The variety of bacterial infections requires a clear identification of the pathogen and the definition of its species. To determine the type of microorganism, microbiologists are helped by its tinctorial properties - the susceptibility of the microbe to staining with various dyes. This method allows you to explore the morphology of the pathogen. The tinctorial properties of bacteria are of great importance for practical and theoretical research in the field of microbiology.
Microbial 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 solving the problems of systematizing the types of microorganisms of general biology and comparative microbiology.
Why paint them
Bacteria are practicallytransparent organisms, and without the use of staining, they are poorly visible for conventional microscopy. You can use special types of microscopy (phase contrast, dark field) to study objects, but the easiest way is to stain, after which the bacteria become visible in a conventional light microscope.
Sample preparation
Regardless of the staining technique used, there are uniform rules for preparing the object under study. The following stages are mandatory:
- Sterile instruments make a smear on a glass slide.
- The sample is being dried. This is done at room temperature or using drying ovens.
- Followed by the fixation stage - microorganisms are attached to the glass with special compounds.
- Proper staining - the sample is covered with 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 dyes are 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). They can be used to stain microorganisms in red (magenta, safranin), violet (methyl violet, thionine), blue (methylene blue), green (malachite green), brown (chrysoidin) and black (indulin) colors.
Dyes, in which the coloring agents are anions, are called acidic (pH less than 7). They will stain the sample red (eosin), yellow (picrin), or black (nigrosin).
There is a group of neutral dyes (for example, rhodamine B), where both cations and anions act as coloring agents.
Culture dead or alive
Staining methods are divided into two groups according to the life form of the test specimen.
- Vital (lifetime) staining. This method of studying the properties of microorganisms is used in the study of living tissues, which makes it possible to observe the vital processes of microbes. For this staining, dyes with low toxicity and high penetrating power are used.
- Post-vital staining. This is the staining of dead or killed microorganisms. Thanks to the tinctorial properties of bacteria, microbiologists determine their structure. It is this staining that is most widely used.
Gram-positive and Gram-negative
It is these characteristics of bacteria that can be found in the instructions for various medicines. This method of studying the tinctorial properties of bacteria is based on the use of gentian violet dye and iodine fixation. This is the technique of Hans Christian Gram, a Danish physician who proposed it in 1884. As a result of this staining, bacteria are divided into two groups:
- Gram (+) - turn blue(staphylococci and streptococci).
- Gram (-) - stain pink to red (enterobacteria, salmonella, E. coli).
Different staining results are due to different tinctorial properties of bacterial walls. The Gram stain method is still the main one in the diagnosis of some infectious diseases.
Other staining techniques
Let's characterize a few more methods widely used in bacteriology.
- Ziehl-Nelson method - determines the acid resistance of bacteria. It identifies the causative agents of tuberculosis and mycobacteriosis.
- Romanovsky-Giemsa technique - stains acidophilic (acetic acid and lactic acid) bacteria red, and basophilic (spirochetes and protozoa) blue.
- Morozov's technique - stains bacteria brown and makes their flagella visible.
Spores can be seen
Tsiel's fuchcin staining allows you to see bacterial spores. Having a pink color after staining, they are clearly visible against the background of blue bacteria. This method is also a tool of bacteriology and is of great practical importance.
