por What is Gram stain?
The gram stain it is the simplest and most useful staining technique in diagnostic microbiology to identify bacteria. This technique was created by the Danish physician Christian Gram in 1884, who classified bacteria as gram-positive (purple) and gram-negative (pink), according to the composition of the cell wall.
The technique underwent certain modifications by Hucker in 1921 to stabilize the reagents and improve the quality of the staining, which is why the Gram stain is also known as Gram-Hucker.
With this technique it is also possible to observe the shape of the microorganisms, that is, if they are cocci, bacilli, cocobacilli, pleomorphic, filamentous, among others. As well as its distribution in space: in a cluster, in a chain, isolated, in pairs, in tetrads, etc.
Basis
It is a technique that presents 4 fundamental steps: staining, fixation with the mordant, bleaching and counterstaining. Therefore, in addition to coloring the bacteria, it also allows them to be differentiated.
Crystal violet is the first dye used. It has an affinity for peptidoglycan and will stain all the bacteria present purple. Lugol is then placed, which acts as a mordant, that is, it will induce the formation of insoluble crystal violet/iodine/ribonuclear protein complexes within the cell. .
Gram-positive bacteria, having a thick wall of peptidoglycan, form more complexes (crystal violet/iodine), therefore they will stain purple.
It also influences that the wall of gram-positive bacteria contain a greater amount of unsaturated acids, which show great affinity for oxidizing agents (Lugol).
Gram-negative bacteria possess a thin layer of peptidoglycan, which makes the bacteria less complex-forming than gram-positive ones.
Next comes the decolorization step, where gram-positive and gram-negative bacteria behave differently.
Gram-negative bacteria contain a lipopolysaccharide-rich outer membrane that forms part of their cell wall. Fats are destroyed by contact with acetone alcohol, so the outer membrane is destabilized, releasing crystal violet.
This is how it is later counter-stained with safranin or basic fuchsin, taking on the red color.
In the case of gram-positive bacteria, they resist bleaching because the bleach works by closing the pores, preventing the crystal violet/iodine complex from leaking out.
Therefore, the staining with crystal violet remains stable, and there is no room for safranin or fuchsin. Because of this, these bacteria stain deep blue or purple.
Materials
Gram’s coloring set is made up of:
– Violet glass.
–Lugol.
– Acetone alcohol.
– Safranin or basic fuchsin.
Preparation of dyes and reagents
crystal violet solution
Solution to:
Violet crystal ————————————- 2 gr
Ethyl alcohol 95% ——————————— 20 cc
Solution B:
Ammonium oxalate ——————————— 0.8 gr
Distilled water————————————– 80 cc
For the final preparation of crystal violet, solution A must be diluted 1:10 with distilled water and mixed with 4 parts of solution B. The mixture is stored for 24 hours before use. Filter into an amber stain bottle using filter paper.
The amount used daily is transferred to an amber bottle with a dropper.
Iodo-Lugol
Weigh and measure the indicated amount of each compound, as follows:
Iodine crystals ————————————- 1 gr
Potassium iodide ———————————— 2 gr
Distilled water ————————————— 300 cc
The potassium iodide is dissolved little by little in the water and later the iodine is added. Transfer the solution to an amber bottle.
The amount used daily is transferred to a smaller amber bottle with a dropper.
bleaching
95% ethyl alcohol ———————————– 50 ml
Acetone ————————————————– 50 ml
It is prepared in equal parts. Cover well, as it tends to evaporate.
Place in a bottle with a dropper.
This preparation provides bleaching in moderate time 5-10 seconds, and is the most recommended.
Beginners prefer to use only 95% ethyl alcohol, where the discoloration is slower, 10-30 sec.
While the more experienced can use pure acetone, where discoloration occurs very quickly from 1 to 5 sec.
Contrast
Safranin stock solution
Safranin ————————————– 2.5 gr
Ethyl alcohol 95% ————————– 100 cc
After weighing the indicated amount of safranin, it is dissolved in 100 cc of 95% ethyl alcohol.
The working safranin solution is prepared from the stock solution.
To do this, measure 10 cc of the mother solution, add 90 cc of distilled water to complete 100 ml.
It is recommended to transfer the amount that is used daily to an amber bottle with a dropper.
Organisms that stain weakly Gram-negative with the Gram-Hucker stain, such as certain anaerobes, Legionella sp., Campylobacter sp. and Brucella sp.can stain much better using Kopeloff’s modification of the Gram-Hucker stain, called the Gram-Kopeloff stain.
This technique changes the safranin dye to basic fuchsin. With this modification, it is possible to effectively stain the aforementioned microorganisms.
Reagent storage
Prepared stains should be stored at room temperature.
Preparation of the smear of the sample to be colored
A sample must contain at least 105 organisms before they are likely to be seen on a smear. The smears can be made from the direct sample or from cultures in solid or liquid media.
The smears should be uniform, well distributed and not too thick, for a better visualization of the structures present.
Gram of direct samples
Uncentrifuged urine gram
The urine is mixed and 10 µl is placed on a glass slide. The observation of at least one bacterium/Immersion field indicates that an infection exists.
This means that the culture will have approximately more than 100,000 CFU/ml (105 CFU/mL) of urine in 85% of the cases.
This method is not useful for colony counts below 100,000 CFU.
CSF gram
The CSF should be centrifuged, the supernatant removed, and the pellet spread on a slide. This fluid is sterile under normal conditions. Observation of bacteria indicates infection.
Gram of respiratory samples
Sputum Gram, bronchial or bronchoalveolar lavage, although there may be a variety of microorganisms, will always guide the diagnosis, as well as being useful for the type of cells observed.
In the case of sputum, the smear should be prepared with the most purulent portions of the sample.
gram of feces
It is not recommended to carry out Gram tests on this type of sample, since it has no diagnostic value.
crop gram
They can be performed in two ways, one from liquid cultures and the other from solid cultures.
liquid cultures
From liquid cultures it is extremely simple: several roasts of the cloudy broth are taken under the burner and placed on a clean and dry slide, making circular movements from the center to the periphery, to distribute the material evenly.
Let it dry spontaneously in the air. Once dry, the material is heat-fixed to the sheet. To do this, with the help of tweezers, pass the sheet 3 to 4 times through the flame of the Bunsen burner, taking care not to burn the material.
The sheet is allowed to cool and is placed on the staining bridge.
solid crops
To make a Gram stain smear from a solid culture, proceed as follows:
Before choosing the colonies to take, the slide must be prepared, placing approximately two drops of sterile physiological saline solution.
If the original culture plate contains several different types of colonies, an isolated colony of each will be chosen to carry out the Gram. Each colony will be taken with the platinum loop to dissolve it in the saline solution previously placed on the slide.
Circular movements are made from the center to the periphery, to distribute the colony evenly on the slide.
Let it dry spontaneously in the air. Once dry, the sheet is fixed with heat, as explained above (flaming the slide sheet with the lighter), taking care not to burn the material.
This procedure must be performed with each different type of colony. On a piece of paper, write down the order of what you observed, for example:
Colony 1: Yellow beta-hemolytic colony: Gram-positive cocci were observed in clusters.
Colony 2: Cream-colored colony, without hemolysis: Gram-negative coccobacilli were observed.
Each sheet must be labeled to know what we are observing.
Technique
The Gram staining technique is extremely simple to perform and relatively inexpensive, and cannot be missing in a microbiology laboratory.
It is done in the following way:
Heat fix the smear and place over the staining bridge.
The slide is completely covered with crystal violet for 1 minute.
Wash with water. do not dry
Cover the slide with Lugol’s solution, leave it to act for 1 minute. Wash with water. do not dry
Decolorize for 5-10 seconds with gentle agitation in alcohol-acetone. Or place the sheet in a vertical position and drop drops of the decolorizer on the surface until the excess of unretained crystal violet is washed away. Do not exceed.
Wash with water. do not dry
Replace the slide on the stain bridge and cover for 30 sec with safranin (Gram-Hucker) or 1 min with basic fuchsin (Gram-Kopeloff).
Wash with water.
Allow to dry spontaneously in the air in an upright position.
Once dry, place 1 drop of immersion oil to observe it under the 100X objective in the optical microscope.
Gram stain uses/applications
– This technique makes it possible to distinguish the morphotintorial differences of most bacteria.
– Yeasts are also distinguished by this coloration. They take crystal violet, that is, they stain Gram-positive.
– Spore-forming gram-positive bacilli can be distinguished, where a clear space is observed within the bacillus where the endospore was formed, although the spores do not stain well. Other techniques are used to stain spores, such as Shaeffer-Fulton.
– Helps determine the type of antibiotic to prepare.
It should be noted that this staining It does not work to stain all types of bacteria, that is, there are cases in which staining does not work.
In these cases, bacteria that lack a cell wall can be mentioned. For example: gender Mycoplasmaspheroplasts, ureaplasmaL forms and protoplasts.
It also stains very poorly bacteria with walls rich in mycolic acids, such as Mycobacteria, and intracellular bacteria, such as Chlamydias and Rickettsias.
It is equally ineffective for staining most…