微生物實(shí)驗(yàn)室必知培養(yǎng)基制備技術(shù)說(shuō)明
發(fā)布時(shí)間：
2022-12-27
作者：
藥廠培養(yǎng)基供應(yīng)商
日水生物關(guān)于微生物培養(yǎng)基的制備技術(shù)的說(shuō)明報(bào)告。一、玻璃器皿的清洗
在制備培養(yǎng)基的過(guò)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(guò)一定的處理，洗刷干凈。有的還要進(jìn)行包裝，經(jīng)過(guò)滅菌等準(zhǔn)備就緒后，才能使用。
1、新購(gòu)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(shí)，使游離的堿性物質(zhì)除去，再以流水沖凈。對(duì)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(dòng)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(guò)的玻璃器皿
凡確無(wú)病原菌或未被帶菌物污染的器皿，使用后可隨時(shí)沖洗，吸取過(guò)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(guò)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(shí)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(qiáng)的腐蝕作用，使用時(shí)應(yīng)特別小心，避免濺到衣服、身體和其他物品上。
二、培養(yǎng)基的類型
在實(shí)驗(yàn)室中配制的適合微生物生長(zhǎng)繁殖或累積代謝產(chǎn)物的任何營(yíng)養(yǎng)基質(zhì)，都叫做培養(yǎng)基(Media)。由于各類微生物對(duì)營(yíng)養(yǎng)的要求不同，培養(yǎng)目的和檢測(cè)需要不同，因而培養(yǎng)基的種類很多。我們可根據(jù)某種標(biāo)準(zhǔn)，將種類繁多的培養(yǎng)基劃分為若干類型。
1、根據(jù)對(duì)培養(yǎng)基組成物質(zhì)的化學(xué)成分是否完全了解來(lái)區(qū)分，可以將培養(yǎng)基分為天然培養(yǎng)基、合成培養(yǎng)基和半合成培養(yǎng)基。
１）天然培養(yǎng)基 天然培養(yǎng)基是指利用各種動(dòng)、植物或微生物的原料，其成分難以確切知道。用作這種培養(yǎng)基的主要原料有：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)配成的培養(yǎng)基雖然不能確切知道它的化學(xué)成分，但一般來(lái)講，營(yíng)養(yǎng)是比較豐富的，微生物生長(zhǎng)旺盛，而且來(lái)源廣泛，配制方便，所以較為常用，尤其適合于配制實(shí)驗(yàn)室常用的培養(yǎng)基。這種培養(yǎng)基的穩(wěn)定性常受生產(chǎn)廠或批號(hào)等因素的影響。
２）合成培養(yǎng)基 合成培養(yǎng)基是一類化學(xué)成分和數(shù)量完全知道的培養(yǎng)基，它是用已知化學(xué)成分的化學(xué)藥品配制而成。這類培養(yǎng)基化學(xué)成分精確、重復(fù)性強(qiáng)，但價(jià)格昂貴，而微生物又生長(zhǎng)緩慢，所以它只適用于做一些科學(xué)研究，例如營(yíng)養(yǎng)、代謝的研究。
３）半合成培養(yǎng)基 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。例如馬鈴薯蔗糖培養(yǎng)基等。這種培養(yǎng)基在生產(chǎn)實(shí)踐和實(shí)驗(yàn)室中使用最多。
２、根據(jù)培養(yǎng)基的物理狀態(tài)來(lái)區(qū)分，可以分為固體培養(yǎng)基、液體培養(yǎng)基和半固體培養(yǎng)基。
１）液體培養(yǎng)基 所配制的培養(yǎng)基是液態(tài)的，其中的成分基本上溶于水，沒(méi)有明顯的固形物，液體培養(yǎng)基營(yíng)養(yǎng)成分分布均勻，易于控制微生物的生長(zhǎng)代謝狀態(tài)。
２）固體培養(yǎng)基 在液體培養(yǎng)基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。固體培養(yǎng)基在實(shí)際中用得十分廣泛。在實(shí)驗(yàn)室中，它被用作微生物的分離、鑒定、檢驗(yàn)雜菌、計(jì)數(shù)、保藏、生物測(cè)定等。
３）半固體培養(yǎng)基 如果把少量的凝固劑加入到液體培養(yǎng)基中，就制成了半固體培養(yǎng)基。以瓊脂為例，它的用量在0.2~1%之間。這種培養(yǎng)基有時(shí)可用來(lái)觀察微生物的動(dòng)力，有時(shí)用來(lái)保藏菌種。
３、根據(jù)培養(yǎng)基的用途來(lái)區(qū)分，可分為選擇培養(yǎng)基、增殖培養(yǎng)基、鑒別培養(yǎng)基等。
１）選擇培養(yǎng)基 在培養(yǎng)基中加入某種物質(zhì)以殺死或抑制不需要的菌種生長(zhǎng)的培養(yǎng)基，稱之為選擇培養(yǎng)基。如鏈霉素、氯霉素等抑制原核微生物的生長(zhǎng)；而制霉菌素、灰黃霉素等能抑制真核微生物的生長(zhǎng)；結(jié)晶紫能抑制革蘭氏陽(yáng)性細(xì)菌的生長(zhǎng)等。
２）增殖培養(yǎng)基 在自然界中，不同種的微生物常生活在一起，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(yíng)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，這種培養(yǎng)基稱為增殖培養(yǎng)基，這種培養(yǎng)基常用于菌種篩選和選擇增菌中。在某種程度上講，增殖培養(yǎng)基也是一種選擇培養(yǎng)基。
３）鑒別培養(yǎng)基 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(kāi)快速鑒別某種微生物。這樣的培養(yǎng)基稱之為鑒別培養(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)培養(yǎng)基就是一種常用的鑒別性培養(yǎng)基。
有些培養(yǎng)基是具有選擇和鑒別雙重作用。例如食品檢驗(yàn)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(mén)氏菌和志賀氏菌）。
另外，根據(jù)培養(yǎng)基的營(yíng)養(yǎng)成分是否“完全”，可以分為基本培養(yǎng)基、完全培養(yǎng)基和補(bǔ)充培養(yǎng)基，這類術(shù)語(yǔ)主要是用在微生物遺傳學(xué)中。根據(jù)培養(yǎng)基用于生產(chǎn)的目的來(lái)區(qū)分，可以分為種子培養(yǎng)基和發(fā)酵培養(yǎng)基。還有專門(mén)用于培養(yǎng)病毒等寄生微生物的活組織培養(yǎng)基，如雞胚等；專門(mén)用于培養(yǎng)自養(yǎng)微生物的無(wú)機(jī)鹽培養(yǎng)基等。
三、培養(yǎng)基制備的基本方法和注意事項(xiàng)
１、培養(yǎng)基配方的選定
同一種培養(yǎng)基的配方在不同著作中常會(huì)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(duì)，再依據(jù)自己的使用目的，加以選用，記錄其來(lái)源。
２、培養(yǎng)基的制備記錄
每次制備培養(yǎng)基均應(yīng)有記錄，包括培養(yǎng)基名稱，配方及其來(lái)源，和各種成份的牌號(hào)，最終pH值、消毒的溫度和時(shí)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)制記錄隨制好的培養(yǎng)基一同存放、以防發(fā)生混亂。
３、培養(yǎng)基成分的稱取
培養(yǎng)基的各種成分必須精確稱取并要注意防止錯(cuò)亂，要一次完成，不要中斷?？蓪⑴浞街糜诎鴤?cè)，每稱完一種成分即在配方面軍做出記號(hào)，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、培養(yǎng)基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培養(yǎng)基中，使細(xì)菌不易生長(zhǎng)。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(dòng)蒸汽消毒器中蒸煮溶化。在鍋中溶化時(shí)、可先用溫水加熱并隨時(shí)擾動(dòng)、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(guò)程中，因蒸發(fā)而丟失的水分，必須加以補(bǔ)足。
５、培養(yǎng)基pH的初步調(diào)正
因培養(yǎng)基在加熱消毒過(guò)程中、pH會(huì)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(huì)有顯著的升高。因此，對(duì)這個(gè)步驟，操作者應(yīng)隨時(shí)注意探索經(jīng)驗(yàn)、以期能掌握培養(yǎng)基的最終PH，保證培養(yǎng)基的質(zhì)量。PH調(diào)整后，還應(yīng)將培養(yǎng)基煮沸數(shù)分鐘，以利培養(yǎng)基沉淀物的析出。
６、培養(yǎng)基的過(guò)濾澄清
液體培養(yǎng)基必須澄清，瓊脂培養(yǎng)基也應(yīng)透明無(wú)顯著沉淀，因此，須要采用過(guò)濾或其它澄清方法以達(dá)到此項(xiàng)要求。一般液體培養(yǎng)基可用濾紙過(guò)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。
瓊脂培養(yǎng)基可用清潔的白色薄絨布趁熱過(guò)濾。亦可用中間夾有薄層吸水棉的雙層紗布過(guò)濾。新制肉、肝、血和土豆等浸液時(shí)、則須先用絨布將碎渣濾去，再用濾紙反復(fù)過(guò)濾。如過(guò)濾法不能達(dá)到澄清要求、則須用蛋清澄清法。即將冷卻至55~60°C的培養(yǎng)基放入大的三角燒瓶?jī)?nèi)，裝入量不得超過(guò)燒瓶容量的1/2，每1000ml培養(yǎng)基加入1~2個(gè)雞蛋的蛋白，強(qiáng)力振搖3~5分鐘，置高壓蒸汽滅菌器中、121°C加熱20分鐘、取出趁熱以絨布過(guò)濾即可。
７、培養(yǎng)基的分裝
培養(yǎng)基的分裝，應(yīng)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(guò)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(shí)最好能使用半自動(dòng)或電動(dòng)的定量分裝器。分裝瓊脂斜面培養(yǎng)基時(shí)，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，以利于培養(yǎng)基的徹底滅菌。每批培養(yǎng)基應(yīng)另外分裝20ml培養(yǎng)基于一小玻璃瓶中，隨該批培養(yǎng)基同時(shí)滅菌，以為測(cè)定該批培養(yǎng)基最終pH之用。
８、培養(yǎng)基的滅菌
一般培養(yǎng)基可采用121°C高壓蒸汽滅菌15分鐘的方法。在各種培養(yǎng)基制備方法中，如無(wú)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(guò)濾或間歇滅菌法消毒，以后再用無(wú)菌操作技術(shù)、定量加于培養(yǎng)基。明膠培養(yǎng)基亦應(yīng)用較低溫度滅菌。血液、體液和抗生素等則應(yīng)以無(wú)菌操作技術(shù)抽取和加入于經(jīng)冷卻約50°C左右的培養(yǎng)基中。
瓊脂斜面培養(yǎng)基應(yīng)在滅菌后立即取出，冷至55℃-60℃時(shí)，擺置成適當(dāng)斜面，待其自然凝固。
９、培養(yǎng)基的質(zhì)量測(cè)試
每批培養(yǎng)基制備好以后，應(yīng)仔細(xì)檢查一遍，如發(fā)現(xiàn)破裂、水分浸入、色澤異常、棉塞被培養(yǎng)基沾染等、均應(yīng)挑出棄去。并測(cè)定其最終pH。
將全部培養(yǎng)基放入36±1°C恒溫箱培養(yǎng)過(guò)夜，如發(fā)現(xiàn)有菌生長(zhǎng)，即棄去。
用有關(guān)的標(biāo)準(zhǔn)菌株接種1~2管或瓶培養(yǎng)基，培養(yǎng)24~48小時(shí)，如無(wú)菌生長(zhǎng)或生長(zhǎng)不好。應(yīng)追查原因并重復(fù)接種一次，如結(jié)果仍同前，則該批培養(yǎng)基即應(yīng)棄去，不能使用。
10、培養(yǎng)基的保存
培養(yǎng)基應(yīng)存放于冷暗處，最好能放于普通冰箱內(nèi)。放置時(shí)間不宜超過(guò)一周，傾注的平板培養(yǎng)基不宜超過(guò)3天。每批培養(yǎng)基均必須附有該批培養(yǎng)基制備記錄副頁(yè)或明顯標(biāo)簽。
Technical instructions for preparation of culture medium for microbiology laboratory
1. Cleaning of glassware
In the process of preparing the medium, we should first use some glassware, such as test tube, triangle bottle, Petri dish, beaker and straw. 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 bought glassware
After removing the contaminated dirt from packaging, brushing with hot soapy water, rinsing water, and soaking in 1 - 2% industrial hydrochloric acid for several hours, remove the free alkaline substance and rinse with water. For the containers with large capacity, such as the large flask, the measuring cylinder, and so on, a little hydrochloric acid is injected after washing, and the inner surface of the vessel is turned into hydrochloric acid. After a few minutes, the hydrochloric acid is tilting to the hydrochloric acid, then the water is washed with water, and the water is dry on the washing rack and can be used.
2. Used glassware
The utensils, which have no pathogenic bacteria or not contaminated by bacteria, can be rinsed at any time after use and absorb the straw of chemical reagents. They can be soaked in clean water first and then be cleaned after a certain amount. Containers that are likely to be contaminated by pathogens must be properly sterilized to remove dirt, wash them with soap solution, and rinse them with 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 lotion are potassium dichromate and concentrated acid, which are used to oxidize organic matter to soluble substances for washing. The lotion has a strong corrosive effect and should be especially careful when used. Avoid splashing onto clothes, body and other articles.
Two. Type of culture medium
In the laboratory, any nutrient matrix suitable for microbial growth, reproduction or accumulation of metabolites is called Media. Because of the different nutritional requirements of all kinds of microorganism, there are many different kinds of culture media for the purpose of cultivation and detection. According to certain criteria, we can divide a large number of culture media into several types.
1. The culture medium can be divided into natural medium, synthetic medium and semi synthetic medium based on the complete understanding of the chemical composition of the medium.
1) natural medium is a natural medium that uses all kinds of raw materials of animals, plants or microorganisms. The main ingredients used for this medium are beef paste, malt juice, peptone, yeast extract, corn flour, bran, various cake powder, potato, milk, and serum. Although the medium made with these substances can not exactly know its chemical composition, in general, the nutrition is rich, the growth of microbes is strong, and the source is wide, the preparation is convenient, so it is more commonly used, especially suitable for the preparation of laboratory medium. The stability of this medium is often influenced by factors such as production plants or batch numbers.
2) synthetic medium is a kind of medium with a complete knowledge of chemical composition and quantity, which is made up of chemical drugs known as chemical components. This kind of medium is accurate and reproducible, but it is expensive, and the microorganism is slow to grow. So it is only suitable for some scientific research, such as nutrition and metabolism.
3) semisynthetic medium is added to a synthetic medium by adding some or several natural ingredients; or in a natural medium, a chemical known as a known component is added to a semisynthetic medium. For example, potato sucrose medium. This medium is the most widely used in production practice and laboratory.
2. According to the physical state of the medium, it can be divided into solid medium, liquid medium and semisolid medium.
1) the medium prepared by the liquid medium is liquid, and the components of the medium are basically dissolved in water, there is no clear solid, the nutrient composition of the liquid medium is evenly distributed, and it is easy to control the growth and metabolism of microorganism.
2) solid medium is added to the liquid medium to form a solid medium with a suitable amount of coagulant. Agar, gelatin and silica gel are commonly used as coagulants. Agar is the most commonly used material. Solid medium is widely used in practice. In the laboratory, it is used for isolation, identification, examination of bacteria, counting, preservation and biometrics.
3) if semisolid medium is added to a liquid medium with a small amount of coagulant, a semisolid medium will be made. Take agar as an example, and its dosage is between 0.2~1%. This medium can sometimes be used to observe the power of microorganisms and sometimes to preserve bacteria.
3. According to the use of culture medium, it can be divided into selective medium, proliferating medium and differential culture medium.
1) choose a medium to add or kill certain substances in the medium to kill or inhibit the growth of an unnecessary strain. Such as streptomycin, chloramphenicol and other inhibition of the growth of prokaryotic microorganisms; and nystatin and chyx can inhibit the growth of eukaryotic microbes; crystal violet can inhibit the growth of Gram-positive bacteria.
2) the proliferating medium often lives together in the natural world. In order to separate the microbes we need, we add some specific microbes to the common culture medium in order to increase the rate of reproduction and gradually eliminate the other microbes. This medium is called increasing. The culture medium is often used in screening and selecting bacteria. To some extent, the proliferation medium is also a selective culture.
3) a variety of reagents or chemicals are added to the medium in the culture medium to make the indistinguishable microorganisms show obvious differences after culture, thus helping to quickly identify a certain microorganism. Such medium is called the differential medium. For example, the eosin methylene blue medium used to check whether water and dairy products contain enteric pathogens is a commonly used differential culture medium.
Some media have 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 have the effect of inhibiting bacteria other than intestinal bacteria (selectivity), lactose and neutral red (indicator) can help distinguish between lactose ferment enterobacteriella (e. E. coli) and intestinal bacteria that can not ferment lactose (such as Salmonella and Shigella).
In addition, according to whether the nutrient components of the medium are "complete", they can be divided into basic medium, complete medium and supplemental medium. These terms are mainly used in microbial genetics. According to the purpose of culture medium, it can be divided into seed medium and fermentation medium. There are also living tissue culture media, such as chicken embryos, specially used to cultivate parasitic microorganisms, such as viruses.
Three. The basic methods and matters needing attention in the preparation of medium.
1. Selection of culture medium formula
There is always some difference in the formula of the same medium in different works. Therefore, in addition to the standard method, it should be made up strictly according to its provisions. Generally, we should collect relevant information as far as possible, check it, and then select and record its source according to the purpose of his own use.
2. Record of culture medium
Each preparation culture medium should have a record, including the name of the medium, the formula and its source, the number of various ingredients, the final pH value, the date and the preparation of the temperature and time of disinfection, the records should be copied, the original records are preserved, and the copy records are stored together with the prepared medium in order to prevent the confusion.
3. The name of the medium composition of the culture medium
The ingredients of the medium must be accurately weighed and attention should be paid to preventing confusion. The formula can be placed beside the side, each of which is called a mark with the square face, and the required medicine is collected at the left, each of which is removed to the right. After the completion of the test, a check should also be made.
4. Mixing and melting of the components of the medium
The chemicals used in the culture medium should be chemically pure. The cooking pot used shall not be copper pots or iron pots, so that no trace copper or iron can be mixed into the medium so that the bacteria are not easy to grow. It is best to use stainless steel pots to be heated and melted, and can be dissolved in a large beaker or large flask in a high pressure steam sterilizer or a mobile steam sterilizer. When melted in a pot, it can be heated with warm water and disturbed at any time to prevent coking. If coke is found, the medium can not be used, and it should be re prepared. 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 correction of culture medium pH
Because the medium will change during the process of heating and disinfection, pH will be changed after the ingredients are completely dissolved. PH should be adjusted preliminarily. For example, beef extract can reduce pH0.2 and intestinal extract pH will increase significantly. Therefore, for this step, the operator should pay attention to exploring experience at any time so as to master the final PH of the medium and ensure the quality of the medium. After PH is adjusted, the medium should be boiled for several minutes to facilitate the precipitation of the media.
6. Filtration and clarification of culture medium
The liquid medium must be absolutely clarified, and the agar medium should also be transparent and no significant precipitation. Therefore, filtering or other clarification methods should be used to achieve this requirement. The general liquid medium can be filtered by filter paper. The filter paper should be folded into a folding fan or funnel shape to avoid the rupture of filter paper caused by uneven hydraulic pressure.
Agar medium 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 making fresh meat, liver, blood and potatoes, it is necessary to filter the residue with flannelette and filter it repeatedly. If the filtration method fails to achieve the clarification requirement, the egg white clarification method must be used. The culture medium that is about to be cooled to 55~60 C is put into a large triangular flask, the loading amount should not exceed the 1/2 of the capacity of the flask, the protein of 1~2 eggs is added to each 1000ml medium, the strength is shaking for 3~5 minutes, and the high pressure steam sterilizer, 121 degree C is heated for 20 minutes, and the heat is removed with the flannelette.
7. The separation of culture medium
The media should be packed in test tubes, flasks and other appropriate containers according to the purpose and requirements of the use. The amount of separation shall not exceed 2/3 of the container's full capacity. The mouth of the container can be blocked by a cotton stopper with wet proof paper, and it must be wrapped with waterproof paper. It is better to use semi-automatic or electric quantified distributor when loading. When the agar slant medium 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 cleaned in advance and dry baked and sterilized, so as to facilitate the complete sterilization of the medium. Each batch of medium should be separately packed with 20ml and cultured in a small glass bottle, and then sterilized with the batch culture medium to determine the final pH of the medium.
8, sterilization of culture medium
The general medium can be sterilized by 121 degree C high pressure steam for 15 minutes. In all kinds of medium preparation methods, without special regulations, this method can be sterilized.
Some of the hot ingredients, such as carbohydrates, should be separated into 20% or more high concentration liquid, which are sterilized by filtration or intermittent sterilization, and then used in aseptic technique and added to the culture medium. The gelatin medium was also used for low temperature sterilization. Blood, body fluids and antibiotics should be extracted and added to the medium with a cooling of about 50 degrees C by aseptic technique.
The agar slant media should be removed immediately after sterilization. When the temperature reaches 55 -60 C, the agar should be placed in a suitable inclined plane until it solidifies naturally.
9, quality test of culture medium
When each batch of medium is prepared, it should be examined carefully, such as breakage, water immersion, color and lustre abnormality, and cotton plugs being stained with culture foundation. And the final pH was determined.
All the medium was incubated in a 36 + 1 degree C incubator overnight.
Inoculate 1~2 tube or bottle medium with standard strains, and cultivate 24~48 hours, such as aseptic growth or poor growth. The cause should be traced and vaccinated repeatedly. If the result is still the same, the batch should be abandoned and not used.
10. Preservation of culture medium
Medium should be stored in cold and dark places, preferably in ordinary refrigerators. The placement time should not exceed one week, and the plate medium should not exceed 3 days. Each batch of culture medium must be attached to the batch medium to prepare a record page or a marked label.