微生物基礎(chǔ)之革蘭氏染色
發(fā)布時間：
2022-12-27
作者：
微生物，本來的意思就是微小的生物（必須通過顯微鏡才能看到）。為了更好的了解微生物的多樣性，人們會從不同的技術(shù)角度對微生物進(jìn)行分類。由于分類是人為的，所以隨著技術(shù)的進(jìn)步，有些微生物的分類會有改變。比如2008 年，Jan Houseknecht 等人通過表型結(jié)合基因型的多相鑒定技術(shù)，確認(rèn)原來的黑曲霉（ATCC16404）為巴西曲霉(Aspergillus brasiliensis)。
上圖是現(xiàn)在主流的微生物的大體分類圖，其中以細(xì)菌的分類研究最為深入，以《伯杰氏細(xì)菌鑒定手冊》第九版收錄內(nèi)容作為細(xì)菌鑒定的金標(biāo)準(zhǔn)。而在我們?nèi)粘９ぷ髦?，用的最多的就是革蘭氏染色。1884年，一位醫(yī)生為了讓肺組織在顯微鏡下更清楚些而發(fā)明了革蘭氏染色。這也說明了臨床檢驗科醫(yī)生做革蘭氏染色做的好也是有源頭的，我們單位有同事就能在同一塊載玻片上同時染6-8個菌。革蘭氏染色主要分為以下四步：結(jié)晶紫初染-碘液媒染-酒精脫色-番紅復(fù)染。
針對初學(xué)者來說，混合染色是最好的考核方式，一般拿金黃色葡萄球菌（Gram+）和大腸埃希菌（Gram-）混合后進(jìn)行染色，按顏色區(qū)分程度評價染色結(jié)果，例圖如下。
但是，進(jìn)入工作崗位后才發(fā)現(xiàn)原來革蘭氏染色技術(shù)并沒有在學(xué)校里用到的那么簡單。
首先，革蘭氏染色染色的對象是細(xì)菌的細(xì)胞壁。不是真菌或者芽孢，其他可能用到其他的染料。菌齡（細(xì)菌的培養(yǎng)時間）很重要，太嫩的細(xì)胞壁還沒有形成，太老的有可能又自融了。如果抑制性平板上的抑制劑剛好抑制的是細(xì)胞壁的話，那你革蘭氏染色也無效。
其次，革蘭氏染色只是分類學(xué)上人為的一個方法，并不是非黑即白（應(yīng)該是非紫即紅）的確切判斷。一些細(xì)菌在染色之后會表現(xiàn)為紫色和紅色的混合，讓人肉眼無法分辨。而且還有一些細(xì)菌在生長過程中細(xì)胞壁的肽聚糖層厚度會逐漸變薄，例如芽孢桿菌、丁酸弧菌和梭菌，導(dǎo)致生長后期會革蘭氏染色結(jié)果發(fā)生改變（由陽性變成陰性）。另外還有一些細(xì)菌，例如分支桿菌、結(jié)核菌和麻風(fēng)桿菌，革蘭氏染色的結(jié)果不可預(yù)見。
下圖就是本人在實際染色中遇到的例子。染色結(jié)果是紅色，屬于革蘭氏陰性。但是在圖中圓圈的位置，你們看到了什么？沒錯，是沒有染上色的芽孢。那有革蘭氏陰性的芽孢桿菌嗎？那只是小概率的存在。主要原因還是菌齡太老了，都產(chǎn)生芽孢了還怎么染色。
總結(jié)起來，在實際操作中，一定注意需要染色的細(xì)菌最好是在基礎(chǔ)平板上(沒有選擇性)的單菌落，最好是新鮮培養(yǎng)物（在生長曲線的指數(shù)期），涂菌要薄。革蘭氏染色結(jié)果幾乎是所有細(xì)菌鑒定技術(shù)的應(yīng)用基礎(chǔ)：生化鑒定卡的選擇，質(zhì)譜涂菌的基質(zhì)選擇等等。要鑒定，請先染好色！
作者|石決明
Microbial foundation - gram staining
Microbes originally mean tiny creatures (which must be seen through a microscope). In order to better understand the diversity of microorganisms, people will classify microorganisms from different technical perspectives. As the classification is artificial, with the progress of technology, the classification of some microbes will change. For example, in 2008, Jan Houseknecht et al. identified Aspergillus brasiliensis as the original Aspergillus Niger (ATCC16404) by phenotypic binding genotyping.
Above is a general taxonomic map of the most prevalent microorganisms, in which bacterial taxonomy is the most in-depth study, with the contents of the ninth edition of the Berger's Bacterial Identification Manual as the gold standard for bacterial identification. And in our daily work, the most used is gram stain. In 1884, a doctor invented Gram staining in order to make lung tissue more clear under microscope. This also shows that clinical laboratory doctors do well in Gram's staining is also a source of our unit colleagues can be on the same slide at the same time infected with 6-8 bacteria. Gram staining is mainly divided into four steps: crystal violet primary dyeing - iodine solution mordant - alcohol decolorization - Plover red.
For beginners, mixed staining is the best way to test, generally take Staphylococcus aureus (Gram +) and Escherichia coli (Gram -) after mixed staining, according to the degree of color differentiation evaluation of staining results, the example is as follows.
However, after entering the job, it was discovered that the original Gram's dyeing technology was not as simple as it was used in school.
First, the object of Gram staining is the cell wall of bacteria. Other fungi may not be used as fungi or spores. Bacterial age is important, too tender cell walls have not yet formed, too old may melt again. If the inhibitor on the inhibitory plate just inhibits the cell wall, then your Gram's staining is also ineffective.
Secondly, Gram's staining is only a taxonomically anthropogenic method, not a precise judgment of whether black or white (or purple or red). Some bacteria, after staining, show a mixture of purple and red, which can not be distinguished from the naked eye. In addition, some bacteria, such as Bacillus, Vibrio butyricum and Clostridium, tend to have thinner peptidoglycan layers on their cell walls during growth, leading to changes in the results of Gram staining (from positive to negative). There are also some bacteria, such as Mycobacterium, tuberculosis and leprosy bacilli, whose Gram staining results are unpredictable.
The following is an example I encountered in actual dyeing. The result is red and belongs to gram negative. But in the picture, what do you see in the position of the circle? Yes, there is no coloring spore. Do they have gram negative bacillus? It's just a small probability. The main reason is that the age of the bacteria is too old to produce spores and how to dye them.
To sum up, in practice, it is important to note that the bacteria that need to be stained should be single colonies on the basic plate (without selectivity), fresh cultures (in the exponential period of the growth curve), and the bacteria should be thin. Gram staining results are the basis of almost all bacterial identification techniques: the selection of biochemical identification cards, matrix selection of mass spectrometry bacteria and so on. For identification, please dye it first.