The variety of bacterial infections requires a clear identification of the pathogen and determination of its species. Microbiologists can determine the type of microorganism by its tinctorial properties - the susceptibility of the microbe to staining with various dyes. This method allows you to study the morphology of the pathogen. The tinctorial properties of bacteria are of great importance for practical and theoretical research in the field of microbiology.
Microbe research
In bacteriology, there are many methods for staining microorganisms. All of them are based on the tinctorial properties of bacteria. Staining allows you to determine their shape, structure, size, relative position. This allows us to solve the problems of systematizing the types of microorganisms of general biology and comparative microbiology.
Why paint them
Bacteria are practically transparent organisms, and without the use of staining, they are poorly visible for ordinary microscopy. You can use special types of microscopy (phase contrast, in a dark field) to study objects, but the simplest way is staining, after which the bacteria become visible in a conventional light microscope.
Sample preparation
Regardless of the applied staining technique, there are uniform rules for the preparation of the studied object. The following stages are mandatory:
- Sterile instruments make a smear on a glass slide.
- The sample is dried. This is done at room temperature or using ovens.
- This is followed by the fixation stage - microorganisms are attached to glass with special compositions.
- Staining itself - the sample is coated with a dye for a fixed period of time, after which it is washed off.
- Final drying - the sample is dried again.
The most common dyes
The most commonly used are dyes based on aniline with different acid values (pH). Most dyes are powders that are diluted in alcohol.
Dyes in which cations are coloring agents are called basic (pH greater than 7). With their help, microorganisms can be stained in red (fuchsin, safranin), violet (methyl violet, thionine), blue (methylene blue), green (malachite green), brown (chrysoidine) and black (inulin).
Dyes in which the coloring agents are anions are called acidic (pH less than 7). They will color the sample in red (eosin), yellow (picrin), or black (nigrosine).
There is a group of neutral dyes (for example, rhodamine B), where cations and anions act as coloring agents.
Culture dead or alive
Staining methods are divided into two groups in accordance with the life form of the test sample.
- Vital (intravital) staining. This method of studying the properties of microorganisms is used in the study of living tissues, which allows you to observe the processes of microbial activity. For such staining, dyes with low toxicity and high penetration are used.
- Postvital staining. This is the staining of dead or euthanized microorganisms. Thanks to the tinctorial properties of bacteria, microbiologists determine their structure. It is this staining that is used most widely.
Gram-positive and gram-negative
It is these characteristics of bacteria that can be found in the instructions for various medications. This method of studying the tinctorial properties of bacteria is based on the use of a gentian violet dye and fixation with iodine. This is the technique of Hans Christian Gram, a Danish doctor who proposed it in 1884. As a result of this staining, the bacteria are divided into two groups:
- Gram (+) - stained blue (staphylococci and streptococci).
- Gram (-) - are colored from pink to red (enterobacteria, salmonella, E. coli).
A different staining result is obtained due to the unequal tinctorial properties of the bacterial walls. The Gram stain method today is the main one in the diagnosis of some infectious diseases.
Other staining techniques
We give a description of several other methods that are widely used in bacteriology.
- Ziel-Nelson method - determines the acid resistance of bacteria. The pathogens of tuberculosis and mycobacteriosis are identified on it.
- Romanovsky-Giemsa technique - stains acidophilic (acetic and lactic) bacteria in red, and basophilic (spirochetes and protozoa) - in blue.
- Morozov's technique - stains bacteria in brown and makes their flagella visible.
You can see the debate
Staining fuchsin Tsilya allows you to see the spores of bacteria. Having a pink color after staining, they are clearly visible against the background of blue bacteria. This method is also a bacteriology tool and is of great practical importance.