實驗一 細菌的培養(yǎng)法

1、實驗一 細菌的培養(yǎng)法 一般細菌均可用人工方法進行培養(yǎng)，使其生長繁殖，以便進一步觀察和研究它們的各種生物學(xué)特性。為了獲得良好的細菌培養(yǎng)物，在分離培養(yǎng)細菌時，除采用適宜的培養(yǎng)基并考慮到其他的培養(yǎng)條件（如溫度、濕度、酸堿度、氣體等）之外，掌握各種分離培養(yǎng)和接種的基本技術(shù)，也是重要環(huán)節(jié)。 在土壤、水、空氣、以及人和動、植物體中，不同種類的細菌混雜地生活在一起。若要研究其中某種細菌，就必須先將各種細菌進行分離，以得到只含有這一種細菌的純培養(yǎng)。分離培養(yǎng)細菌的方法有多種，平板劃線法即是其中之一。該法主要是借劃線而將混雜的細菌在瓊脂平板表面分散開，使單個細菌能固定在某一點，生長繁殖后形成單個的細菌集團（即菌落

2、）以達到分離純種的目的。 當(dāng)細菌分離成純種后，常需要接種到有關(guān)的培養(yǎng)基中，以測試其各種生物學(xué)性狀。一般可用斜面、液體和半固體培養(yǎng)基來檢驗細菌的培養(yǎng)特征，因此接種方法可相應(yīng)的分為三種。 斜面培養(yǎng)基接種法 常用于細菌的大量繁殖，保存菌種，或觀察其某些生化特性。瓊脂斜面、尿素培養(yǎng)基、雙糖鐵培養(yǎng)基、檸檬酸鹽培養(yǎng)基等具有斜面外形的固體培養(yǎng)基均可用此法接種。 液體培養(yǎng)基接種法 可用于觀察細菌不同的生長狀況，有的呈均勻混濁，有的呈沉淀生長，還有的在液體表面形成菌膜；另外還可以供測定細菌生化特性之用。凡是肉湯、葡萄糖蛋白胨水以及各種單糖發(fā)酵管等液體培養(yǎng)基均用此法接種。穿刺接種法 常用于半固體瓊脂培養(yǎng)基、醋酸鉛

3、培養(yǎng)基、雙糖鐵培養(yǎng)基等的接種，前者用于測定細菌的動力，后二者則用于觀察細菌的生化反應(yīng)。目的要求 1學(xué)習(xí)和掌握細菌分離和培養(yǎng)的各種基本技術(shù)。 2進一步熟悉和掌握微生物的無菌操作技術(shù)。材料 1菌種和培養(yǎng)基平板劃線接種法斜面培養(yǎng)基接種法液體培養(yǎng)基接種法穿刺接種法菌種葡萄球菌和大腸桿菌的混合菌液大腸桿菌培養(yǎng)物大腸桿菌培養(yǎng)物枯草桿菌培養(yǎng)物變形桿菌培養(yǎng)物痢疾桿菌培養(yǎng)物培養(yǎng)基*普通瓊脂平板瓊脂斜面培養(yǎng)基普通肉湯培養(yǎng)基半固體瓊脂培養(yǎng)基 *各培養(yǎng)基的組成及配置方法參見書后附錄。 2接種環(huán)、接種針、酒精燈、記號筆、試管架等方法1 平板劃線接種法 （1）標(biāo)記 在培養(yǎng)皿底玻璃上，用記號筆注明接種的菌名、接種者姓名、班

4、級、日期等。 （2）滅菌接種環(huán) 點燃酒精燈，右手以持筆式握持接種環(huán)并放置火焰中燒灼滅菌，如圖1-1，先將接種環(huán)的接種絲部分置于火焰中，待金屬絲燒紅并蔓延至金屬環(huán)端，再直接燒灼金屬環(huán)直至燒紅，然后由金屬環(huán)至金屬桿方向快速通過火焰，隨后再反方向通過火焰，如此23次。圖1-1 接種環(huán)的滅菌操作然后將接種環(huán)移開火焰，待其冷卻。注意，接種環(huán)不能距離火焰過遠，一般應(yīng)在距火焰10cm范圍之內(nèi)（視此范圍為無菌環(huán)境）；滅菌后的接種環(huán)不能再碰及他物。 （3）取菌種 左手持裝有葡萄球菌和大腸桿菌混合菌液的試管，用持有接種環(huán)的右手手掌及小指拔取試管棉塞，將試管管口迅速通過火焰23次進行滅菌。 將已滅菌且已冷卻的接種環(huán)

5、伸入菌種管中，取一接種環(huán)的混合菌液，然后退出菌種管。將菌種管管口再次通過火焰23次滅菌，塞好棉塞，放至原來的位置。 （4）分離劃線接種細菌（見圖3-1） 左手持瓊脂平板培養(yǎng)基（將皿蓋反放在桌上酒精燈附近），盡量使之直立以免空氣中的細菌落入其中，并靠近火焰。右手持接種環(huán)在瓊脂平板上端來回劃線，涂成一細菌薄膜（約占平板面的1/10），視為一區(qū)。劃線時使接種環(huán)與平板面成3040度角，以腕力在平板表面行輕而快地來回滑動動作。切記，接種環(huán)不應(yīng)嵌進培養(yǎng)基內(nèi)，避免將瓊脂表面戳破。 旋轉(zhuǎn)瓊脂平板90度。燒灼接種環(huán)，以殺滅環(huán)上的殘留細菌，將接種環(huán)觸及培養(yǎng)基表面以使其冷卻。滅菌接種環(huán)通過薄膜處作連續(xù)平行劃線（約占

6、平板1/5），此為二區(qū)。注意接種環(huán)只通過薄膜12次，以獲取薄膜處少量的細菌。 旋轉(zhuǎn)瓊脂平板90度。燒灼接種環(huán)滅菌并使之冷卻。將滅菌接種環(huán)接二區(qū)連續(xù)平行劃線（約占平板1/4），此為三區(qū)。接種環(huán)只通過二區(qū)12次以獲得少量細菌。 旋轉(zhuǎn)瓊脂平板90度。接種環(huán)不必再滅菌，接三區(qū)連續(xù)平行劃線，劃滿平板其余部分，此為四區(qū)。 注意各區(qū)接種線間盡量互不交接，以達到細菌逐漸稀釋的目的。 （5）劃線完畢，將瓊脂平板放進皿蓋，將培養(yǎng)皿倒放（這樣可避免培養(yǎng)過程中凝結(jié)水自皿蓋滴下，沖散菌落），送進37溫箱培養(yǎng)。 （6）培養(yǎng)1824小時后將培養(yǎng)皿取出。觀察瓊脂平板表面生長的各種菌落，注意其大小、形狀、邊緣、表面結(jié)構(gòu)、透明度

7、、顏色等性狀。 1 2 3 4圖1-2 四區(qū)劃線接種法2 斜面培養(yǎng)基接種法 （1）用記號筆在待接種的培養(yǎng)管上寫明標(biāo)記。 （2）點燃酒精燈。 （3）左手拇指、食指、中指及無名指分別握持菌種與待接種的培養(yǎng)基管，使菌種管位于左側(cè)，斜面部均應(yīng)向上，勿成水平，以免凝結(jié)水浸潤培養(yǎng)基表面，甚至沾濕棉塞。 （4）以右手拇指和食指捏持轉(zhuǎn)動兩管棉塞，以便在接種時易于拔取。 （5）右手持接種環(huán)，在火焰上燒灼滅菌。 （6）以右手手掌及小指，小指及無名指分別拔取并夾持兩管棉塞，將兩管管口滅菌。 （7）將已滅菌且已冷卻的接種環(huán)伸入菌種管內(nèi)，從斜面上輕輕挑取少量菌苔退出菌種管（注意，一要防止取菌過多；二要防止弄破培養(yǎng)基表面

8、）。再伸進待接種的培養(yǎng)基管，如圖3-2進行斜面培養(yǎng)基接種，從斜面底部輕輕向頂端彎曲劃線，不要觸破培養(yǎng)基表面。沾有細菌的接種環(huán)進出試管時不應(yīng)觸及到試管內(nèi)壁和試管口。 （8）接種完畢，滅菌兩試管的管口，塞好棉塞，并放至原來的位置上。重新燒灼接種環(huán)，滅菌后放回試管架上。接種好的試管放37溫箱培養(yǎng)，1824小時后觀察生長情況。圖1-3 斜面培養(yǎng)基接種法3 液體培養(yǎng)基接種法 （1）用記號筆在待接種培養(yǎng)基上寫明標(biāo)記。 （2）如斜面培養(yǎng)基的接種方法一樣，握持菌種管及待接種的肉湯管。（3）接種環(huán)滅菌冷卻后，伸入菌種管取少量細菌再伸入肉湯管內(nèi)，在接近液面管壁處輕輕研磨，蘸取少量肉湯調(diào)和，使菌混于肉湯中（見圖3-

9、3）。塞好試管棉塞后，搖動液體，使細菌在液體中均勻分布。 （4）接種完畢，將接種環(huán)滅菌后放回試管架上。肉湯管放37溫箱培養(yǎng)，1824小時后觀察生長情況。圖1-4 液體培養(yǎng)基接種法4 穿刺接種法（1）用記號筆在待接種培養(yǎng)基上寫明標(biāo)記。（2）如斜面培養(yǎng)基接種法，握持菌種管及待接種的半固體瓊脂培養(yǎng)基。（3）右手持接種針，滅菌冷卻后，以針挑取菌苔，如圖3-4垂直刺入半固體瓊脂培養(yǎng)基的中心，刺達近管底部，但不必及于管底，然后循原路退出。（4）接種完畢，接種針重行滅菌后放至試管架上，塞好棉塞，37培養(yǎng)1824小時后取出觀察細菌的生長情況。圖1-5 穿刺接種法 實驗二 細菌的形態(tài)與結(jié)構(gòu) 各種細菌在一定環(huán)境條

10、件下，有相對恒定的形態(tài)與結(jié)構(gòu)。了解細菌的形態(tài)與結(jié)構(gòu)是鑒別細菌的重要方法之一。此外，對分析細菌的致病性和免疫發(fā)生的機理等方面，也有一定的意義。目的要求 觀察細菌的基本形態(tài)和一些細菌的特殊結(jié)構(gòu)。一、細菌的基本形態(tài) 細菌在適宜的生長繁殖條件下所顯示的形態(tài)可分為三大類：球菌、桿菌和螺形菌。不同的細菌又可表現(xiàn)出不同的排列方式，在細菌的鑒別上有一定的參考價值。材料1 球菌示教片 葡萄球菌、鏈球菌和肺炎鏈球菌2 桿菌示教片 大腸桿菌和炭疽桿菌3 螺形菌示教片 霍亂弧菌方法1 使用顯微鏡的油鏡觀察球菌、桿菌和螺形菌的示教片。2 注意各菌的形狀、大小、排列方式等特點（注意：細菌染成什么顏色不是本實驗要求，染色方

11、法將在以后實驗中實際操作）。結(jié)果記錄實驗結(jié)果葡萄球菌鏈球菌肺炎鏈球菌大腸桿菌炭疽桿菌霍亂弧菌細菌形狀細菌排列方式二、細菌的特殊結(jié)構(gòu) 細菌的特殊結(jié)構(gòu)僅為某些細菌所具有，而且特殊結(jié)構(gòu)的形成受到一定條件的限制。雖然特殊結(jié)構(gòu)不是細菌生存所必須的，但他們的存在將賦予細菌一定的功能，在致病性、抗原性、以及對細菌的鑒別上都有一定的意義。材料1 破傷風(fēng)梭菌示教片（示芽胞）2 肺炎鏈球菌示教片（示莢膜）3 變形桿菌示教片（示鞭毛）方法1 使用顯微鏡的油鏡，觀察細菌的芽胞、莢膜和鞭毛的示教片。2 注意芽胞在菌體上的位置和大??；莢膜的大小及其與菌體的關(guān)系；注意鞭毛形態(tài)、數(shù)量及其位置。將所觀察到的細菌特殊結(jié)構(gòu)的形態(tài)特

12、點記錄于下表中。結(jié)果記錄 細菌破傷風(fēng)梭菌肺炎鏈球菌變形桿菌特殊結(jié)構(gòu)實驗三 革蘭染色法 活的細菌為無色半透明狀，在普通光學(xué)顯微鏡下不易觀察清晰。若用染色的方法可使菌體表面及內(nèi)部結(jié)構(gòu)著色與背景形成鮮明對比，可在顯微鏡下清晰地觀察其形態(tài)。 細菌的染色方法很多，其中最為廣泛使用的一種鑒別染色法是由丹麥醫(yī)生Christian Gram于1884年創(chuàng)建的革蘭（Gram）染色法。利用此法可將細菌分為革蘭陽性細菌和革蘭陰性細菌兩大類。革蘭陽性菌因細胞壁中含有大量的肽聚糖和磷壁酸，可保留結(jié)晶紫與碘的復(fù)合物，并不被酒精脫色而顯示為藍紫色。革蘭陰性菌因其細胞壁中肽聚糖含量少，脂類物質(zhì)含量高而被酒精脫色，經(jīng)復(fù)染呈紅色

13、。目的要求 了解革蘭染色的原理，掌握革蘭染色的方法。材料1 菌種 葡萄球菌、大腸桿菌培養(yǎng)物2 染液 結(jié)晶紫染液、盧戈（Rugol）碘液、95%酒精和沙黃染液3 其他 玻片、接種環(huán)、酒精燈、無菌生理鹽水等方法 1涂片 取潔凈玻片1張，作好標(biāo)記后置實驗臺上。 點燃酒精燈，右手以持筆式握持接種環(huán)并放置火焰中燒灼滅菌。用滅菌的接種環(huán)取無菌生理鹽水2環(huán)，分別置于玻片左右兩處。然后左手持培養(yǎng)物瓊脂平板；右手仍以持筆式將接種環(huán)再次放在火焰上滅菌，待接種環(huán)冷卻后，挑取單一金黃色葡萄球菌菌落適量培養(yǎng)物，將培養(yǎng)物瓊脂平板放回皿蓋。然后將挑取的細菌混合于其中一處的鹽水中，涂抹均勻使成一層薄膜（若檢材是液體，則不必加

14、鹽水），薄膜涂抹的面積約1.5×2.0cm2。按上法制備大腸桿菌涂膜。 2于室溫中自然干燥。 3固定 涂片在酒精燈火焰上快速通過34次以固定細菌，并使其不易從玻片上脫落。注意不要將涂片直接放在火焰上烤，以免破壞細菌結(jié)構(gòu)。4 染色 （1）初染 在涂膜上滴加結(jié)晶紫染液12滴，染1分鐘，水沖洗，并輕輕傾去玻片上的積水。 （2）媒染 加盧戈碘液12滴，染1分鐘，水沖洗。將表面積水甩干。 （3）脫色 用95%酒精數(shù)滴滴于玻片上，頻頻搖晃以脫色，半分鐘左右，立即用水沖洗（若涂膜較厚，可延長脫色時間；必須隨時觀察，以免脫色過度）。 （4）復(fù)染 最后滴加沙黃染液12滴，復(fù)染1分鐘后用水沖洗。最后用吸

15、水紙吸干。 5用顯微鏡的油鏡觀察染色結(jié)果，將實驗結(jié)果記錄于下表中。結(jié)果記錄 細菌染色性形態(tài)排列方式葡萄球菌大腸桿菌實驗四 抗酸染色法 結(jié)核分枝桿菌是引起結(jié)核病的病原體，菌體細長略彎曲，有分枝生長趨勢，因其細胞壁含有大量脂質(zhì)，一般不易著色，若經(jīng)加溫或延長染色時間或提高染液濃度而著色后能抵抗鹽酸酒精的脫色，故又稱抗酸桿菌。目的要求 學(xué)習(xí)抗酸染色方法。材料 1開放型肺結(jié)核病人痰標(biāo)本涂片和枯草桿菌涂片 2抗酸染色劑（石炭酸復(fù)紅染液、3鹽酸酒精、堿性美藍染液），載玻片方法 1. 染色在已固定的涂片上滴加石炭酸復(fù)紅液數(shù)滴，染色810分鐘，水沖洗。滴加3鹽酸酒精脫色，至復(fù)紅的顏色不再繼續(xù)脫下為止，時間為半分

16、鐘至1分鐘，水沖洗。滴加堿性美藍液23滴，1分鐘后水沖洗。用吸水紙把玻片吸干，油鏡檢查。 2. 抗酸染色標(biāo)本鏡檢 結(jié)核桿菌為抗酸染色陽性，在藍色背景下可見染成紅色的細長或略帶彎曲的桿菌，有分枝生長趨向，有時菌體內(nèi)可含濃染顆粒，呈念珠狀。Experiment 1 Handling and Examining CulturesIntroduction: To obtain information about bacteria biologic characteristics, it is necessary to observe microorganisms in culture. If we a

17、re to cultivate them successfully in the laboratory, we must provide them with suitable nutrients, such as protein components, carbohydrates, minerals, vitamins, and moisture in the right composition. This mixture is called a culture medium. It may be prepared in liquid form, as a broth, or solidifi

18、ed with agar. Agar medium may be used in tubes as a solid column or as slants. They are also commonly used in Petri dishes or plates. Solid medium is essential for the isolation and separation of bacteria growing together in a specimen. If a mixture of bacteria is spread across the surface of a plat

19、ed agar medium, individual organisms will multiply at individual sites until a visible aggregate called a colony forms. One colony of a single species can then be separated from the rest and transferred to another medium, where it will grow as a pure culture, and can be studied as such. The appearan

20、ce of colony can be very distinctive for individual species, such as color, density, consistency, surface texture, shape, and size of colonies. They can provide clues for the identification of an organism, although final identification can not be made by morphology alone. In liquid medium, some bact

21、eria may grow diffusely, producing uniform clouding. Others may look very granular in broth. Observation of such features can also be helpful in recognizing types of organisms. There is another kind of culture medium called semisolid medium. It is used to detect motility of bacteria. Non-motile bact

22、eria will grow only along the line of inoculation, whereas motile bacteria will migrate throughout the medium. In this experiment, you will learn how to make aseptic transfers of pure culture and to examine them for important gross features.Materials:1.Culture Medium and Bacterial Strains:Culture Me

23、diumBacteria StrainsPlatea mixture of S.aureus & E.coliLiquid MediumE.coli, B.subtitisSlantE.coliSemisolid MediumS.dysenteriae, Proteus2.Inoculating loop, Inoculating needle, Alcohol lamp, Matches, Marking pencil.Methods: A. Liquid Medium Inoculation: Procedures:1. Take up the loop by the handle

24、 and hold it as holding a pencil, loop down. Put the wire in the flame of the alcohol lamp (see Fig.1-1) until it turns red. Remove the loop from the flame and hold it until it becomes cool (Do not put it down or touch it to anything).Fig.1-1 Flaming the loop 2. Pick up the slant culture of E. coli

25、( or B. Subtilis) with your left hand, and use the little finger of the loop hand to remove the cotton plug off the culture tube. Keep your little finger curled around this cotton plug. Note: Dont place it on the table.3. Pass the neck of the open tube rapidly through the flame two or three times. T

26、his flaming sterilizes the air in and immediately around the mouth of the tube.4. Insert the loop into the open tube (holding both horizontally). Touch the loop to the growth on the slant and remove a loopful of culture. Note: Don't dig the loop into the agar. (see Fig. 1-2) Fig. 1-2. Get some c

27、ulture out of a slant. Note: the tube is held horizontally. 5. Withdraw the loop slowly and steadily, being careful not to touch it to the mouth of the tube. Keep it steadily, and do not touch anything while you replace the tube closure. Put the tube back in the rack. 6. Use the other hand to pick u

28、p a tube of sterile nutrient broth, remove the tube closure. Flame the neck of the tube; insert the loop into the tube and into the broth. Gently rub the loop against the wall of the tube. 7. Withdraw the loop, flame the tube neck, replace the closure, put the tube back in the rack. Then carefully f

29、lame the loop, holding it first in the coolest part of the flame (yellow), then in the hot blue cone until it glows. When the wire becomes cool, the loop can be placed on the bench top. 8. Label the tube you have just inoculated with your name, the name of the microorganism, and the date.9. Incubate

30、 the tubes overnight at 37. B. Slant Inoculation: Procedures: 1. Flame the loop. 2. Pick up the slant culture of E. coli, open it, flame, and take some growth with the sterile loop. 3. Pick up a nutrient agar slant. Open and flame it as above. 4. Introduce the loop into the tube. Streak the surface

31、of the plate from bottom to top. Then withdraw the loop from the tube without touching its inner surface. 5. Flame, close, and replace the inoculated tube in the rack, then sterilize the loop as above. 6. Label the freshly inoculated tube and then incubate it overnight at 37. C. Semisolid Medium Ino

32、culation:Procedures: 1. Flame the inoculating needle. 2. Pick up the slant culture of S.dysenteriae (or Proteus), open and flame it, then take some growth with the sterile needle. 3. Pick up a tube. Open and flame it as above. 4. Stab the needle into the semisolid medium without touching the bottom

33、of the tube, then retrace the way back. 5. Flame, close and replace the inoculated tube in the rack.6. Label the tube and incubate it overnight at 37.D. Streak Plate: A pure culture contains a single kind of microorganism. The pure culture of bacteria alone can be widely used in the following studie

34、s: morphologic; cultural; physiological or biochemical; pathogenic; serologic; genetic; and bacteriophage typing. A pure culture can be obtained from mixture culture or clinical specimens by plating. In this experiment you will prepare a streak plate with four-area streaking pattern. (see Fig. 1-3)

35、1.With a sterile loop, transfer 2.Rotate the plate 90°. Streak a loopful of culture to a corner from the first quadrant into of the first quadrant. Spread the the second quadrant. This is culture over 1/10 of the plate streak area 2. It accounts for surface. This is streak area 1. 1/5 of the pl

36、ate. 3.Flame the loop and rotate the 4.Rotate the plate 90°again and plate 90°. Repeat the streaking continue the streaking from from the second quadrant into the streak area 3 into the rest area third quadrant. This is streak of the plate. This is streakarea 3. It occupies 1/4 of the area

37、 4. plate.Fig. 1-3 Method to obtain pure culture Procedures: 1. Label plate. 2. Streak the plate according to the steps above. 3. Incubate the plate overnight in an inverted position at 37.After incubation, isolated colonies should be seen in the last area of the streaked plate.Experiment 2 Observat

38、ion Morphology of BacteriaMaterials and Equipment:1.Smears: Basic shape of the Bacteria: 1) Cocci Staphylococci, Streptococci, Streptococcus pneumoniae 2) Bacilli Escherichia coli, Bacillus subtilis 3) Spirilla Vibrio choleraSpecial Structures of the bacteria 1) Capsule Streptococcus pneumoniae, Bac

39、illus anthrax 2) Spore (or Endospore) Clostridium tetani 3) Flagella Proteus2.Instrament:Light Microscope (oil-immersion lens)Experiment 3 Gram StainIntroduction: Gram stain has become one of the most useful tools of microbiology. In the diagnostic laboratory, it is used not only for the study of mi

40、croorganisms in cultures, but it is also often applied to smears made directly from clinical specimens. Direct, Gram-stained smears are read promptly to determine the relative number and morphology of bacteria in the specimen. This information is often of value to the physician in planning the patie

41、nt's treatment before culture results are available. It is also of value to the microbiologist, who can plan his/her culture procedures on the basis of his/her knowledge of the bacterial forms he/she has seen in the specimen. Purpose of this experiment is to learn the Gram Stain technique and to

42、 understand its value in the study of bacterial morphology.Gram Stain Reagents:ReagentFunctionCrystal violetPrimary stain: stains all bacteria blue to purple.Grams iodineMordant: enhances reaction between cell wall and primary stain.Ethyl alcohol oracetoneDecolorization: Gram-positive bacteria retai

43、n the primary stain because of the peptidoglycan and teichoic acid cross links. Gram-negative bacteria lose the primary stain because of the large amount of lipopolysaccharide in the cell wall. Safranin orcarbolfuchsinCounterstain: no effect on Gram-positive bacteria, stain Gram-negative bacteria pi

44、nk to red.Materials: 1. Plate culture of Staphylococcus aureus and Escherichia coli 2. Crystal violet solution 3. Iodine solution 4. Ethyl alcohol, 95% 5. Safranine solution 6. Normal saline 7. Slides and bibulous paper 8. Marking pencil and label paper 9. Alcohol lampProcedures: 1. Prepare a smear:

45、 Emulsify a colony in normal saline on a slide to make a thin layer of smear. Air dry and then pass the smear over flame for three or four times (Note: don't overheat it). On the underside of each slide make a penciled code mark so that you can identify the slide after staining. 2. Stain each sm

46、ear by the following procedures: 1) Overlay smear with crystal violet solution. Allow it to stand for one minute. 2) Gently wash with tap water. 3) Flood with iodine solution. Leave it for one minute. 4) Gently wash with tap water. 5) Decolorize with alcohol (95%) for about half a minute until no color being washed off. This is a most critical step. Be careful not to overdecolorize it, as many Gram-positive bacteria may lose the primary stain and