無菌操作、消毒滅菌及培養(yǎng)基制備等微生物實驗注意事項有哪些？
發(fā)布時間：
2022-12-24
作者：
無菌操作、消毒滅菌及培養(yǎng)基制備等微生物實驗注意事項有哪些？
一、無菌操作要求
1. 接種細(xì)菌時必須穿工作服、戴工作帽。
2. 進(jìn)行接種食品樣品時，必須穿專用的工作服、帽及拖鞋，應(yīng)放在無菌室緩沖間， 工作前經(jīng)紫外線消毒后使用。
3. 接種食品樣品時，應(yīng)在進(jìn)無菌室前用肥皂洗手，然后用75％酒精棉球?qū)⑹植粮蓛簟?
4. 進(jìn)行接種所用的吸管，平皿及培養(yǎng)基等必須經(jīng)消毒滅菌，打開包裝未使用完的器皿，不能放置后再使用，金屬用具應(yīng)高壓滅菌或用95%酒精點(diǎn)燃燒灼三次后使用。
5. 從包裝中取出吸管時，吸管尖部不能觸及外露部位，使用吸管接種于試管或平皿時，吸管尖不得觸及試管或平皿邊。
6. 接種樣品、轉(zhuǎn)種細(xì)菌必須在酒精燈前操作，接種細(xì)菌或樣品時，吸管從包裝中取 出后及打開試管塞都要通過火焰消毒。
7. 接種環(huán)和針在接種細(xì)菌前應(yīng)經(jīng)火焰燒灼全部金屬絲，必要時還要燒到環(huán)和針與桿的連接處，接種結(jié)核菌和烈性菌的接種環(huán)應(yīng)在沸水中煮沸5min，再經(jīng)火焰燒灼。
8. 吸管吸取菌液或樣品時，應(yīng)用相應(yīng)的橡皮頭吸取，不得直接用口吸。
二、無菌間使用要求
1、無菌間通向外面的窗戶應(yīng)為雙層玻璃，并要密封，不得隨意打開，并設(shè)有與無菌間大小相應(yīng)的緩沖間及推拉門，另設(shè)有0.5-0.7平方米的小窗，以備進(jìn)入無菌間后傳遞物品。
2、無菌間內(nèi)應(yīng)保持清潔，工作后用2%-3%煤酚皂溶液消毒，擦拭工作臺面，不得存放與實驗無關(guān)的物品。
3、無菌間使用前后應(yīng)將門關(guān)緊， 打開紫外燈，如采用室內(nèi)懸吊紫外燈消毒時，需30W紫外燈，距離在1.0m處，照射時間不少于30min，使用紫外燈，應(yīng)注意不得直接在紫外線下操作，以免引起損傷，燈管每隔兩周需用酒精棉球輕輕擦拭，除去上面灰塵和油垢，以減少紫外線穿透的影響。
4、處理和接種食品標(biāo)本時，進(jìn)入無菌間操作，不得隨意出入，如需要傳遞物品，可通過小窗傳遞。
5、在無菌間內(nèi)如需要安裝空調(diào)時，則應(yīng)有過濾裝置。
三、消毒滅菌要求
微生物檢測用的玻璃器皿、金屬用具及培養(yǎng)基、被污染和接種的培養(yǎng)物等，必須經(jīng)滅菌后方能使用。
（一）干熱和濕熱高壓蒸氣鍋滅菌方法
1、滅菌前準(zhǔn)備
（1）所有需要滅菌的物品首先應(yīng)清洗晾干，玻璃器皿如吸管、平皿用紙包裝嚴(yán)密，如 用金屬筒應(yīng)將上面通氣孔打開。
（2）裝培養(yǎng)基的三角瓶塞，用紙包好，試管蓋好蓋，注射器須將管芯抽出，用紗布包 好。
2、裝放
（1）干熱滅菌器：裝放物品不可過擠，且不能接觸箱的四壁。
（2）大型高壓蒸氣鍋：放置滅菌物品分別包扎好，直接放入消毒筒內(nèi)，物品之間不能過擠。
3、設(shè)備檢查
（1）檢查門的開關(guān)是否靈活，橡皮圈有無損壞，是否平整。
（2）檢查壓力表蒸氣排盡時是否停留在零位，關(guān)好門和蓋，通蒸氣或加熱后，觀察是手提式高壓鍋或立式壓力蒸氣滅菌器的使用應(yīng)按下列步驟進(jìn)行：
①手提式高壓鍋在主體內(nèi)加入3L清水，立式高壓鍋加水16L（重復(fù)使用時應(yīng)將水 量補(bǔ)足，水變混濁需更換）；
②手提式壓力鍋將頂蓋上的排氣管插入消毒桶內(nèi)壁的方管中（無軟管或軟管銹蝕破裂的滅菌器不得使用）；
③蓋好頂蓋擰緊，勿使漏氣；置滅菌器于火源上加熱，立式壓力鍋通上電源，并打開頂蓋上的排氣閥放了冷氣（水沸騰后排氣10-15min）；
④關(guān)閉排氣閥，使蒸氣壓上升到規(guī)定要求，并維持規(guī)定時間（按滅菌物品性質(zhì)與有關(guān)情況而定）；
⑤達(dá)到規(guī)定時間后，對需干燥的物品，立即打開排氣閥排出蒸氣，待壓力恢復(fù)到零時，自然冷卻至 60℃后開蓋取物，如為液體物品，不要打開排氣閥，而應(yīng)立即將鍋去除熱源，待自然冷卻，壓力恢復(fù)至零，溫度降到60℃以下再開蓋取物，以防突然減壓液體劇烈沸騰或容器爆破。
（3）臥式壓力鍋蒸氣滅菌器的使用按下列步驟進(jìn)行：
①關(guān)緊鍋門，打開進(jìn)氣閥，將蒸氣引入夾層進(jìn)行預(yù)熱，夾層內(nèi)冷空氣經(jīng)阻氣器自動排出；
② 夾層達(dá)到預(yù)定溫度后，打開鍋室進(jìn)氣閥，將蒸氣引入鍋室，鍋室內(nèi)冷空氣經(jīng)鍋室阻氣器自動排出；
③待鍋室達(dá)到規(guī)定的壓力與溫度時，調(diào)節(jié)進(jìn)氣閥，使保持恒定至滅菌完成；
④自然或人工降溫至60℃再開門取物，不得使用快速排出蒸氣法，以防突然降壓，液體劇烈沸騰或容器爆破；
⑤使用自動程序控制式壓力蒸氣滅菌器，在放好物品關(guān)緊門后，應(yīng)根據(jù)物品類別按 動相應(yīng)開關(guān)，以便按要求程序自動進(jìn)行滅菌，滅菌時必須利用附設(shè)儀表記錄溫度與時間以備查，操作要求應(yīng)嚴(yán)格按照廠家說明書進(jìn)行。
5、滅菌溫度與時間
（1）干熱滅菌器滅菌溫度160℃，1.5-2h。
（2）壓力蒸氣滅菌鍋滅菌溫度與時間一般為121℃,15min。
（二）間歇滅菌方法
1、滅菌方法系利用不加壓力的蒸氣滅菌，某些物質(zhì)經(jīng)高壓蒸氣滅菌容易破壞，可用此法滅菌。
（1）將欲滅菌物品置于鍋內(nèi)，蓋上頂蓋，打開排水口，使器內(nèi)余水排盡；
（2）關(guān)閉排水口，打開進(jìn)氣門，根據(jù)需要消毒10～20min；
（3）滅菌完畢關(guān)閉進(jìn)氣門，取出物品待冷至室溫溫度，放入37℃溫箱過夜，次日仍按上述方法消毒，如此三次，即可達(dá)到滅菌目的。
2、血清凝固器使用方法，培養(yǎng)基中含有血清或雞蛋特殊成份時，因高熱會破壞其營 養(yǎng)成份，故用低溫，可使血清凝固，又可達(dá)到滅菌目的。
（1）在使用該法滅菌的血清等分裝時，需嚴(yán)格遵守?zé)o菌操作，試管、平皿也經(jīng)滅菌后使用；
（2）將培養(yǎng)基按要求使成斜面或高層，加足水后，接上電源，升溫75～90℃1h滅菌，放37℃溫箱過夜，再如此滅菌三次。
3、煮沸消毒：可用煮鍋或煮沸消毒器，水沸騰后再煮5～15 min，也可在水中加入2%石炭酸煮沸5 min，加入0.02%甲醛，80℃煮60 min均可達(dá)到滅菌目的， 但選用 煮沸消毒的增消劑時， 應(yīng)注意對物品的腐蝕性。
4、滅菌處理：滅菌后物品，按正常情況已屬無菌，從滅菌器中取出應(yīng)仔細(xì)檢查放置，以免再度污染。
（1）物品取出，隨即檢查包裝的完整性，若有破壞或棉塞脫掉，不可作為無菌物品使用；
（2）取出的物品，如為包裝有明顯的水浸者，不可作為無菌物品使用；
（3）培養(yǎng)基或試劑等，應(yīng)檢查是否符合達(dá)到滅菌后的色澤或狀態(tài)，未達(dá)到者應(yīng)廢棄；
（4）啟閉式容器，在取出時應(yīng)將篩孔關(guān)閉；
（5）取出的物品掉落在地或誤放不潔這處，或沾有水液，均視為受到污染，不可作為無菌物品使用；
（6）取出的合格滅菌物品，應(yīng)存放于貯藏室或防塵柜內(nèi)，嚴(yán)禁與未滅菌物品混放；
（7）凡屬合格物品，應(yīng)標(biāo)有滅菌日期及有效期限；
（8）每批滅菌處理完成后，記錄滅菌品名、數(shù)量、溫度、時間、操作者。
四、有毒有菌污物處理要求
微生物實驗所用實驗器材、培養(yǎng)物等未經(jīng)消毒處理，一律不得帶出實驗室。
1、經(jīng)培養(yǎng)的污染材料及廢棄物應(yīng)放在嚴(yán)密的容器或鐵絲筐內(nèi)，并集中存放在指定地點(diǎn)，待統(tǒng)一進(jìn)行高壓滅菌。
2、經(jīng)微生物污染的培養(yǎng)物，必須經(jīng)121℃ 30min高壓滅菌。
3、染菌后的吸管，使用后放入5%煤酚皂溶液或石炭酸液中，最少浸泡24h（消毒液體不得低于浸泡的高度）再經(jīng)121℃ 30 min高壓滅菌。
4、涂片染色沖洗片的液體，一般可直接沖入下水道，烈性菌的沖洗液必須沖在燒杯 中，經(jīng)高壓滅菌后方可倒入下水道，染色的玻片放入5%煤酚皂溶液中浸泡24h后，煮沸洗滌。做凝集試驗用的玻片或平皿，必須高壓滅菌后洗滌。
5、打碎的培養(yǎng)物，立即用5%煤酚皂溶液或石炭酸液噴灑和浸泡被污染部位，浸泡半小時后再擦拭干凈。
污染的工作服或進(jìn)行烈性試驗所穿的工作服、帽、口罩等，應(yīng)放入專用消毒袋內(nèi)，經(jīng)高壓滅菌后方能洗滌。
五、培養(yǎng)基制備要求
培養(yǎng)基制備的質(zhì)量將直接影響微生物生長。因為各種微生物對其營養(yǎng)要求不完全相同，培養(yǎng)目的的不同，各種培養(yǎng)基制備要求如下：
1、根據(jù)培養(yǎng)基配方的成分按量稱取，然后溶于蒸餾水中，在使用前對應(yīng)用的試劑藥 品應(yīng)進(jìn)行質(zhì)量檢驗。
2、PH 測定及調(diào)節(jié)：PH測定要在培養(yǎng)基冷至室溫時進(jìn)行，因在熱或冷的情況下，其PH有一定差異，當(dāng)測定好時，按計算量加入堿或酸混勻后，應(yīng)再測試一次。培養(yǎng)基 PH值一定要準(zhǔn)確，否則會影響微生物的生長或影響結(jié)果的觀察。但需注意因高壓滅菌可 影響一些培養(yǎng)基的 PH 降低或升高，故不宜滅菌壓力過高或次數(shù)太多，以免影響培養(yǎng)基 的質(zhì)量，指示劑、去氧膽酸鈉、瓊脂等一般在調(diào)完P(guān)H后再加入。
3、培養(yǎng)基需保持澄清，便于觀察細(xì)菌的生長情況，培養(yǎng)基加熱煮沸后，可用脫脂棉 花或絨布過濾，以除去沉淀物，必要時可用雞蛋白澄清處理，所用瓊脂條要預(yù)先洗凈晾干后使用，避免因瓊脂含雜質(zhì)而影響透明度。
4、盛裝培養(yǎng)基不宜用鐵、銅等容器，使用洗凈的中性硬質(zhì)玻璃容器為好。
5、培養(yǎng)基的滅菌既要達(dá)到完全滅菌目的，又要注意不因加熱而降低其營養(yǎng)價值，一般121℃15min即可，如為含有不耐高熱物質(zhì)的培養(yǎng)基如糖類、血清、明膠等，則應(yīng)采用低溫滅菌或間歇法滅菌，一些不能加熱的試劑如亞碲酸鉀、卵黃、TTC、抗菌素等，待基礎(chǔ)瓊脂高壓滅菌后涼至50℃左右再加入。
6、每批培養(yǎng)基制備好后，應(yīng)做無菌生長試驗及所檢菌株生長試驗。如果是生化培養(yǎng)基，使用標(biāo)準(zhǔn)菌株接種培養(yǎng)，觀察生化反應(yīng)結(jié)果，應(yīng)呈正常反應(yīng)，培養(yǎng)基不應(yīng)貯存過久，必要時可置4℃冰箱存放。
7、目前各種干燥培養(yǎng)基較多，每批需用標(biāo)準(zhǔn)菌株進(jìn)行生長試驗或生化反應(yīng)觀察，各種培養(yǎng)基用相應(yīng)菌株生長試驗良好后方可應(yīng)用，新購進(jìn)的或存放過久的干燥培養(yǎng)基，在配制時也應(yīng)測PH，使用時需根據(jù)產(chǎn)品說明書用量和方法進(jìn)行。
8、每批制備的培養(yǎng)基所用化學(xué)試劑、滅菌情況及菌株生長試驗結(jié)果、制作人員等應(yīng)做好記錄，以備查詢。
六、樣品采集及處理要求
1、所采集的檢驗樣品一定要具有代表性，采樣時應(yīng)首先對該批食品原料、加工、運(yùn)輸、貯藏方法條件、周圍環(huán)境衛(wèi)生狀況等進(jìn)行詳細(xì)調(diào)查，檢查是否有污染源存在。
2、根據(jù)食品的種類及數(shù)量，采樣數(shù)量及方法應(yīng)按標(biāo)準(zhǔn)檢驗方法的要求進(jìn)行。
3、采樣應(yīng)注意無菌操作，容器必須滅菌，避免環(huán)境中微生物污染，容器不得使用煤 酚皂溶液，用新潔爾滅、酒精等消毒藥物滅菌，更不能含有此類消毒藥物或抗生素類藥物，以避免殺死樣品中的微生物，所用剪、刀、匙用具也需滅菌方可應(yīng)用。
4、樣品采集后應(yīng)立即送往檢驗室進(jìn)行檢驗，送檢過程中一般不超過3h，如路程較遠(yuǎn)，可保存在1～5℃環(huán)境中，如需冷凍者，則在凍存狀態(tài)下送檢。
5、檢驗室收到樣品后，進(jìn)行登記（樣品名稱、送檢單位、數(shù)量、日期、編號等），觀察樣品的外觀，如果發(fā)現(xiàn)有下列情況之一者，可拒絕檢驗。
（1）樣品經(jīng)過特殊高壓、煮沸或其他方法殺菌者，失去代表原食品檢驗意義者。
（2）瓶、袋裝食品已啟開者，熟肉及其制品、熟禽等食品已折碎不完整者，即失去原食品形狀者（食物中毒樣品除外）。
（3）按規(guī)定采樣數(shù)量不足者。
對送檢符合要求的樣品， 檢驗室收到后，應(yīng)立即進(jìn)行檢驗，如果條件不具備，應(yīng)置4℃冰箱存放，及時準(zhǔn)備創(chuàng)造條件，然后進(jìn)行檢驗。
6、樣品檢驗時，根據(jù)其不同性狀，進(jìn)行適當(dāng)處理。
（1）液體樣品接種時，應(yīng)充分混合均勻，按量吸取進(jìn)行接種。
（2）固體樣品，用滅菌刀、剪取其不同部位共25g，置于225ml 滅菌生理鹽水或其他溶液中，用均質(zhì)器攪碎混勻后，按量吸取接種。
（3）瓶、袋裝食品應(yīng)用滅菌操作啟開，根據(jù)性狀選擇上述方法處理后接種。
七、記錄和報告的要求
1、檢驗室收到樣品后，首先進(jìn)行外觀檢驗，及時按照國家標(biāo)準(zhǔn)檢驗方法進(jìn)行檢驗， 檢驗過程中要認(rèn)真、負(fù)責(zé)、嚴(yán)格進(jìn)行無菌操作，避免環(huán)境中微生物污染。
2、樣品檢驗過程中所用方法、出現(xiàn)的現(xiàn)象和結(jié)果等均要用文字寫出試驗紀(jì)錄，以作為對結(jié)果分析、判定的依據(jù)，記錄要求詳細(xì)、清楚、真實、客觀、不得涂改和偽造。
青島日水生物：
無菌操作、消毒滅菌及培養(yǎng)基制備等微生物實驗注意事項有哪些？
Matters needing attention in microbiological experiments such as aseptic operation, sterilization and preparation of culture medium
I. sterile operation requirements
1. Work clothes and work caps must be worn when inoculation of bacteria.
2. When conducting food samples for inoculation, special working clothes, hats and slippers must be worn, which should be placed in the sterile room buffer room, and used after ultraviolet disinfection before work.
3. Wash hands with soap before entering the sterile room, and then wipe hands with 75% alcohol cotton balls.
4. Used for inoculation of straws, AGAR and medium must be disinfection sterilization, such as open the packing vessel has not used up, can't placed before using, metal utensils should be high pressure sterilization or used after burning for three times with 95% alcohol.
5. When taking the straw out of the package, the tip of the straw should not touch the exposed part. When using the straw to inoculate in the test tube or plate, the tip of the straw should not touch the edge of the test tube or plate.
6. The inoculation samples and transgenic bacteria must be operated in front of the alcohol lamp. When the bacteria or samples are inoculated, the straw shall be disinfected by flame after being removed from the package and the tube plug is opened.
7. Inoculation loops and needle before inoculation of bacteria should be fully wire by the flame burning, burn when necessary to ring and pin and the joint of rod, n/med tuberculosis bacterium and virulent bacteria inoculated ring should be in boiling water boil 5 min, then through the flame burning.
8. When sucking the bacteria solution or sample through the straw, the corresponding rubber head shall be applied to absorb the product, and the product shall not be sucked directly by the mouth.
Ii. Requirements for use in sterile rooms
1, sterile towards the outside of the window should be double deck glass, and I will seal, do not get optional open, and offers corresponding to the size of the buffer between sterile room and sliding door, the other is equipped with 0.5 to 0.7 square meters of small window, for after entering between sterile items.
2. The sterile room should be kept clean. After work, 2-3% cresol solution should be used to disinfect and wipe the working table.
3, sterile between before and after use should shut the door, open the uv lamp, such as using indoor hanging lamp uv disinfection, need 30 w uv lamp, the distance at 1.0 m, the irradiation time of not less than 30 min, the use of uv lamp, may not directly under ultraviolet light should be paid attention to the operation, lest cause damage, lamp every two weeks need alcohol sponge wiped gently, remove dirt and oil dirties, above in order to reduce the influence of ultraviolet rays penetrate.
4. When handling and inoculation of food samples, enter the sterile room for operation, and do not enter or leave at will. If the goods need to be delivered, they can be delivered through a small window.
5. If air conditioning is required to be installed in the sterile room, there shall be a filter device.
Iii. Sterilization requirements
The glassware, metal utensils and media, contaminated and inoculated culture materials used for microbial testing must be sterilized before they can be used.
(1) sterilization method for high pressure steam boilers with dry heat and moist heat
1. Preparation before sterilization
(1) all articles that need sterilization should be cleaned and dried first. Glassware such as straw and plate paper should be tightly packed. If metal tube is used, the top vent should be opened.
(2) the triangular bottle stopper of the culture medium shall be wrapped in paper, the tube cover shall be covered, the syringe shall take out the tube core and wrap it in gauze.
2, put
(1) dry heat sterilizer: it is not allowed to over squeeze the articles in the container and contact the four walls of the box.
(2) large high pressure steam boiler: place sterilized articles wrapped separately and directly put into the sterilizing drum, and do not over squeeze between articles.
3. Equipment inspection
(1) check whether the switch of the door is flexible, whether the rubber band is damaged, and whether it is smooth.
(2) check the pressure gauge when the steam exhaust all stay at zero, and door cover, after the steam or heat, observation is the use of portable pressure cooker or vertical pressure steam sterilizer should be according to the following steps:
Water is added into the main body of the hand-held pressure cooker with 3L water, and 16L water is added to the vertical pressure cooker (the amount of water should be sufficient for repeated use, and the water needs to be replaced for turbidity).
Circulating pressure cooker inserts the exhaust pipe on the top cover into the square pipe of the inner wall of the sterilizing drum (sterilizer without hose or hose corrosion cracking shall not be used);
Tighten the upper cover of the clamping cover to prevent air leakage. The sterilizer is heated on the fire source, the vertical pressure cooker is connected to the power supply, and the exhaust valve on the top cover is opened and the cool air is released (after the water boils, the air is discharged for 10-15 minutes).
Closing the exhaust valve to raise the vapor pressure to the specified requirements and to maintain the specified time (as per the nature and circumstances of sterilization goods);
(5) after reaching time, to need to dry goods, immediately open the discharge valve discharge steam, pressure recovery to zero, after natural cooling to 60 ℃ open content, such as liquid items, do not open the exhaust valve, and shall immediately remove pan heat source, natural cooling, pressure buildup to zero, the temperature dropped below 60 ℃ to open cover fetch, in case of sudden relief blasting violent boiling liquid or container.
(3) the use of steam sterilizer for horizontal pressure cooker shall follow the following steps:
Close the door of the boiler, open the inlet valve, introduce the steam into the interlayer for preheating, and the cold air in the interlayer will be automatically discharged by the choke.
After reaching the predetermined temperature, turn on the air inlet valve of the boiler chamber and introduce the steam into the furnace chamber.
When the boiler room reaches the specified pressure and temperature, adjust the inlet valve to keep constant until sterilization is completed.
(4) natural or artificial cooling and 60 ℃ and then open the door, quick discharge steam method, shall not be used in case of sudden depressurization, violent boiling liquid or container blasting;
(5) the use of automatic program control type pressure steam sterilizer, after put good items, shut the door, should be made according to the categories according to the corresponding switch, so that the program automatically according to the requirement of sterilization, sterilization must be using the attached instrument to record the temperature and time for future reference, operation requirements shall be in strict accordance with the manufacturer instructions.
5. Sterilization temperature and time
(1) hot air sterilizer sterilization temperature 160 ℃, 1.5 2 h.
(2) general pressure steam sterilization pot sterilization temperature and time is 121 ℃, 15 min.
(2) intermittent sterilization
1. Sterilization method is to sterilize certain substances by steam without pressure. Some substances are easy to be destroyed by high-pressure steam sterilization, which can be sterilized by this method.
(1) place the sterilized articles in the pot, cover the top cover, open the drain outlet, and drain the remaining water in the pot;
(2) close the drain outlet, open the inlet valve, and disinfect for 10-20min as required;
Close the inlet valve (3) sterilization, remove the items to be cold temperature to room temperature, in 37 ℃ warm box for the night, the next day, still according to the above method of disinfection, so three times, can achieve sterilization.
2, serum photocoagulator use method, culture medium containing serum specific ingredients or eggs, because of the high temperature damage the battalion nurturance, reason with low temperature, can make the serum levels of coagulation, and can achieve sterilization.
(1) when using the sterilized serum of this method, the sterile operation shall be strictly observed, and the test tube and plate shall also be used after sterilization;
(2) the medium escarp as required or top, add enough water, turn on the juice and 1 h sterilization temperature 75 ~ 90 ℃, with 37 ℃ temperature box for the night, and so the sterilization three times.
3, boil disinfection, can use the pot or boiling sterilizer, cook for 5 ~ 15 min after the water boils, and 5 min 2% carbolic acid can be added in the water is boiling, add 0.02% formaldehyde, 80 ℃ boiled 60 min all can achieve sterilization, but when you boil disinfection of elimination agent should pay attention to corrosion of the item.
4. Sterilization treatment: after sterilization, the product is sterile according to normal conditions. When taken out of the sterilizer, it should be carefully checked and placed to avoid re-contamination.
(1) when the goods are taken out, the integrity of the packaging shall be checked immediately. If there is any damage or the cotton stopper removed, it shall not be used as sterile goods;
(2) articles taken out, such as those with obvious water immersion in the package, shall not be used as sterile articles;
(3) the culture medium or reagent shall be checked to see if it conforms to the color or state after sterilization, and if it fails, it shall be discarded;
(4) the opening and closing container should close the screen hole when it is taken out;
(5) the items taken out shall be deemed to be contaminated and shall not be used as sterile articles if they fall on the ground or are improperly placed or are contaminated with water;
(6) the qualified sterilized articles taken out shall be stored in the storage room or dustproof cabinet. It is strictly prohibited to mix them with unsterilized articles.
(7) for qualified goods, the sterilization date and expiry date shall be marked;
(8) after each batch of sterilization treatment is completed, the name, quantity, temperature, time and operator of sterilization products are recorded.
Iv. Requirements for the treatment of toxic and bacteria-contaminated substances
No experimental equipment or culture materials used in microbiological experiments shall be taken out of the laboratory without disinfection treatment.
1. The cultivated pollution materials and wastes shall be placed in strict containers or wire baskets and stored in designated places for high pressure sterilization.
2, the microbial contamination of cultures, must by 121 ℃ for 30 min high pressure sterilization.
3, dyed bacteria after straws, after using in 5% lysol or carboniferous acid, at least for 24 h (soaking disinfection liquid shall not be lower than the height of the) then through 121 ℃ for 30 min high pressure sterilization.
4, smear dyeing washing liquid, generally can be directly into the sewer, virulent bacteria rinses must be charged in a beaker, via high pressure sterilization rear can pour into the sewer, stained glass in the 5% lysol 24 h after soaking, boiling washing. The glass or plate used for agglutination test must be washed after sterilization under high pressure.
5. For the broken culture, immediately spray and soak the contaminated area with 5% cresol soap solution or carbonic acid solution, and then wipe clean after soaking for half an hour.
The work clothes, hats and face masks worn by the contaminated work clothes or the strength test shall be put into special disinfection bags, which can be washed after high pressure sterilization.
Requirements for preparation of culture medium
The quality of culture medium preparation will affect the growth of microorganism directly. Because the nutritional requirements of various microorganisms are not exactly the same and the purpose of culture is different, the preparation requirements of various media are as follows:
1. Take the ingredients according to the amount of the medium formula and dissolve them in distilled water, and conduct quality inspection of the reagent drugs before use.
2, PH measurement and adjust: PH measurement to cool to room temperature in the medium, as in the case of hot or cold, its PH have certain differences, when, when the good is measured according to the amount of calculation in acid or alkali, after blending should be test again. The PH value of the culture medium must be accurate, otherwise it will affect the growth of microorganisms or the observation of results. But need to pay attention to because of the high pressure sterilization can affect some of the culture medium PH lower or higher, therefore, should not be sterilized pressure too high or too many times, so as not to affect the quality of the medium, indicator, deoxidation cholic acid sodium, AGAR, etc in commonly after adjust the PH to join again.
3, culture needs to keep clear, easy to observe the growth of bacteria, medium heat to boil, absorbent cotton lint filter or flowers are available, and to remove sediment, egg white clarification if necessary processing, the article AGAR to wash to dry after use, to avoid affect transparency by AGAR containing impurities.
4. The medium should not be filled with iron, copper and other containers. It is better to use a clean neutral hard glass container.
5, sterilization of culture medium to achieve complete sterilization, and pay attention to not because of the heat and reduce its nutritional value, generally 121 ℃ for 15 min, such as culture medium containing no heat resistant material such as sugar, serum, gelatin, etc., should adopt low temperature sterilization or intermittent sterilization method, some can't heating reagents such as potassium tellurite, egg yolk, TTC, antibiotics, etc., after being based AGAR autoclave cooler to 50 ℃ or so and then join in.
6. After each batch of culture medium is prepared, sterile growth test and growth test of tested strains should be conducted. If it is a biochemical culture medium, the use of standard strain inoculation training, observing biochemical reactions as a result, should be a normal reaction, medium should not be stored for too long, can buy 4 ℃ refrigerator storage when necessary.
7, at present all kinds of drying medium is more, each batch need standard strain growth test or biochemical reactions observed, all kinds of culture medium with corresponding growth test is good application, new purchases or for too long drying medium, should also be measured PH during preparation, when