Gram stain is important in microbiology. It is used to diagnose infectious diseases, identify the pathogenicity of bacteria. The method allows us to determine bacteria similar in shape and size, which belong to different species and genera.
Coloring features
Gram stain of bacteria is a complex method in which two types of color are used: primary and secondary. In the process of performing the manipulation, bleaching agents are used, which include alcohols and acids.
In microbiology, Gram stain is carried out by the triphenylmethane group of dyes. It includes gentian, methyl blue, crystal violet. Different types of bacteria give different colors, forming strong compounds with dyes.
Colors
In microbiology, Gram stain is of two types: gram-positive and gram-negative. In the first case, microorganisms give strong compounds with dyes and iodine. During the analysis, they do not discolour when exposed to alcohol, which is why they do not change their original purple hue.
Gram-negative colors are formed when exposed to iodine and are easily destroyed by the action of iodine. As a result, microbes become discolored, and then acquire a red tint.
Material preparation
According to the rules of microbiology, Gram stain requires preliminary preparation of materials. It must be spread with a thin layer on the surface of the glass. Then the smear is dried and after complete drying is fixed. In this case, the smear is fixed on the glass, which eliminates the washing off of bacteria with dyes. In addition, dead microorganisms stain better than living ones.
After preparing the material, a staining method is selected: physical or chemical. The first involves exposure to high temperature on the microbial cell. During the chemical method, various chemicals are used that cause coagulation of cytoplasmic proteins.
Prepare a set for Gram stain, slides with bacteria. They are taken with tweezers or gently with your fingers by the ribs with a smear up, then they are carried out a couple of times above the top of the burner flame. The process should take a couple of seconds.
The chemical fixation process involves the use of acetone, alcohol, special mixtures, Carnoy fluid. A slide with a dried smear is immersed in a fixing substance for fifteen minutes, then dried in air.
Dyeing
Gram smears are stained according to a specific algorithm.
- First, a basic dye is applied to a fixed smear for a couple of minutes. To avoid sedimentation, the procedure is performed using filter paper.
- The dye is removed (merged), the paper is removed. The smear is immersed in a Lugol solution for two minutes or in a gram gram iodine solution until the preparation is blackened.
- The smear is rinsed with an alcohol solution or acetone, pouring and draining it until the smear is discolored and the liquid becomes clean. It takes about 30 to 60 seconds.
- Glasses are thoroughly washed with distilled water.
To determine if there are gram-negative bacteria in the smear, additional staining with fuchsin or safranin is carried out. The drugs are applied for two minutes. At the end of the procedure, the glass is washed, dried.
results
All gram-positive microorganisms are stained in dark purple, and gram-negative in red. Spherical forms usually refer to gram-positive species, and crimped forms are negatively colored. Rod-shaped bacteria can be gram-negative and gram-positive.
To obtain the most reliable research results, it is recommended to use daily cultures, as well as freshly prepared solutions for staining.
The staining mechanism allows one to see and evaluate the physicochemical structural features of the cytoplasm, the cell walls of gram-positive and gram-negative bacteria. The former contain a lot of ribonucleic acid, proteins in the cytoplasm, and peptidoglycan in the cell walls. Because of this, during the treatment of smears with gentian violet and Lugol, a strong combination of iodine and dye ions forms in gram-positive bacteria.
When treated with alcohols, gram-positive microorganisms retain the dye, and gram-negative microorganisms discolor and stain with a red diluted fuchsin. Some bacteria are able to stain only at the growth stage.
Application
Gram stain is carried out when analyzing the type of microorganisms in smears from the vagina and saliva. This method allows you to determine the type of microorganisms in the feces, in the pleural, peritoneal fluid and not only. Any liquids that may contain bacteria are examined by this method. After staining, doctors select a treatment method, prescribing drugs that can affect gram-positive or gram-negative bacteria.