微生物培養(yǎng)基的制備過(guò)程是怎么的？
發(fā)布時(shí)間：
2022-12-24
作者：
國(guó)內(nèi)比較好的微生物培養(yǎng)基生產(chǎn)公司
不同類(lèi)型培養(yǎng)基制備的程序也不盡相同，一般制備過(guò)程為：配料，溶化，矯正pH，澄清過(guò)濾，分裝，滅菌及檢定等8個(gè)步驟。
1，配料：按培養(yǎng)基處方準(zhǔn)確稱(chēng)取各種成分，先在三角燒瓶中加入少量蒸餾水，再加入各種成分，以防蛋白胨等粘附于瓶底，然后再以剩余的水沖洗瓶壁。
2，溶化：將各種成分混勻于水中，最好以流通蒸氣溶化半小時(shí)，如在電爐上溶化應(yīng)隨時(shí)攪拌，如有瓊脂成分時(shí)更應(yīng)注意防止外溢。溶化完畢，補(bǔ)足失去的水分。
3，矯正pH：1.pH測(cè)定：取與標(biāo)準(zhǔn)管同口徑的試管[通常用華氏試管]3支，于第1，3管各加入欲測(cè)定pH的的培養(yǎng)基5ml，并于第一管中加入0.2g／L的酚紅0.25ml作為測(cè)定管，混勻于第2管加入蒸餾水5ml，第4管為pH標(biāo)準(zhǔn)比色管。
pH的校正：若測(cè)定管過(guò)酸或過(guò)堿可用0.1mol／L氫氧化鈉或0.1mol／L鹽酸溶液矯正，直至顏色與標(biāo)準(zhǔn)管相同為止，加堿或加酸時(shí)要精確緩慢，每加1滴后要充分混勻，比色后再加第2滴[有時(shí)僅加半滴]準(zhǔn)確記錄加入的量。
計(jì)算：設(shè)5ml培養(yǎng)基矯正pH至7.4時(shí)需0.1mol／L氫氧化鈉0.15ml，現(xiàn)有培養(yǎng)基4990ml，需加氫氧化鈉的量可按下列方法計(jì)算：5∶4990＝0.15∶X
X＝0.15×4990/5＝149.7[ml]如將此0.1mol／L的氫氧化鈉改用1mol／L的氫氧化鈉時(shí)，則需14.9ml即可。
4，過(guò)濾澄清：培養(yǎng)基配制后一般都有沉渣或混濁出現(xiàn)，需過(guò)濾成清晰透明后方可使用，常用的過(guò)濾方法如下：
液體培養(yǎng)基必須清晰，以便觀察細(xì)菌的生長(zhǎng)情況，常用濾紙過(guò)濾，亦可在加熱前加入用水稀釋的雞蛋白[1000ml培養(yǎng)基用1個(gè)雞蛋白]在100℃加熱后保持60～70℃40～60分鐘，使其不溶性物質(zhì)附于凝固的蛋白上而沉淀，然后再用虹吸法吸出上清液或以濾紙過(guò)濾。
固體培養(yǎng)基：于加熱融化后需趁熱以絨布或兩層紗布中夾脫脂棉過(guò)濾；亦可用自然沉淀法，即將瓊脂培養(yǎng)基盛人鋁鍋或廣口搪瓷容器內(nèi)，以高壓[103.43kPa]蒸汽融化15分鐘后，靜置高壓鍋內(nèi)過(guò)夜，次日將瓊脂傾出，用刀將底部沉渣切去，再融化即可收清晰的瓊脂培養(yǎng)基。
5，分裝：根據(jù)需要將培養(yǎng)基分裝于不同容量的三角燒瓶，試管中。分裝的量不宜超過(guò)容器的2／3以免滅菌時(shí)外溢。瓊脂斜面分裝量為試管容量的1／5，滅菌后須趁熱放置成斜面，斜面長(zhǎng)約為試管長(zhǎng)的2／3。半固體培養(yǎng)基分裝量約為試管長(zhǎng)的1／3，滅菌后直立凝固待用。高層瓊脂分裝量約為試管的1／3，滅菌后趁熱直立，待冷后凝固待用。液體培養(yǎng)基分裝于試管中，約是試管長(zhǎng)度的1／3。瓊脂平板：將滅菌[或加熱融化]后的培養(yǎng)基冷至50℃左右，以無(wú)菌手續(xù)傾人滅菌平皿內(nèi)，內(nèi)徑9cm的平皿傾注培養(yǎng)基約13～15ml，輕搖平皿底平鋪于平皿底部，待凝固后即成，傾注培養(yǎng)基時(shí)，切勿將皿蓋全部啟開(kāi)，以免空氣中塵埃及細(xì)菌落入。
新制成的平板培養(yǎng)基[簡(jiǎn)稱(chēng)平板]，表面水分較多，不利于細(xì)菌的分離，通常應(yīng)將平皿倒扣擱置于37℃培養(yǎng)箱內(nèi)約30分鐘待平板平面干燥后使用。
6，滅菌：不同成分，性質(zhì)的培養(yǎng)基，可采用不同的滅菌方法。高壓蒸汽滅菌法：高壓滅菌的溫度與時(shí)間隨種類(lèi)及數(shù)量的不同有所差別，
一般培養(yǎng)基少量分裝時(shí)高壓[103.43kPa]滅菌15分鐘即可，分裝量較大時(shí)，可高壓[103.43kPa]滅菌30分鐘，含糖的培養(yǎng)基高壓[55.16kPa]滅菌15分鐘。以免糖類(lèi)被破壞。
7，檢定：每批培養(yǎng)基制成后須經(jīng)檢定方可使用，檢定時(shí)將培養(yǎng)基放37℃溫箱內(nèi)培養(yǎng)24小時(shí)后，證明無(wú)菌，同時(shí)用已知菌種檢查在此培養(yǎng)基上生長(zhǎng)繁殖及生化反應(yīng)情況，符合要求者方可使用。
8，保存：制好的培養(yǎng)基產(chǎn)品，不宜保存過(guò)久，以少量勤做為宜。每批應(yīng)注明名稱(chēng)，分裝量，制作日期等，放在4℃冰箱內(nèi)備用。
What is the process of the preparation of the microbial medium?
There are different procedures for the preparation of medium. However, the procedures of general medium can be divided into 8 steps: batching, melting, correcting pH, clarifying filtration, packing, sterilizing and verifying.
1, ingredients: a variety of ingredients are accurately called according to the prescription of the medium. First, a small amount of distilled water is added to the triangular flask, and then a variety of ingredients are added to prevent peptone from sticking to the bottom of the bottle and then rinse the bottle wall with the remaining water.
2, dissolve: mix all ingredients in water, it is better to melt steam for half an hour, such as melting in the electric furnace should be stirred at any time, if there is agar composition, more attention should be paid to prevent spillover. After dissolving, the loss of water is made up.
3, the correction pH:1.pH determination: take the test tube with the standard tube with the same caliber [usually with the Fahrenheit test tube]3 branch, first, 3 tubes each add the pH culture medium 5ml, and add the 0.2g / L phenol red 0.25ml as the determination tube in the first tube, mix in the second tube into the distilled water 5ml, the fourth tube is the pH standard colorimetric tube.
PH correction: if the determination of tube peric acid or overalkali can be corrected with 0.1mol / L sodium hydroxide or 0.1mol / L hydrochloric acid solution until the same color is the same as the standard tube, it should be precise and slow when adding alkali or acid, and should be fully mixed after 1 drops, and then add second drops [sometimes only a half drop] after color comparison.
Calculation: when the 5ml medium is set up to correct pH to 7.4, it needs 0.1mol / L sodium hydroxide 0.15ml and the existing medium 4990ml, and the amount of sodium hydroxide can be calculated according to the following methods: 5: 4990 = 0.15: X
X = 0.15 x 4990/5 = 149.7[ml]. If the sodium hydroxide of 0.1mol / L is converted to sodium hydroxide of 1mol / L, 14.9ml is needed.
4, filtration and clarification: after the media are prepared, sediment or turbidity usually appears and needs to be filtered for clarity and transparency.
Liquid medium: liquid culture medium must be clear in order to observe the growth of bacteria, filter paper often, and add water diluted chicken protein [1000ml medium before heating with 1 chicken proteins at 100 C to keep 60~70 centigrade for 40~60 minutes to precipitate the insoluble matter on the coagulated protein. Then siphon out the supernatant or filter paper.
Solid medium: if the agar medium is used as an agar medium, the heat is melted before the heat is melted in the flannelette or two layers of gauze. It is also available by natural precipitation. The agar medium will be filled with aluminum pot or wide open enamel container. After melting with high pressure [103.43kPa] steam for 15 minutes, the high pressure pot will stay in the high pressure pot for the night, and the agar is tilted out the next day. Cut the bottom sediment with a knife, and then melt to clear the agar medium.
5, sub packing: according to the need, the medium is divided into different capacity triangular flasks and test tubes. The amount of packing should not exceed 2 / 3 of the container to avoid spillover during sterilization. The amount of agar slant is 1 / 5 of the test tube capacity. After sterilization, it must be placed on the slope of heat and the length of the slope is about 2 / 3 of the length of the tube. The semisolid medium was packed at about 1 / 3 of the length of the tube, and was used for upright coagulation after sterilization. The level of agar in high level is about 1 / 3 of the test tube. After sterilization, it will be hot and upright. The liquid medium was packed in a test tube, about 1 / 3 of the length of the tube. Agar plate: the culture medium after sterilization [or heating melting] is cold to about 50 degrees C, in the sterilized flat plate with aseptic formalities, the plate of inner diameter 9cm is poured about 13 ~ 15ml, and the medium is rolled at the bottom of the plate, and then the plate will not be completely opened to avoid air when it is pour into the medium. Egyptian bacteria fall into the middle dust.
The newly made plate culture medium [abbreviated as flat plate] has more surface moisture and is not conducive to the separation of bacteria. Usually, it should be put on the flat plate in the incubator of 37 C for about 30 minutes after the flat plane is dried.
6, sterilization: different ingredients and properties of the medium can be used with different sterilization methods. High pressure steam sterilization: the temperature and time of autoclaving vary with the type and quantity of medium.
The high pressure [103.43kPa] can be sterilized for 15 minutes when a small amount of culture medium is divided. When the medium is large, the high pressure [103.43kPa] can be sterilized for 30 minutes, and the sugar containing medium of high pressure [55.16kPa] is sterilized for 15 minutes. Lest the sugar is destroyed.
7, verification: each batch of culture medium must be used by the calibrated side. After the test, the culture base is placed in the incubator of 37 C for 24 hours, and the asepsis is proved. At the same time, the growth propagation and biochemical reaction on this medium are checked with the known strains, which can be used in accordance with the requirements.
8, preservation: the medium should not be preserved for too long, and it should be done with a small amount of diligence. Each batch should specify the name, the amount of packing, the date of production, etc., and reserve it in the refrigerator at 4 degrees.