GRAM STAIN: RATIONALE, MATERIALS, TECHNIQUE AND USES - BIOLOGY - 2024

The Gram stain is the simplest and most useful staining technique in diagnostic microbiology. This technique was created by the Danish doctor Hans Christian Gram in 1884, who managed to classify bacteria as Gram positive and Gram negative, 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.

Various smears stained with Gram stain. A. Gram positive cocci. B. Gram negative rods. C. Gram-positive, polymorphonuclear and mononuclear bacilli. D. Yeasts.

With this technique it is also possible to observe the shape of the microorganisms, that is, if they are cocci, bacilli, coccobacilli, pleomorphic, filamentous, among others. As well as its distribution in space: in a cluster, in a chain, isolated, in pairs, in tetrads, etc.

When a bacterial infection is suspected, most of the samples received should be smeared on a slide and Gram stained for microscopic examination.

The Gram report will guide the doctor on what type of microorganism may be the cause of the infection, before obtaining the final culture result.

In some cases the life of the patient is very compromised, therefore doctors urgently need the Gram report to place an empirical treatment, while they wait for the identification of the microorganism.

For example, if the Gram reveals that there are Gram positive cocci in the cerebrospinal fluid, the doctor will guide the initial therapy with antibiotics that eliminate this type of bacteria, according to the protocols established for this.

Once the final result arrives with the name of the isolated microorganism and its respective antibiogram, the doctor will evaluate whether or not to change the therapy. This decision will be made according to the study of susceptibility of the microorganism to the antibiotics he is receiving and the evolution of the patient.

Basis

This is a technique that has 4 fundamental steps: staining, fixation with the mordant, discoloration and counterstain. Therefore, this technique, in addition to coloring the bacteria, also allows them to be differentiated.

Crystal violet is the first colorant used. It has an affinity for peptidoglycan and will stain all the bacteria present purple, subsequently the lugol is placed, which acts as a mordant, that is, it will induce the formation of insoluble crystal violet-iodine complexes - ribonuclear proteins within the cell..

Gram positive bacteria, having a thick wall of peptidoglycan, form more complexes (crystal violet-iodine), therefore they retain the dye.

In addition, it also influences that the wall of Gram positive bacteria contains a greater amount of unsaturated acids, which show great affinity for oxidizing agents (Lugol).

Meanwhile, Gram negative bacteria have a thin layer of peptidoglycan, which causes the bacteria to form less complexes than Gram positive ones.

Then comes the discoloration step, where Gram positive and Gram negative bacteria behave differently.

Gram negative bacteria contain an outer membrane rich in lipopolysaccharides that is part of their cell wall. Fats are destroyed by contact with acetone alcohol, so the outer membrane becomes destabilized, releasing the violet crystal.

This is how it is then countered with safranin or basic fuchsin, turning red.

In the case of Gram positive bacteria, they resist fading because the bleach works by closing the pores, preventing the crystal violet / iodine complex from leaking out.

Therefore, the coloration with crystal violet remains stable, and there is no room for safranin or fuchsin. This is why these bacteria stain deep blue or purple.

materials

Gram's staining set consists 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% ----------- 20cc

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 staining bottle using filter paper.

The amount to be used daily is transferred to an amber dropper bottle.

Iodo-Lugol

Weigh and measure the indicated amount of each compound, as follows:

Iodine crystals ------------- 1gr

Potassium iodide ------------- 2gr

Distilled water ------------- 300 cc

Potassium iodide dissolves little by little in the water and then the iodine is added. The solution is shaved into an amber bottle.

The amount to be 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 dropper bottle.

This preparation provides a discoloration in moderate time 5-10 seconds and is the most recommended.

Beginners prefer to use only 95% ethyl alcohol, where fading is slower than 10 to 30 sec.

While the more experienced can use pure acetone, where discoloration occurs very quickly from 1 to 5 sec.

Contrast

Safranin Stock Solution

Safranina -------------– 2.5 gr

Ethyl alcohol 95% --------– 100 cc

After weighing the indicated amount of safranin, it is dissolved in 100 ml of 95% ethyl alcohol.

The working safranin solution is prepared from the stock solution.

To do this, measure 10 cc of the stock solution, add 90 cc of distilled water to make 100 ml.

It is recommended to transfer the amount to be used daily to an amber bottle with a dropper.

Microorganisms that stain weakly Gram-negative with the Gram-Hucker stain, such as certain anaerobes, Legionella sp, Campylobacter sp, and Brucella sp, can be stained much better using Kopeloff's modification of the Gram-Hucker stain, called Gram-Kopeloff stain.

This technique changes the safranin dye to basic fuchsin. With this modification it is possible to effectively color the aforementioned microorganisms.

Reagent storage

Prepared colorants should be stored at room temperature.

Preparation of the smear of the sample to be colored

A sample must contain at least 10 ⁵ microorganisms before observation of the microorganism in a smear is likely. 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

Gram of uncentrifuged urine

The urine is mixed and 10 µl is placed on a slide. The observation of at least one bacteria / Dip field indicates that there is an infection.

This means that the culture will have approximately more than 100,000 CFU / ml (10 ⁵ 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 liquid is sterile under normal conditions; observation of bacteria indicates infection.

Gram of respiratory samples

The sputum, bronchial or bronchoalveolar lavage Gram, although there may be a variety of microorganisms, will always guide the diagnosis, in addition to being useful 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 stool

It is not recommended to perform a Gram on this type of samples, since it has no diagnostic value.

-Gram of crops

They can be done 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 towards the periphery, to distribute the material evenly.

Let it dry spontaneously in the air. Once dry, the material is fixed to the sheet with heat. To do this, with the help of a tweezers, the sheet is passed 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 coloring bridge.

Solid crops

To perform a smear for Gram stain from a solid culture, proceed as follows:

Before choosing the colonies to be taken, the slide should 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 perform the Gram. Each colony will be taken with the platinum loop to dissolve in the saline solution previously placed on the slide.

Circular movements are made from the center towards the periphery, to homogeneously distribute the colony on the slide.

Let it dry spontaneously in the air. Once dry, the sheet is fixed with heat, as explained previously (by flaming the slide with the lighter), taking care not to burn the material.

This procedure must be done with each different type of colony. On a piece of paper, the order of what is observed should be noted, for example:

Colony 1: Beta-haemolytic yellow colony: Gram positive cocci were observed in clusters

Colony 2: Cream colored colony, without hemolysis: Gram negative coccobacilli were observed.

Each slide must be labeled to know what we are observing.

Technique

The Gram staining technique is extremely easy to perform and relatively inexpensive and cannot be missed in a microbiology laboratory.

It is done as follows:

1. Fix the smear with heat and place on the staining bridge.
2. Cover the slide completely with crystal violet for 1 minute.
3. Wash off with water Do not dry
4. Cover the sheet with lugol solution, leave to act for 1 minute. Wash off with water Do not dry.
5. Bleach for 5-10 seconds with gentle shaking 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 violet glass is removed. Do not exceed.
6. Wash off with water Do not dry.
7. Replace the slide on the staining bridge and cover for 30 sec with safranin (Gram-Hucker) or 1 min with basic fuchsin (Gram-Kopeloff).
8. Wash with water
9. Let it air dry spontaneously in an upright position.

Once dry, place 1 drop of immersion oil to observe it under the 100X objective in the light microscope.

Utility

This technique allows to distinguish the morphotintorial differences of most bacteria.

Yeasts are also distinguished by this coloration. They take the crystal violet, that is, they stain Gram positive.

On the other hand, spore-forming Gram-positive bacilli can be distinguished, in which a clear space is observed within the bacillus, where the endospore formed, although the spores do not stain well. Other techniques such as Shaeffer-Fulton are used to stain spores.

It should be noted that this staining is not used to color all types of bacteria, that is, there are cases in which the staining does not work.

In this case bacteria lacking a cell wall can be mentioned. For example: genus Mycoplasma, spheroplasts, ureaplasma, L-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 Rickettsia.

It is also ineffective in staining most spirochetal bacteria.

There are bacteria of the same genus that can be observed in the same sample as Gram positive and Gram negative. When this happens it is called variable Gram stain, which can be due to alteration in nutrients, temperature, pH or electrolyte concentration.

Common mistakes

Exaggerated bleaching

Exaggeration in the discoloration step can lead to the observation of false Gram negative microorganisms.

Not waiting long enough drying time to add the immersion oil

It can cause Gram positive bacteria to stain Gram negative (false Gram negative). This happens because in old cultures there are likely to be dead or spoiled bacteria and under these conditions the bacteria do not retain the crystal violet.

Use very old lugol solution:

Over time the lugol loses its properties and its color fades. If the already degenerated reagent is used, it will not fix the crystal violet well, therefore there is a possibility of obtaining a visualization of falsely Gram negative microorganisms.

Blue background

A properly discolored background will be red. A blue background indicates that the discoloration was insufficient.

References

1. Ryan KJ, Ray C. 2010. Sherris. Medical Microbiology, 6th edition McGraw-Hill, New York, USA
2. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. (5th ed.). Argentina, Editorial Panamericana SA
3. Forbes B, Sahm D, Weissfeld A. 2009. Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA
4. Casas-Rincón G. 1994. General Mycology. 2nd Ed. Central University of Venezuela, Library Editions. Venezuela Caracas.
5. "Gram stain." Wikipedia, The Free Encyclopedia. 4 Oct 2018, 23:40 UTC. 9 Dec 2018, 17:11. Taken from es.wikipedia.org.
6. González M, González N. 2011. Manual of Medical Microbiology. 2nd edition, Venezuela: Directorate of media and publications of the University of Carabobo.
7. López-Jácome L, Hernández-Durán M, Colín-Castro C, Ortega-Peña S, Cerón-González G, Franco-Cendejas F. Basic stains in the microbiology laboratory. Research in Disability. 2014; 3 (1): 10-18.