微生物基础之革兰氏染色

发布时间:

2022-12-27

作者:


微生物,本来的意思就是微小的生物(必须通过显微镜才能看到)。为了更好的了解微生物的多样性,人们会从不同的技术角度对微生物进行分类。由于分类是人为的,所以随着技术的进步,有些微生物的分类会有改变。比如2008 年,Jan Houseknecht 等人通过表型结合基因型的多相鉴定技术,确认原来的黑曲霉(ATCC16404)为巴西曲霉(Aspergillus brasiliensis)。
 
上图是现在主流的微生物的大体分类图,其中以细菌的分类研究最为深入,以《伯杰氏细菌鉴定手册》第九版收录内容作为细菌鉴定的金标准。而在我们日常工作中,用的最多的就是革兰氏染色。1884年,一位医生为了让肺组织在显微镜下更清楚些而发明了革兰氏染色。这也说明了临床检验科医生做革兰氏染色做的好也是有源头的,我们单位有同事就能在同一块载玻片上同时染6-8个菌。革兰氏染色主要分为以下四步:结晶紫初染-碘液媒染-酒精脱色-番红复染。
 
针对初学者来说,混合染色是最好的考核方式,一般拿金黄色葡萄球菌(Gram+)和大肠埃希菌(Gram-)混合后进行染色,按颜色区分程度评价染色结果,例图如下。
金黄色葡萄球菌(Gram+)和大肠埃希菌(Gram-)混合后进行染色图
但是,进入工作岗位后才发现原来革兰氏染色技术并没有在学校里用到的那么简单。
 
首先,革兰氏染色染色的对象是细菌的细胞壁。不是真菌或者芽孢,其他可能用到其他的染料。菌龄(细菌的培养时间)很重要,太嫩的细胞壁还没有形成,太老的有可能又自融了。如果抑制性平板上的抑制剂刚好抑制的是细胞壁的话,那你革兰氏染色也无效。
 
其次,革兰氏染色只是分类学上人为的一个方法,并不是非黑即白(应该是非紫即红)的确切判断。一些细菌在染色之后会表现为紫色和红色的混合,让人肉眼无法分辨。而且还有一些细菌在生长过程中细胞壁的肽聚糖层厚度会逐渐变薄,例如芽孢杆菌、丁酸弧菌和梭菌,导致生长后期会革兰氏染色结果发生改变(由阳性变成阴性)。另外还有一些细菌,例如分支杆菌、结核菌和麻风杆菌,革兰氏染色的结果不可预见。
 
下图就是本人在实际染色中遇到的例子。染色结果是红色,属于革兰氏阴性。但是在图中圆圈的位置,你们看到了什么?没错,是没有染上色的芽孢。那有革兰氏阴性的芽孢杆菌吗?那只是小概率的存在。主要原因还是菌龄太老了,都产生芽孢了还怎么染色。
染色结果是红色,属于革兰氏阴性图
总结起来,在实际操作中,一定注意需要染色的细菌最好是在基础平板上(没有选择性)的单菌落,最好是新鲜培养物(在生长曲线的指数期),涂菌要薄。革兰氏染色结果几乎是所有细菌鉴定技术的应用基础:生化鉴定卡的选择,质谱涂菌的基质选择等等。要鉴定,请先染好色!
 
作者|石决明

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.