(yǎng)基平皿單位是國民黨中央防疫處，即中華人民共和國成立后組建的昆明生物制品研究所，其制備工藝為1：2牛肉湯，加蛋白胨、氯化鈉、條狀瓊脂，加熱充分溶解，補水定容；再用20%氫氧化鈉調節(jié)PH值為7.6，然后加熱煮沸，分裝于500ml的克氏瓶中，每瓶分裝250ml，分裝結束后將其進行121度，15分鐘高溫滅菌；滅菌完成后冷卻至50度，在潔凈環(huán)境且在火焰保護下分裝于已滅菌的玻璃平皿中，待冷卻凝固后放置在37度培養(yǎng)箱中培養(yǎng)3天，若無污染視為合格產(chǎn)品。
隨著市場經(jīng)濟的發(fā)展及手工制備的平皿具有效期短、成本高等弊端，小作坊式的手工生產(chǎn)已難以滿足工業(yè)大生產(chǎn)的需要，這樣的時代背景為成品預裝培養(yǎng)基平皿商品化創(chuàng)造了可行之路。20世紀80年代，中國市場上出現(xiàn)了國產(chǎn)商品化的成品預裝培養(yǎng)基平皿，且目前使用越來越廣泛，但是至今尚無市場管理措施和統(tǒng)一的質量標準及控制方法，致使商品化培養(yǎng)基平皿的質量缺乏穩(wěn)定性及規(guī)范性，直接影響潔凈區(qū)微生物檢測結果的準確性和可靠性。
隨著《藥品生產(chǎn)質量管理規(guī)范》和《中國藥典》不斷進步和優(yōu)化，潔凈區(qū)微生物的檢測操作方法也隨之規(guī)范?？傊_到藥品預期的質量要求，必須確保藥品生產(chǎn)過程中潔凈區(qū)微生物污染水平的真實性，從而就必須選擇符合質量要求的培養(yǎng)基平皿對潔凈區(qū)環(huán)境進行全過程的管理控制。
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Practical technical manual of finished preloaded culture medium plate (serialized 3) : development history of finished preloaded culture medium plate
The history of development of finished preloaded culture medium plate
The finished prepared petri dish, also known as ready-to-use medium, is a medium that has been sterilized and packaged, and can be used directly without weighing the sterilized medium.
The end of the 19th century, well-known German bacteriologist Koch (Robert Koch, 1843 ~ 1910) solid medium was prepared successfully, and the glass plate, to have achieved a significant change of bacteriology, greatly promote the bacteria isolation, culture and identification technology, and also promoted the development of the finished product with culture medium plate, meet the needs of the microbial detection.
In order to directly observe the shape and growth of microbes, scientists hope to grow them on solid surfaces, just as microbes grow on orange peels or potatoes. Robert Koch used boiled potatoes to grow bacteria. After that, he tried gelatinizing the culture medium with gelatin. He added gelatin to a liquid medium to melt it, then slowly poured the evenly mixed liquid onto the surface of a glass plate. When the gelatin cools and solidifies, a solid medium is formed on the surface of the glass plate. To prevent contamination with bacteria in the air, Koch also used a glass cover to separate the glass from the surrounding environment.
However, it was soon discovered that gelatin solidified very weakly. The gelation temperature varies with the concentration and the type, concentration and pH of the coexisting salts. Melting temperature and setting temperature difference is very small, gelatin in 20 ℃ above will become soft, isolation of microorganisms operation very difficult. When temperature above 25 ℃, gelatin liquefaction, and most bacteria cultivation temperature below 25 ℃.
Robert Koch's colleague, Walter Hesse, frets about the same problem. At one point, Walter Hesse's wife, Fannie, suggested that he try agar-agar as a coagulant because Fannie did a good job of making jello out of AGAR. Walter Hesse took his wife's advice and found that agar-agar was better than gelatin as a coagulant for the medium, a big step towards improving the technology of solid media.
In 1887, Richard Petri made further improvements in the Koch plate technology. Petri has designed a round double dish with a round edge, with the large plate as the lid and the small plate as the bottom. When preparing the solid medium, pour the melted medium into a small plate and cover the plate with a large plate. This is the culture medium plate that we use today.
Since then, the world famous media production companies such as Difco in the us and Oxoid in the UK have emerged. The Difco in the United States has a history of more than one hundred years. In 1979, commercial production of finished preloaded culture medium plate began. The product entered the Chinese market in 1994.
China began to develop dry medium at the end of 1950s. Up to now, there have been more than 100 medium manufacturers and more than 300 dry medium varieties. In recent decades, China's media industry has developed rapidly, and great progress has been made in the research, development and production and use of raw materials, greatly shortening the gap with developed countries.
The data shows, the earliest production of medium plate unit in China is the kuomintang central and epidemic prevention, which formed after the founding of the People's Republic of kunming institute of biological products, the preparation technology of 1:2 beef soup, add peptone AGAR, sodium chloride, strip, heating dissolves, hydrating constant volume; Add 20% sodium hydroxide to adjust the PH value to 7.6, then heat and boil it, and divide it into 500ml bottles. Each bottle is 250ml. Sterilization after the completion of the cooled to 50 degrees, in a clean environment and jars in fire protection in sterilized glass plate, wait for after cooling solidification placed at 37 degrees cultivation in 3 days, if no pollution as qualified products.
With the development of the market economy and the manual preparation on the AGAR with valid in short, the cost is higher, the production of small cottage by hand has been difficult to meet the needs of industrial production, such as time background for the finished product with culture medium plate commercialization created a feasible way. Appeared in the 1980 s, the Chinese market of domestic commercial products with medium plate, and now more and more widely used, but so far there is no market management measures and unified quality standards and control method, the commercialization of medium plate quality lack of stability and normative, directly influence the veracity and reliability of clean areas microbial test results.
With the continuous progress and optimization of the standards for the quality management of drug production and the Chinese pharmacopoeia, the detection and operation methods of microbes in clean areas are also standardized. In a word, to achieve the desired drug quality requirement, must ensure that the pharmaceutical production process clean areas microbial contamination level of authenticity, thereby must choose to meet the requirements of the quality of medium plate control over the management of the whole process of the clean area environment.