培養(yǎng)基的配置、滅菌及無(wú)菌檢測(cè)技術(shù)
發(fā)布時(shí)間：
2022-12-27
作者：
做培養(yǎng)基好的廠家
一、實(shí)驗(yàn)?zāi)康?/span>
1.了解培養(yǎng)基的配置原理
2.掌握其制備過(guò)程和方法
二、實(shí)驗(yàn)原理
培養(yǎng)基是人們按照不同微生物生長(zhǎng)繁殖或積累代謝產(chǎn)物的需要而配置的營(yíng)養(yǎng)基質(zhì)。用于微生物的分離純化培養(yǎng)鑒定和保存菌種。培養(yǎng)基的種類(lèi)有很多，按對(duì)培養(yǎng)基的組成物質(zhì)的化學(xué)成分是否完全了解可以分為天然培養(yǎng)基和合成培養(yǎng)基。按照不同微生物對(duì)營(yíng)養(yǎng)的要求，選擇可被迅速利用的碳源、氮源、無(wú)機(jī)鹽及其他營(yíng)養(yǎng)成分可制成不同的培養(yǎng)基。
三、實(shí)驗(yàn)器材
1.牛肉膏、蛋白胨、可溶性淀粉、NaCI、K2HPO4、KNO3、MgSO4FeSO4馬鈴、蔗糖、瓊脂、10%NaOH和10%HCI。
2.刻度搪瓷杯、天平、牛角匙、小燒杯、稱量紙、分裝漏斗、試管、棉花、鐵絲筐、電爐、玻棒、紗布。
四、實(shí)驗(yàn)操作
1.稱量：按照培養(yǎng)基配方，準(zhǔn)確稱取各種成分。
（1）牛肉膏蛋白胨瓊脂培養(yǎng)基
牛肉膏 3克 蛋白胨 5克
NaCl 5克 瓊脂15-20克
水 1000毫升
PH調(diào)至7.2-7.4 0.1MPa壓力下滅菌30分鐘
（2）馬鈴薯蔗糖瓊脂培養(yǎng)基（PDA）
馬鈴薯 200克 蔗糖(白糖) 20克
瓊脂 15-20克 水1000毫升
去皮洗凈的馬鈴薯按量稱取，切成小塊，放在水中煮沸，并維持20一30分鐘，用雙層紗布過(guò)濾得馬鈴薯汁，補(bǔ)足到定量的水。自然pH值0.1MPa壓力下滅菌30分鐘。
（3）高氏一號(hào)培養(yǎng)基
可溶性淀粉 20克 KNO3 1克 K2HPO4'3H2O 0.5克 NaCl 0.5克 MgSO4'7H2O 0.5克 FeSO4'7H2O
pH調(diào)至7.2~~7.4先用少量的水將淀粉調(diào)成糊狀后再加水和其他的鹽類(lèi)。0.1MPa壓力下滅菌30分鐘
2.溶化：
依配方順序，用少量的水溶解各成分，有時(shí)需加熱溶化，最后補(bǔ)足所需水量。在暴沸時(shí)添加少量的水抑制沸騰，融化過(guò)程需不斷攪拌。
3.調(diào)整pH：
用PH試紙或酸酸度計(jì)先試測(cè)初制備培養(yǎng)基的的PH值，然后用10%NaOH或10%HCⅠ調(diào)整至所需的pH范圍。
4.過(guò)濾：
在配制固體培養(yǎng)基時(shí)，事先將瓊脂稱好用冷水浸泡，洗凈擠干后加人液體培養(yǎng)基中，瓊脂融化的過(guò)程中，需不斷攪拌，并控制火力，不要使培養(yǎng)基溢出或燒焦，待完全融化后，補(bǔ)足水分。
5.分裝：根據(jù)不同的需要，可將配制的培養(yǎng)基分裝在試管或三角瓶?jī)?nèi)，注意不要將培養(yǎng)基沾染在管口和瓶口上，以免粘住棉塞，引起污染。
6.裝棉塞或硅晈塞，棉塞粗細(xì)、長(zhǎng)短要求合適，外面再包一層報(bào)紙避免造成污染。
7.滅菌：
培養(yǎng)基的滅菌時(shí)間和溫度，需按各種規(guī)定進(jìn)行，保證滅菌數(shù)果和不損失培養(yǎng)基的必要成分。滅菌后的培養(yǎng)基要趁熱擺成斜面，半固體要垂直放置，待冷卻即可成為斜面培養(yǎng)基和半固體深層瓊脂培養(yǎng)基，而且必須放在37℃溫箱培養(yǎng)24小時(shí)，無(wú)菌生長(zhǎng)方可使用。
五、注意事項(xiàng)
1.牛肉膏的稱取：
用玻璃棒蘸取適量牛肉膏，抹于稱量紙上，勿取過(guò)量，不足時(shí)，再蘸取少量，逐步添加；然后將牛肉膏連同稱量紙放入水中，稍等片刻牛肉膏即從稱量紙上脫落，用玻璃棒將紙撈出即可。
2.加瓊脂：
瓊脂一般應(yīng)在調(diào)好了pH后加入，瓊脂的加入不會(huì)影響pH，加入瓊脂后要控制火力并不斷攪拌，以免沸騰溢出或瓊脂糊底燒焦。加熱過(guò)程中因水分蒸發(fā)而可能使體積減小，應(yīng)補(bǔ)充水分至所需量。
3.滅菌前一定要用報(bào)紙包扎好。
日水培養(yǎng)基qdrishui.cn
First, the purpose of the experiment
1. understand the configuration principle of culture medium
2. master the process and method of its preparation
Two. The principle of the experiment
The culture medium is a nutrient matrix which is distributed according to the needs of different microorganisms to grow, reproduce or accumulate metabolites. It is used for isolation, purification, cultivation, identification and preservation of microorganisms. There are many kinds of culture medium, which can be divided into natural medium and synthetic medium according to the complete understanding of the chemical composition of the components of the medium. According to the requirements of different microbes for nutrition, the carbon sources, nitrogen sources, inorganic salts and other nutrients that can be used quickly can be made into different media.
Three. Experimental equipment
1. beef paste, peptone, soluble starch, NaCI, K2HPO4, KNO3, MgSO4FeSO4 horse bell, sucrose, agar, 10%NaOH and 10%HCI.
2. scale enamel cup, balance, ox horn horn, small beaker, weighing paper, loading hopper, test tube, cotton, wire basket, electric furnace, glass rod, gauze.
Four. Experimental operation
1. weighing: according to the media formula, accurately weigh various ingredients.
(1) beef paste Peptone Agar Medium
Beef Cream 3 grams of peptone 5 grams
NaCl 5 gram agar 15-20 grams
1000 milliliters of water
PH to 7.2-7.4 0.1MPa pressure to sterilize for 30 minutes
(2) the sucrose agar medium (PDA) of potato
200 grams of sucrose (white sugar) 20 grams of potato
Agar 15-20 grams of water 1000 ml
The peeled and washed potatoes are weighed in quantity, cut into small pieces and boiled in water, and are maintained for 20 and 30 minutes. The double double gauze is used to filter potato juice and fill up to the quantity of water. The natural pH value is 0.1MPa under pressure for 30 minutes.
(3) the No.1 culture medium of Kao's
Soluble starch 20 g KNO3 1 g K2HPO4'3H2O 0.5 g NaCl 0.5 g MgSO4'7H2O 0.5 g FeSO4'7H2O
When pH is transferred to 7.2~~7.4, the starch is pasted into paste with a small amount of water before adding water and other salts. 0.1MPa sterilization for 30 minutes under pressure
2. dissolve:
According to the formula, dissolve each component with a small amount of water, sometimes it needs heating and melting, and finally make up the required water. When boiling, add a small amount of water to inhibit boiling. The process of melting must be constantly stirred.
3. adjust pH:
The pH value of the medium was initially measured by PH test paper or acidity meter, then adjusted to the required pH range with 10%NaOH or 10%HC I.
4. filter:
In the preparation of solid medium, the agar is soaked in cold water in advance, and after washing and squeezing the liquid culture medium. During the melting process of agar, the agar must be constantly stirred and control fire, and the medium will not be spilled or burnt, and the moisture is filled after the melt is completely melted.
5. Division: according to the different needs, the preparation of the culture medium can be installed in the test tube or the bottle, not to stain the culture medium on the mouth and bottle mouth, so as to avoid adhesion of cotton plug, causing pollution.
6. packing cotton stopper or silicon stopper, cotton stopper thickness and length requirements are suitable, and another layer of newspaper should be wrapped outside to avoid pollution.
7. sterilization:
The sterilization time and temperature of the medium must be regulated according to various media, ensuring that the number of fruits is absorbed and the essential ingredients of the medium are not lost. After sterilizing, the culture medium should be inclined to the slope, and the semisolid should be placed vertically. The culture medium of the inclined surface and the semi solid deep agar medium should be made by cooling, and it must be placed at the temperature box of 37 C for 24 hours, and the sterile growth can be used.
Five. Precautions
1. the name of the beef extract:
Dip a proper amount of beef paste on a glass stick and put it on the weighing paper. Do not overdose it. When it is insufficient, dip it in a small amount and gradually add it. Then, add beef paste and weighing paper into the water.
2. plus agar:
Agar should be added after pH is well adjusted. Agar addition does not affect the pH of the medium. After adding agar, it is necessary to control fire and stir constantly, so as to avoid boiling overflow or agar paste bottom burning. During the heating process, the volume of the medium may be reduced due to the evaporation of water, and the moisture should be supplied to the required amount.
3. must be bandaged with newspapers before sterilization.