實驗室制備培養(yǎng)基的基本操作方法和注意事項
發(fā)布時間：
2022-12-27
作者：
一，玻璃器皿的清洗
在制備培養(yǎng)基的過程中，首先要使用一些玻璃器皿，如試管，三角瓶，培養(yǎng)皿，燒杯和吸管等。這些器皿在使用前都要根據(jù)不同的情況，經(jīng)過一定的處理，洗刷干凈。有的還要進(jìn)行包裝，經(jīng)過滅菌等準(zhǔn)備就緒后，才能使用。
1，新購的玻璃器皿
除去包裝沾染的污垢后，先用熱肥皂水刷洗，流水沖凈，再浸泡于１～２％的工業(yè)鹽酸中數(shù)小時，使游離的堿性物質(zhì)除去，再以流水沖凈。對容量較大的器皿，如大燒瓶，量筒等，洗凈后注入濃鹽酸少許，轉(zhuǎn)動容器使其內(nèi)部表面均沾有鹽酸，數(shù)分鐘后傾去鹽酸，再以流水沖凈，倒置于洗滌架上將水空干，即可使用。
2，用過的玻璃器皿
凡確無病原菌或未被帶菌物污染的器皿，使用后可隨時沖洗，吸取過化學(xué)試劑的吸管，可先浸泡于清水中，待到一定數(shù)量后再集中進(jìn)行清洗。有可能被病原菌污染的器皿，必須經(jīng)過適當(dāng)消毒后，將污垢除去，用皂液洗刷，再用流水沖洗干凈。若用皂液未能洗凈的器皿，可用洗液浸泡適當(dāng)時間后再用清水洗凈。洗液的主要成份是重鉻酸鉀和濃流酸，其作用是將有機(jī)物氧化成可溶性物質(zhì)，以便沖洗。洗液有很強的腐蝕作用，使用時應(yīng)特別小心，避免濺到衣服，身體和其他物品上。
二，在實驗室中配制的適合微生物生長繁殖或累積代謝產(chǎn)物的任何營養(yǎng)基質(zhì)，都叫做培養(yǎng)基(Media)。由于各類微生物對營養(yǎng)的要求不同，培養(yǎng)目的和檢測需要不同，可根據(jù)某種標(biāo)準(zhǔn)，劃分為若干類型。
1，根據(jù)組成物質(zhì)的化學(xué)成分是否完全了解來區(qū)分：
天然培養(yǎng)基是指利用各種動，植物或微生物的原料，其成分難以確切知道。主要原料有：牛肉膏，麥芽汁，蛋白胨，酵母膏，玉米粉，麩皮，各種餅粉，馬鈴薯，牛奶，血清等。用這些物質(zhì)配成雖然不能確切知道它的化學(xué)成分，但一般來講，營養(yǎng)是比較豐富的，微生物生長旺盛，而且來源廣泛，配制方便，所以較為常用，尤其適合于配制實驗室常用的培養(yǎng)基。這一類產(chǎn)品穩(wěn)定性常受生產(chǎn)廠或批號等因素的影響。
合成培養(yǎng)基是一類化學(xué)成分和數(shù)量完全知道的peiyangji，它是用已知化學(xué)成分的化學(xué)藥品配制而成。這類產(chǎn)品化學(xué)成分精確，重復(fù)性強，但價格昂貴，而微生物又生長緩慢，所以它只適用于做一些科學(xué)研究，例如營養(yǎng)，代謝的研究。
c.在合成培養(yǎng)基中加入某種或幾種天然成分；或者在天然培養(yǎng)基中加入一種或幾種已知成分的化學(xué)藥品即成半合成培養(yǎng)基。這一類產(chǎn)品在生產(chǎn)實踐和實驗室中使用最多。
2，根據(jù)物理狀態(tài)來區(qū)分：
a.液體培養(yǎng)基所配制的是液態(tài)的，其中的成分基本上溶于水，沒有明顯的固形物，營養(yǎng)成分分布均勻，易于控制微生物的生長代謝狀態(tài)。
b.在液體中加入適量的凝固劑即成固體培養(yǎng)基。常用作凝固劑的物質(zhì)有瓊脂，明膠，硅膠等，以瓊脂最為常用。在實驗室中，它被用作微生物的分離，鑒定，檢驗雜菌，計數(shù)，保藏，生物測定等。
c.如果把少量的凝固劑加入到液體中，就制成了半固體培養(yǎng)基。以瓊脂為例，它的用量在0.2至1%之間。有時可用來觀察微生物的動力，有時用來保藏菌種。
3，根據(jù)用途來區(qū)分：
a.在其中加入某種物質(zhì)以殺死或抑制不需要的菌種生長的培養(yǎng)基，如鏈霉素，氯霉素等抑制原核微生物的生長；而制霉菌素，灰黃霉素等能抑制真核微生物的生長；結(jié)晶紫能抑制革蘭氏陽性細(xì)菌的生長等。
b.為了分離我們所需要的微生物，在普通培養(yǎng)基中加入一些某種微生物特別喜歡的營養(yǎng)物質(zhì)，以增加這種微生物的繁殖速度，逐漸淘汰其它微生物，這種被稱為增殖培養(yǎng)基，常用于菌種篩選和選擇增菌中。
c.在培養(yǎng)基中加入某種試劑或化學(xué)藥品，使難以區(qū)分的微生物經(jīng)培養(yǎng)后呈現(xiàn)出明顯差別，因而有助開快速鑒別某種微生物。這樣的被稱之為鑒別培養(yǎng)基。例如用以檢查飲水和乳品中是否含有腸道致病菌的伊紅美藍(lán)就是常用的一種。
有些是具有選擇和鑒別雙重作用。例如食品檢驗中常用的麥康凱培養(yǎng)基是一例。它含有膽鹽，乳糖和中性紅。膽鹽具有抑制腸道菌以外的細(xì)菌的作用選擇性，乳糖和中性紅指示劑能幫助區(qū)別乳糖發(fā)酵腸道菌如大腸桿菌和不能發(fā)酵乳糖的腸道致病菌如沙門氏菌和志賀氏菌。
三，制備培養(yǎng)基的基本方法和注意事項
1，配方的選定：同一種配方在不同著作中常會有某些差別。因此，除所用的是標(biāo)準(zhǔn)方法，應(yīng)嚴(yán)格按其規(guī)定進(jìn)行配制外，一般均應(yīng)盡量收集有關(guān)資料，加以比較核對，再依據(jù)自己的使用目的，加以選用，記錄其來源。
2，制備記錄：每次制備均應(yīng)有記錄，包括名稱，配方及其來源，和各種成份的牌號，最終ph值，消毒的溫度和時間制備的日期和制備者等，記錄應(yīng)復(fù)制一份，原記錄保存?zhèn)洳?，?fù)制記錄隨制好的一同存放，以防發(fā)生混亂。
3，成分的稱取：各種成分必須精確稱取并要注意防止錯亂，最好一次完成，不要中斷。可將配方置于傍側(cè)，每稱完一種成分即在配方面軍做出記號，并將所需稱取的藥品一次取齊，置于左側(cè)，每種稱取完畢后，即移放于右側(cè)。完全稱取完畢后，還應(yīng)進(jìn)行一次檢查。
4，各成份的混合和溶化：培養(yǎng)基所用化學(xué)藥品均應(yīng)是化學(xué)純的。使用的蒸煮鍋不得為銅鍋或鐵鍋，以防有微量銅或鐵混入其中，使細(xì)菌不易生長。最好使用不銹鋼鍋加熱溶化，可放入大燒杯或大燒瓶中置高壓蒸汽滅菌器或流動蒸汽消毒器中蒸煮溶化。在鍋中溶化時，可先用溫水加熱并隨時擾動，以防焦化，如發(fā)現(xiàn)有焦化現(xiàn)象，該培養(yǎng)基即不能使用，應(yīng)重新制備。待大部分固體成分溶化后，再用較小火力使所有成分完全溶化，迄至煮沸。如為瓊脂溶化，用另一部分水溶化其它成分，然后將兩溶液充分混合。在加熱溶化過程中，因蒸發(fā)而丟失的水分，最后必須加以補足。
5，ph的初步調(diào)正：在加熱消毒過程中，ph會有所變化，培養(yǎng)基各成分完全溶解后，應(yīng)進(jìn)行ph的初步調(diào)正。例如，牛肉浸液約可降低ph0.2，而腸浸液ph卻會有顯著的升高。因此，對這個步驟，操作者應(yīng)隨時注意探索經(jīng)驗，以期能掌握最終ph來保證質(zhì)量。ph調(diào)整后，還應(yīng)煮沸數(shù)分鐘，以利沉淀物的析出。
6，過濾：瓊脂培養(yǎng)基應(yīng)透明無顯著沉淀，因此，須要采用過濾或其它澄清方法以達(dá)到此項要求。一般液體的可用濾紙過濾法，濾紙應(yīng)折疊成折扇或漏斗形，以避免因液壓不均勻而引起濾紙破裂。
它可用清潔的白色薄絨布趁熱過濾。亦可用中間夾有薄層吸水棉的雙層紗布過濾。新制肉，肝，血和土豆等浸液時，則須先用絨布將碎渣濾去，再用濾紙反復(fù)過濾。如過濾法不能達(dá)到澄清要求，則須用蛋清澄清法。即將冷卻至55至60攝氏度的培養(yǎng)基放入大的三角燒瓶內(nèi)，裝入量不得超過燒瓶容量的1/2，每1000ml里加入1至2個雞蛋的蛋白，強力振搖3至5分鐘，置高壓蒸汽滅菌器中，121攝氏度加熱20分鐘，取出趁熱以絨布過濾即可。
7，分裝：按使用的目的和要求，分裝于試管，燒瓶等適當(dāng)容器內(nèi)。分裝量不得超過容器裝盛量的2/3。容器口可用墊有防濕紙的棉塞封堵，其外還須用防水紙包扎現(xiàn)試管一般多有用螺旋蓋者。分裝時最好能使用半自動或電動的定量分裝器。分裝瓊脂斜面時，分裝量應(yīng)以能形成2/3底層和1/3斜面的量為洽當(dāng)。分裝容器應(yīng)預(yù)先清洗干凈并經(jīng)干烤消毒，以利于徹底滅菌。每批應(yīng)另外分裝20ml于一小玻璃瓶中，隨該批同時滅菌，以為測定該批最終ph之用。
8，培養(yǎng)基的滅菌：一般可采用121攝氏度高壓蒸汽滅菌15分鐘的方法。在各種制備方法中，如無特殊規(guī)定，即可用此法滅菌。
某些畏熱成分，如糖類，應(yīng)另行配成20%或更高的濃液，以過濾或間歇滅菌法消毒，以后再用無菌操作技術(shù)，定量加于培養(yǎng)基。
瓊脂斜面應(yīng)在滅菌后立即取出，冷至55攝氏度-60攝氏度時，擺置成適當(dāng)斜面，待其自然凝固。
9，質(zhì)量測試：每批制備好以后應(yīng)仔細(xì)檢查一遍，如發(fā)現(xiàn)破裂，水分浸入，色澤異常，棉塞被產(chǎn)品沾染等，均應(yīng)挑出棄去。并測定其最終ph。放入36±1攝氏度恒溫箱培養(yǎng)過夜，如發(fā)現(xiàn)有菌生長，即棄去。
用有關(guān)的標(biāo)準(zhǔn)菌株接種1至2管或瓶培養(yǎng)基，培養(yǎng)24至48小時，如無菌生長或生長不好。應(yīng)追查原因并重復(fù)接種一次，如結(jié)果仍同前，則該批即應(yīng)棄去，不能使用。
10，保存：應(yīng)存放于冷暗處，最好能放于普通冰箱內(nèi)。放置時間不宜超過一周，傾注的平板不宜超過3天。每批培養(yǎng)基均必須附有該批制備記錄副頁或明顯標(biāo)簽。
Cleaning of glassware
In the process of preparing the medium, some glassware such as test tube, triangle bottle, Petri dish, beaker and straw should be used first. These containers should be cleaned and cleaned according to different situations before they are used. Some have to be packed and sterilized before they can be used.
1, newly purchased glassware.
After removing the contaminated dirt from the package, wash it with hot soap water, rinse it with running water, then soak it in 1-2% industrial hydrochloric acid for several hours to remove the free alkaline substance, and then rinse it with running water. For large capacity containers, such as large flasks, measuring barrels, etc., after washing, a little concentrated hydrochloric acid is injected into the container, turning the container so that its internal surface is stained with hydrochloric acid, a few minutes later to pour out hydrochloric acid, then rinse with running water, pour it on the washing rack and dry the water.
2, used glassware
If there is no pathogenic bacteria or contaminated utensils, they can be washed at any time after use. The straws that have absorbed chemical reagents can be immersed in clear water, and then washed in a certain amount. Containers that may be contaminated by pathogens must be properly disinfected, dirt removed, soaped and rinsed with running water. If you can't wash the soap with liquid soap, wash it with suitable lotion and wash it with clean water. The main components of the lotion are potassium dichromate and concentrated acid, which oxidize organic matter into soluble substances for washing. The lotion has a strong corrosive effect and should be used with special care to avoid splashing on clothes, bodies and other items.
Second, any nutrient substrate prepared in the laboratory for microbial growth, reproduction or accumulation of metabolites is called a media.
Due to the different nutritional requirements of various microorganisms, different culture purposes and detection needs, according to a certain standard, can be divided into several types.
1, distinguish whether the chemical composition of the constituent substance is completely understood.
Natural medium refers to the ingredients of various plants, animals and microorganisms, and its components are hard to know. The main raw materials are: beef paste, wort, peptone, yeast extract, corn flour, bran, all kinds of cake powder, potato, milk, serum and so on. Although the chemical composition of these substances can not be accurately known, but generally speaking, nutrition is relatively rich, microorganisms grow vigorously, and a wide range of sources, easy preparation, so more commonly used, especially suitable for the preparation of laboratory commonly used medium. The stability of such products is often affected by factors such as factories or batch numbers.
The synthetic medium is a kind of peiyangji with well-known chemical constituents and quantities. It is prepared from chemicals with known chemical constituents. These products are chemically accurate, repeatable, but expensive, and microorganisms grow slowly, so they are only suitable for doing scientific research, such as nutrition and metabolism.
C. Adding one or more natural ingredients to the synthetic medium, or adding one or more known chemicals to the natural medium to form a semi-synthetic medium. This kind of product is the most widely used in production practice and laboratory.
Culture map of laboratory microorganism
2, distinguish according to physical state.
A. The liquid medium is made up of liquid, which is basically soluble in water, without obvious solids, nutrient distribution is uniform, easy to control the growth and metabolism of microorganisms.
B. added a suitable amount of coagulant in the liquid to form a solid medium. Agar, gelatin and silica gel are commonly used as coagulants. Agar is the most commonly used material. In the laboratory, it is used for microbial isolation, identification, detection of miscellaneous bacteria, counting, preservation, biological determination and so on.
C. If a small amount of coagulant is added to the liquid, a semisolid medium will be made. Take agar as an example. Its dosage ranges from 0.2 to 1%. Sometimes it can be used to observe the power of microorganisms, and sometimes to preserve bacteria.
3, according to the purpose, we should distinguish:
A. Some substances were added to the medium to kill or inhibit the growth of unnecessary bacteria, such as streptomycin and chloramphenicol, which inhibited the growth of prokaryotic microorganisms; nystatin and griseofulvin could inhibit the growth of eukaryotic microorganisms; crystal violet could inhibit the growth of Gram-positive bacteria, etc.
B. In order to isolate the microorganisms we need, some nutrients which some microorganisms particularly like are added to the ordinary medium to increase the propagation rate of this microorganism and gradually eliminate other microorganisms. This is called the proliferation medium, which is often used in the selection and enrichment of strains.
C. Adding a reagent or chemical to the medium makes the indistinguishable microorganisms show distinct differences after culture, thus facilitating the rapid identification of certain microorganisms. This is called discriminating medium. Eosin methylene blue, for example, is commonly used to detect intestinal pathogens in drinking water and dairy products.
Some have the dual functions of selection and identification. For example, Makanke culture medium, commonly used in food inspection, is an example. It contains bile salts, lactose and neutral red. Bile salts selectively inhibit bacteria other than intestinal bacteria, and lactose and neutral red indicators help distinguish between lactose-fermenting intestinal bacteria such as Escherichia coli and intestinal pathogens such as Salmonella and Shigella that do not ferment lactose.
Three, the basic methods and matters needing attention in the preparation of culture medium.
1, the selection of recipes: there is always some difference between the same formula in different works. Therefore, in addition to the standard method, should be strictly compounded in accordance with its provisions, should generally try to collect relevant information, to compare and check, and then according to their own use purposes, to select and record its source.
2. Preparation records: Each preparation should have a record, including the name, formula and source, and various components of the brand, the final pH value, disinfection temperature and time preparation date and the preparator, the record should be duplicated, the original record kept for reference, duplicated records stored with the system in order to prevent confusion.
3. Components: All components must be accurately weighed and attention should be paid to prevent confusion, it is best to complete one time, do not interrupt. The recipe can be placed on the side, after each ingredient is weighed, that is, the recipe army makes a mark, and the required medicines are taken one at a time, placed on the left, after each weighing is completed, that is, moved to the right. After the completion of the test, a check should also be made.
4, the mixing and dissolving of each component: the chemicals used in the culture medium should be chemically pure. The cooking pot used should not be copper or iron pot, in case there is a trace of copper or iron mixed into it, so that bacteria do not grow easily. It is best to melt in a stainless steel pan. It can be boiled and melted in a large beaker or flask in a high-pressure steam sterilizer or a flowing steam sterilizer. When melting in a pot, the medium can be heated with warm water and disturbed at any time to prevent coking. If coking is found, the medium can not be used and should be reproduced. After most of the solid components are melted, use less firepower to dissolve all the components until boiling. If it is dissolved for agar, dissolve some other components with another part of water, and then mix the two solution together. In the process of heating and melting, the water lost by evaporation must be supplemented finally.
5. Preliminary adjustment of ph: in the process of heating and disinfection, the pH will change. After the components of the medium are completely dissolved, the initial adjustment of pH should be carried out. For example, beef extract can reduce ph0.2 and intestinal extract pH will increase significantly. Therefore, for this step, the operator should always pay attention to exploring experience, in order to master the final pH to ensure quality. After adjustment, pH should be boiled for several minutes to facilitate the precipitation of precipitates.
6, filtration: agar medium should be transparent without significant precipitation, therefore, it is necessary to use filtration or other clarification methods to achieve this requirement. The filter paper should be folded into folding fan or funnel shape to avoid the filter paper breaking because of the non-uniform hydraulic pressure.
It can be filtered with clean white flannelette. It can also be filtered by double-layer gauze sandwiched with thin absorbent cotton sandwiched in the middle. When fresh meat, liver, blood and potatoes are soaked, the crumbs must be filtered out with flannel, and then filtered repeatedly with filter paper. If the filtration method fails to meet the clarification requirements, the egg white clarification method must be used. The medium will be cooled to 55 to 60 degrees Celsius into a large triangular flask, not more than 1/2 of the flask capacity, every 1000 ml to add 1 to 2 eggs of protein, strong shake 3 to 5 minutes, placed in a high-pressure steam sterilizer, 121 degrees Celsius heated for 20 minutes, take out while hot cloth filtration can be.
7, sub packing: according to the purpose and requirements of the use, it will be packed in the appropriate containers such as test tube and flask. The loading capacity should not exceed 2/3 of container loading capacity. The container can be sealed with a cotton plug padded with moisture-proof paper, and the test tube should be wrapped with water-proof paper. It is better to use semi-automatic or electric quantified distributor when loading. When the agar slant is packed separately, the amount of packing should be consistent with the amount of 2/3 bottom and 1/3 slope. The separated containers should be pre cleaned and dry baked for disinfection. Each batch should be separated into 20 ml in a small glass bottle and sterilized simultaneously with the batch for the purpose of determining the final pH of the batch.
8, sterilization of culture medium: generally, it can be sterilized by 121 degree centigrade high pressure steam for 15 minutes. In various preparation methods, if there is no special provision, this method can be used for sterilization.
Some heat-resistant components, such as carbohydrates, should be separately prepared into 20% or higher concentrated solution to be sterilized by filtration or intermittent sterilization, and then added to the medium quantitatively by aseptic operation.
The slopes of agar should be taken out immediately after sterilization. When it is cooled to 55 - 60 degrees Celsius, it should be placed on a proper slope until it solidifies naturally.
9. Quality testing: After each batch is prepared, it should be carefully checked, such as cracking, water immersion, abnormal color, cotton plug quilt products stained, etc., should be picked out and discarded. The final pH was determined. Incubate in incubators at 36 + 1 degrees Celsius for overnight.
Inoculate 1 to 2 tubes or bottles of the standard strains and incubate them for 24 to 48 hours, if they are sterile or do not grow well. The cause should be traced and repeated for one time. If the result is still the same, the batch should be abandoned and cannot be used.
10, preservation: should be stored in cold dark places, preferably in ordinary refrigerators. The placement time should not exceed one week, and the flat plate should not exceed 3 days. Each batch of medium must be attached to the batch preparation record page or obvious label.