無菌間消毒滅菌方法與要求內(nèi)容說明
發(fā)布時間：
2022-12-27
作者：
預灌裝終端滅菌培養(yǎng)基
在微生物實驗操作中，環(huán)境的無菌度對實驗結(jié)果以及實驗的有效性有決定性的影響。所以對于微生物工作者來說，無菌室的消毒滅菌是一項非常重要的工作。下面將常用的無菌室消毒滅菌方法進行匯總：
1、紫外線消毒：
在室溫20℃～25℃時220V，30W紫外燈下方垂直位置1.0m處，253.7nm紫外線輻射強度應≥70μW/cm2 ，低于此值時應更換。適當數(shù)量的紫外燈，確保平均每立方米應不少于1.5W。
紫外線消毒時，無菌室內(nèi)應保持清潔干燥。
在無人條件下，可采取紫外線消毒，作用時間應≥30min。室內(nèi)溫度＜20℃或＞40℃、相對濕度大于60%時，應適當延長照射時間到足夠的照射劑量。
人員在關(guān)閉紫外燈至少30min后方可入內(nèi)作業(yè)。
2.消毒劑噴灑：
用0.1%新潔爾滅或2%甲酚液或其他適宜消毒液（常用消毒劑的品種有：5-20倍稀釋的碘伏水溶液、0.1%新潔爾滅溶液、1：50的84消毒液、75%乙醇溶液、3%碘酒溶液、5%石炭酸（來蘇兒）消毒溶液、2%戊二醛水溶液、尼泊金乙醇消毒液（處方：對羥基苯甲酸甲酯21.5g；對羥基苯甲酸丙酯8.6g，75%乙醇10ml）等，所用的消毒劑品種與使用要進行有效性驗證方可使用，并定期更換消毒劑的品種）擦拭操作臺及可能污染的死角。
方法是用無菌紗布浸漬消毒溶液清潔超凈臺的整個內(nèi)表面、頂面、及無菌室、人流、物流、緩沖間的地板、傳遞窗、門把手。清潔消毒程序應從內(nèi)向外，從高潔凈區(qū)到低潔凈區(qū)。逐步向外退出潔凈區(qū)域。然后開啟無菌空氣過濾器及紫外燈殺菌1-2h，以殺滅存留微生物。在每次操作完畢，同樣用上述消毒溶液擦拭工作臺面，除去室內(nèi)濕氣，用紫外燈殺菌30min。
3、熏蒸：
3.1甲醛熏蒸
按照甲醛（40%）10毫升／立方米、高錳酸鉀5克／立方米計算用量。不同情況下，用量有所不同，但比例為2：1。盛藥容器要大、耐熱、耐腐蝕；一般用陶瓷或玻璃容器，因為高錳酸鉀和甲醛都具有腐蝕性，且混合后反應劇烈，釋放熱量，是將甲醛倒入高錳酸鉀溶液內(nèi)。
房間要密閉：這樣熏蒸效果才會好。容器應盡量靠近門，以便操作人員迅速撤離；先將溫水倒入容器內(nèi)，后加入高錳酸鉀，攪拌均勻；再加入甲醛；加入甲醛后人立即離開，密閉房間。消毒時間一般為20-30分鐘，消毒后要打開門窗通風換氣。
3.2乳酸熏蒸
按照蒸餾水：乳酸為1:1的比例配置乳酸溶液，1-2mL/m3，熏蒸2小時，密閉門窗12h。對器物腐蝕性較強。
4、臭氧發(fā)生器
臭氧是世界公認的廣譜高效殺菌消毒劑。采用空氣或氧氣為原料利用高頻高壓放電產(chǎn)生臭氧。臭氧比氧分子多了一活潑的氧原子臭氧，化學性質(zhì)特別活潑，是一種強氧化劑，在一定濃度下可迅速殺滅空氣中的細菌。沒有任何有毒殘留，不會形成二次污染，被譽為“最清潔的氧化劑和消毒劑”。
用臭氧對物體表面消毒，由于作用緩慢，其濃度應≥60mg/m3，相對濕度≥70%,作用時間60min-120min。
The principle of preservation of bacteria species:
According to the physiological and biochemical characteristics of microorganism, the conditions of artificial creation are mainly three cases of low temperature, dry and anoxic. The metabolism of microorganism is in a state of inactivity and inhibition of growth and reproduction, in order to keep the fine traits of pure species.
Introduction of common bacteria preservation methods:
Regular transplantation (also known as subculture preservation)
After inoculation, the strain was inoculated in suitable culture medium and stored at 4 -6 C for a long time. Including oblique culture, puncture culture, liquid culture and so on.
Liquid paraffin method (also known as mineral oil preservation)
It is an auxiliary method for the regular preservation of the preservation method. The bacteria were inoculated into the suitable slope culture medium, and under the optimum conditions, the liquid paraffin was incubated to the strain of the bacteria to infuse the sterile liquid, so that it covered the whole slope and was placed at 4 -6 C for preservation.
Preservation of porcelain beads
The cultured microbiological cells or spores are made into suspension and transferred into aseptic bottles containing sterile porous glass beads (or porcelain beads) to adsorb on the surface of glass beads and remove superfluous suspension and cryopreservation of a kind of bacteria preservation method.
Low temperature preservation at -80 C
A method of preserving bacteria in refrigerator at -80 C. The principle is that low temperature conditions can slow down the metabolism of microorganisms in order to achieve long-term and effective preservation of microorganisms.
Common bacteria preservation methods:
Liquid paraffin method (also known as mineral oil preservation)
Inoculate the desired species to the most suitable medium.
Choose pure and high quality paraffin oil, sterilize at 121 degrees, 30 minutes, and put it in dry oven (about 160 degrees Celsius) after sterilization.
When the wax oil becomes clear and transparent, it can be added into the inclined plane when cooling. The bubbles can not appear on the inclined surface. The amount of liquid wax added is about 1cm higher than the top of the slope.
After sealing, the storage temperature is required at 4~6 degrees C.
Preservation of porcelain beads
The direct 2mm-3mm ceramic beads were packed into a small nut tube and made into a freezing tube for autoclaving.
Mark the freezing pipe and open the freezing pipe under sterile conditions.
The fresh culture of the preserved strain was inserted into the culture medium, and the suspension was then made into the freezing pipe.
Tighten the cap and turn four to five times back and forth. Do not use a vortex oscillator;
After the bacteria were adsorbed on the porcelain beads, the superfluous bacteria suspension was sucked out.
Low temperature preservation at -80 C
The cultured strains were made into high concentration suspension (108 ~ 1010CFU/mL).
The glycerol concentration was about 20%.
After mixing, it is stored in a freezing test tube or ampoule and stored in a -80 low temperature refrigerator.
Precautions for the preservation of bacteria species:
Suitable medium (regular transplant method and liquid paraffin method):
Bacteria - nutritive agar;
Fungi --PDA;
The --MRS medium of lactic acid bacteria;
Anaerobes - Pao meat medium;
Vibrio halophilus (Vibrio halophilus) - TSA or nutritive agar containing 3%NaCl;
Suitable temperature:
It is preserved under suitable temperature according to the characteristics of the strain. For example, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae and other Vibrio at room temperature (below 25 degrees Celsius) can not be refrigerated.
Suitable oxygen conditions:
According to the characteristics of the strain, suitable oxygen conditions, such as micro aerobic or anaerobic conditions, are provided.
4. The ozone generator
Ozone is widely recognized as a broad-spectrum and highly effective disinfectant in the world. Ozone is produced by high frequency and high voltage discharge using air or oxygen as raw material. Ozone is more active than oxygen, which is a strong oxidizing agent. It is a strong oxidizing agent, which can kill bacteria in the air at a certain concentration. There is no toxic residue and no two pollution will be formed. It is known as "the cleanest oxidant and disinfectant".
Ozone is used to disinfect the surface of the object. Due to its slow action, its concentration should be more than 60mg/m3, the relative humidity is more than 70%, and the time of action is 60min-120min.