制備微生物檢測(cè)培養(yǎng)基的細(xì)節(jié)問(wèn)題
發(fā)布時(shí)間：
2022-11-16
作者：
成品培養(yǎng)基廠家
用精確度1/100的電子天平稱取培養(yǎng)基所需藥品。先根據(jù)配方計(jì)算出配制一定量培養(yǎng)基所需各種成分藥品的量，然后用天平準(zhǔn)確稱取。稱量時(shí)用稱量紙折疊成簸箕狀盛放藥品。稱量紙折疊方法如圖所示。
二、溶化
培養(yǎng)基放入燒杯或搪瓷缸中，慢慢加入少量所需水，邊加入邊用玻棒攪拌。如果培養(yǎng)基中不含有瓊脂，培養(yǎng)基不需要加熱；如果含有瓊脂，則需要用本生燈或電磁爐加熱煮沸，完全溶解后，再補(bǔ)齊所需水，并攪拌均勻。如果配制的培養(yǎng)基量很大，可用不銹鋼鍋加熱溶化，可先用溫水加熱并隨時(shí)攪動(dòng)，防止焦化，如有焦化現(xiàn)象，配制的培養(yǎng)基就不能使用，要重新配制。
配制培養(yǎng)基時(shí)不可用銅或鐵的容器溶化，因銅或鐵制容器可能會(huì)使培養(yǎng)基中的銅和鐵的含量超標(biāo)，影響實(shí)驗(yàn)(培養(yǎng)基中銅含量大于0.3mg/L，細(xì)菌不宜生長(zhǎng)，鐵含量超過(guò)0.14mg/L，妨礙細(xì)菌產(chǎn)毒素)。對(duì)容易發(fā)生反應(yīng)，產(chǎn)生沉淀的藥品，要分開(kāi)溶解，最后加入培養(yǎng)基，如磷酸氫二鉀和硫酸鎂。
三、調(diào)pH
雖然培養(yǎng)基中含有緩沖物質(zhì)成分，能使培養(yǎng)基的pH盡可能的保持在要求的范圍內(nèi)，但是配出的培養(yǎng)基若不符合要求，就要進(jìn)行必要的調(diào)整。如果有已校準(zhǔn)pH計(jì)，可用pH計(jì)，如果沒(méi)有，可用精密的pH試紙，再根據(jù)需要用1 mol/L氫氧化鈉或1mol/L鹽酸(微調(diào)可用0.1氫氧化鈉或0.1mol/L鹽酸)調(diào)制所需的pH。培基的pH一般為7.4～7.6，也有酸性或堿性的。用氫氧化鈉調(diào)整的需要高壓滅菌的培養(yǎng)基，在調(diào)整pH時(shí)要調(diào)至高出所需0.1個(gè)～0.2個(gè)單位，因用氫氧化鈉調(diào)整時(shí)，高壓滅菌后，培養(yǎng)基的pH要降低0.1~0.2。如培養(yǎng)基中含有碳酸鈣成分，可不pH。
四、過(guò)濾
配成的培養(yǎng)基，若無(wú)特殊要求，可省略此步。若有沉淀或渾濁.需要澄清的.液體培養(yǎng)基可用油紙過(guò)濾。而固體培養(yǎng)基可用中間夾一薄層脫脂棉的雙層紗布過(guò)濾。如果過(guò)濾法還不能達(dá)到澄清的要求，則可用蛋清澄清法，即培養(yǎng)基加熱冷卻到50~60℃，裝入三角瓶?jī)?nèi)(不超過(guò)容量的二分之一)，按1000mL加人1個(gè)～2個(gè)雞蛋的蛋清，用力搖晃3-5min，高壓蒸汽滅菌121℃、20min，取出趁熱過(guò)濾即可。
五、分裝
配制好的培養(yǎng)基根據(jù)不同的用途分裝在三角瓶、試管等容器中，分裝試管，如果試管量很大，可用自動(dòng)分液器，如果試管量少，可用漏斗分裝。分裝量不超過(guò)容器容積的三分之二。三角瓶以不超過(guò)容積的二分之一為宜；瓊脂斜面以不超過(guò)試管長(zhǎng)度的五分之一為宜，滅菌后，擺成的斜面為培養(yǎng)基量的三分之一，底層為三分之二的量為宜；半固體瓊脂分裝量為試管長(zhǎng)度的三分之一；用來(lái)接種或保菌的高層瓊脂，分裝量為試管長(zhǎng)度的四分之一或三分之一，用來(lái)接種厭氧菌的要達(dá)到三分之二；瓊脂平板，內(nèi)徑為90mm的以13mL~15 mL為宜，內(nèi)徑70mm的平板為8mL~10 mL，制成的瓊脂平板若表面水分較多，可將平板倒扣，放置在37℃培養(yǎng)箱內(nèi)30min，干燥后再用。每批培養(yǎng)基應(yīng)另外分裝一小玻璃瓶(約20mL)與該批培養(yǎng)基同時(shí)滅菌，為測(cè)定該批培養(yǎng)基最終pH。
六、滅菌
分裝好的培養(yǎng)基應(yīng)立即進(jìn)行滅菌。滅菌的方法種類如下：
1. 高壓蒸汽滅菌法
大多耐熱培養(yǎng)基都可用此法，小量分裝時(shí)，用121℃，15min；滅菌量大時(shí)，用121℃，30min滅菌；含糖類的培養(yǎng)基只能113~115℃，15min滅菌，避免糖類遭破壞。
2.煮沸滅菌法
對(duì)含有不耐高溫物質(zhì)的培養(yǎng)基，可使用此方法。
3.過(guò)濾除菌
以過(guò)濾的方法除菌，用無(wú)菌操作技術(shù)，定量加入培養(yǎng)基。血液、抗生素可用無(wú)菌操作技術(shù)抽取，加入冷卻到50℃左右的培養(yǎng)基中。培養(yǎng)基含有不耐熱的物質(zhì)時(shí)，可用此法。
婚慶 心 動(dòng)態(tài)分割線
對(duì)LST培養(yǎng)基進(jìn)行滅菌時(shí)，有時(shí)發(fā)酵管內(nèi)會(huì)有氣泡。為防止發(fā)酵管內(nèi)產(chǎn)生氣泡，可以采取以下幾項(xiàng)措施：
1. 小倒管浸滿培養(yǎng)基(不留氣泡)后再加入到盛LST的試管中。
2.滅菌鍋關(guān)閉放氣閥前，將鍋內(nèi)氣體排干凈。
3.試管塞不要塞得太緊(使用硅膠塞的時(shí)候)，勿使用橡皮塞。
4不要過(guò)早打開(kāi)滅菌鍋，要等滅菌鍋內(nèi)氣壓和溫度都降到與室溫一致或相差不大時(shí)再打開(kāi)滅菌鍋。
如果以上情況都做到還有氣泡，可用水做培養(yǎng)基組的對(duì)照試驗(yàn)，若培養(yǎng)基組有氣泡，而對(duì)照組沒(méi)有氣泡可確定是培養(yǎng)基自身的原因。
七、倒平板
滅菌融化的培養(yǎng)基冷卻到50℃后，倒入無(wú)菌干燥的培養(yǎng)皿中。培養(yǎng)基的溫度不能過(guò)高，否則容易在培養(yǎng)皿的內(nèi)蓋上形成太多的冷凝水；溫度太低，培養(yǎng)基又容易凝固成塊，無(wú)法制成平板。倒平板時(shí)，應(yīng)在靠近酒精燈火焰進(jìn)行，以免外界的雜菌落入平板內(nèi)，左手拿培養(yǎng)皿，右手拿三角瓶的底部，用左手的小指和手掌部位把三角瓶的棉塞拔下，灼燒瓶口，用大拇指和食指把培養(yǎng)皿蓋打開(kāi)一條縫，至瓶口剛好伸入，倒入培養(yǎng)基，以鋪滿底為限，不超過(guò)培養(yǎng)皿高度的三分之一，迅速蓋好蓋，放在桌面上，輕輕地轉(zhuǎn)動(dòng)培養(yǎng)皿，使培養(yǎng)基分布均勻，冷凝后即可。
八、擺斜面
裝在試管中的瓊脂培養(yǎng)基，在滅菌完成后，趁熱立即擺放在木棒(或玻璃棒)上，并成適當(dāng)?shù)男倍?，冷卻后，瓊脂凝固即成斜面。斜面長(zhǎng)度不用超過(guò)試管二分之一。
九、質(zhì)量檢查
培養(yǎng)基滅菌后仔細(xì)檢查一遍，發(fā)現(xiàn)有破裂、水分浸入、色澤異常、棉塞被培養(yǎng)基沾染，都要丟棄，不能再用。并測(cè)定其最終pH。還需進(jìn)行無(wú)菌檢查和效果檢查。無(wú)菌檢查是將滅菌后的培養(yǎng)基取1管(瓶)~2管(瓶)，37℃恒溫培養(yǎng)1d～2d，確定無(wú)雜菌生長(zhǎng)；效果檢查是用標(biāo)準(zhǔn)菌株接種在相關(guān)的培養(yǎng)基上檢查檢查菌的生長(zhǎng)、形態(tài)及生化情況，與已知情況相符。兩種情況都合格，配制的培養(yǎng)基方可使用。
Details of preparation of "microbe" detection media
Use an electronic balance of accuracy 1/100 to weigh the medicine needed for the culture medium. According to the formula, the amount of various ingredients and drugs needed to prepare a certain amount of culture medium was calculated, and then accurately weighed with a balance. When weighing, fold the weighing paper into a dustpan to hold the medicine. The folding method of weighing paper is shown in the figure.
Second, the melt
The culture medium is placed in a beaker or enamel bowl, and a small amount of water is added slowly. If AGAR is not present in the medium, the medium does not need to be heated. If it contains agar-agar, it needs to be heated and boiled by Bunsen burner or induction cooker. After it is completely dissolved, the water needed is filled up and stirred evenly. If the amount of medium preparation is very big, can use stainless steel pot heating, melting heat and stir at any time, can use first warm water to prevent coking and coking phenomenon, if any, preparation of culture medium can't use, need to make.
Compound medium not use copper or iron when the container of melting, because of the copper or iron container may lead to excessive copper and iron content in culture medium, the impact experiment (copper content in the culture medium is greater than 0.3 mg/L, should not be bacteria growth, iron content is more than 0.14 mg/L, prevent bacteria produce toxins). The precipitated drugs, which react easily, are dissolved separately and then added to the culture media, such as dipotassium hydrogen phosphate and magnesium sulfate.
Third, the pH
Although the medium containing buffer material composition, to keep as much as possible of the pH value of the medium within the scope of the requirements, but if the medium is not in conformity with the requirements, will make the necessary adjustments. If you have already calibrated pH meter, pH meter are available, and if not, the precision of the pH test paper are available, and then according to the need to use 1 mol/L sodium hydroxide or 1 mol/L HCL (trimming or 0.1 to 0.1 mol/L sodium hydroxide or hydrochloric acid) modulation the required pH. The pH of culture medium is 7.4 ~ 7.6, also have acid or alkaline. Medium, sodium hydroxide, is used to adjust the needs of high pressure sterilization, needed to move to higher when adjusting the pH 0.1 ~ 0.2 units, by sodium hydroxide, high pressure sterilization, medium pH to reduce 0.1 ~ 0.2. If the medium contains calcium carbonate, it does not have a pH.
Four, filtering,
This step may be omitted if the prepared medium has no special requirements. If there is precipitation or turbidity, it needs to be clarified. The liquid medium can be filtered by oil paper. The solid medium can be filtered by a double layer gauze with a thin layer of absorbent cotton in the middle. If the clarification filtering method can't meet the requirements, usable egg white clarification method, namely the culture medium and 50 ~ 60 ℃, the cooling/heating load within the triangle bottle (no more than half the capacity of), according to 1000 ml and 1 ~ 2 egg whites, forcibly shake 3-5 min, high pressure steam sterilization, 121 ℃ and 20 min, take out hot filtering.
Five, the partial shipments
Make good medium according to the different USES partial shipments in triangle bottles, tubes and other containers, packing tube, if the tube is heavy, automatic liquid points are available, and if the tube quantity is little, available funnel partial shipments. The loading capacity shall not exceed two-thirds of the container volume. The triangular bottle should not exceed half of the volume. AGAR slant should be no more than one fifth of the length of the test tube. After sterilization, the slant should be one third of the amount of medium and two thirds of the amount of bottom layer. The semisolid agar-agar content is one third of the tube length. High AGAR used for inoculation or sterilization, containing 1/4 or 1/3 of the length of the test tube, and two-thirds of the amount used for anaerobic inoculation; AGAR plate, inner diameter is 90 mm with mL 13 ~ 15 mL advisable, a diameter of 70 mm plate 8 mL ~ 10 mL, if made of AGAR plate surface water more, buckle the tablet, can be placed at 37 ℃ incubator 30 min, reoccupy after drying. Each batch of medium shall be separately filled with a small glass bottle (about 20mL) and sterilized at the same time with the batch of medium, so as to determine the final pH of the batch of medium.
Six, sterilization
The prepared media should be sterilized immediately. Sterilization methods are as follows:
1. High pressure steam sterilization
Most of the heat medium is available, small packaging, with 121 ℃, 15 min; Large amount of sterilization, with 121 ℃, 30 min sterilization; Sugary medium can only 113 ~ 115 ℃, 15 min sterilization, avoid sugar destruction.
2. Boiling sterilization
This method can be used for medium containing non - heat resistant substances.
3. Filtration and sterilization
The culture medium was added quantitatively by means of filtration and sterilization. Blood of sterile operation, antibiotics available technology to extract, add cooling medium to about 50 ℃. This method can be used when the medium contains non - heat - resistant substances.
Wedding heart dynamic dividing line
When sterilizing LST medium, bubbles sometimes appear in the fermentation tube. To prevent bubbles from forming in the fermentation tube, the following measures can be taken:
1. The small tube was filled with the medium (no bubbles left) and added to the LST tube.
2. Drain the gas from the sterilizer before closing the air release valve.
3. The tube plug should not be too tight (when using the silicone plug), do not use the rubber plug.
4. Do not open the sterilizer too early, and do not open the sterilizer until the air pressure and temperature in the sterilizer are in line with or not much different from the room temperature.
If bubbles are present in all of the above cases, use water as a control test for the medium group. If there are bubbles in the medium group and no bubbles in the control group, the reason for the medium itself can be determined.
Vii. Pour plate
Sterilization after melting medium cooled to 50 ℃, into a sterile dry in a petri dish. The temperature of the medium should not be too high, or too much condensed water could be formed on the inner cover of the dish. The temperature is too low, and the medium is easy to solidify into lumps and cannot be made into flat plates. Fall flat, should be near the alcohol lamp flame, so as to avoid external miscellaneous colonies into the tablet, left hand petri dishes, triangle bottle at the bottom of the right hand, left hand little finger and palm part put triangle bottle cotton unplug, burning flask, with their thumb and forefinger to the petri dish cover open a crack, to just into the bottle, pour into the culture medium, is limited to covered the bottom, no more than a third of the plate height, cover cover quickly, on the desktop, gently turn the petri dishes, uniform distribution of medium, after condensation.
Swing the bevel
When sterilization is completed, the AGAR medium installed in the test tube will be placed on the wooden rod (or glass rod) as soon as it is hot. The AGAR will set to the inclined plane after cooling. The bevel should not exceed half the length of the tube.
Quality inspection
After sterilization, the culture medium was carefully examined and found to have cracks, water immersion, color abnormality, cotton plug was contaminated by the culture medium, all were discarded and could not be used again. And its final pH was measured. Sterility and efficacy tests are also required. Asepsis check is medium after sterilization will be taken 1 pipe (bottle) ~ 2 (bottle), 37 ℃ constant temperature culture 1 d ~ 2 d, make sure no bacterial growth; The effect test was to test the growth, morphology and biochemistry of the bacteria by inoculation of the standard strain on the relevant medium, which was consistent with the known situation. In both cases, the prepared medium can only be used.