Gram Staining – Principle, Procedure, Purpose and Examples

Gram’s method is also called gram staining and gram stain. Usually, it is used to differentiate between different types of bacteria i.e., gram-positive bacteria and gram-negative bacteria. Hans Christian Gram is a Danish bacteriologist who named this stain and developed this method. The basic function of this technique is to differentiate between bacteria on the basis of the chemical and physical properties of the cell walls. The difference of the cells can be identified by the cell wall as the gram-negative bacteria has a thin cell wall due to which the violet stain gets washed out with ethanol whereas the cell wall of gram-positive bacteria is thicker because of which violet stain stays out and give pink color to the bacteria. Normally fuchsine or safranin are used to stain the bacteria. There’s an iodine solution i.e., Lugol’s iodine which is poured after violet stain to make a strong bond between the dye and the cell membrane. Whenever any bacterial identification is carried out the basic primary step is to check the type of bacteria it is which is done by gram staining. As if in terms of research and clinical field this technique is very important but all bacteria can’t get differentiated by this technique. New groups i.e., gram variable and gram-intermediate groups are formed through this technique only. This technique was developed in Berlin while working in the morgue at a city hospital by Hans Christian Gram and Carl Friedlander. Initially, it was developed to see bacteria more clearly on the lung tissue and not for differentiating bacteria. He first published a report in 1884 on this method concluding that the stain doesn’t stay in typhus bacillus. The most common, most usable, and important technique for the differentiation of bacteria is gram staining. Also helpful in the classification and differentiation of microorganisms.

Gram Negative Bacteria

If the bacteria is colored pink or red that shows Gram-negative bacteria. Gram-negative bacteria lose the strain because the cell wall is not present in -ve bacteria.

Gram-Positive Bacteria

The gram +ve bacteria retain the blue color because the thick cell wall is present.

Principle

The basic principle behind this technique is that some bacteria have the ability to retain the dye while the other bacteria don’t have the ability to retain the dye after staining with crystal violet dye. This differentiation is possible due to the difference in the cell wall of bacteria. Gram-negative bacteria have a high content of lipid and low content of peptidoglycan which is a protein-sugar content due to this composition the cell wall of gram-negative bacteria is less thick than gram-positive bacteria and it doesn’t retain the color of the dye. On the other side, the cell wall composition of gram-positive bacteria consists of high content of peptidoglycan and low content of lipid and so it has a thicker cell wall than negative bacteria and so it retains the color of the dye. The pores of the cell wall get closed as the cell gets shrink and dehydrated due to decolorizing and because of this the dye gets fixed in the cell wall and then it doesn’t move out of the cell which gives a permanent color to the bacteria. This is how the violet-iodine complex gets attached to the cell wall and gives blue, pink, or purple color to bacteria and this is how bacteria gets differentiated on the basis of dye. The gram-negative bacteria have thinner cell walls as we all know due to which they can not retain the color otherwise this bacterium also picks up the dye and it goes into the cell membrane but when decolorizing is done its color of dye gets washed off as the cell membrane cannot hold it. The role of decolorizer and alcohol is to cleanse the cell which has extra dye that will be cleared off. In the last when safranin is applied to give color to the bacteria.

Procedure

Normal reagent which is used in this technique are as follows-

• The mordant, Iodine
• Decolorizer (consist of acetone and (95%) alcohol).
• The counterstain, Safranin.
• The primary clean, Crystal Violet.

There are different steps in this procedure which is needed to be followed step by side to successfully complete this technique. Those steps are given below-

• Firstly, get a free slide which should be clean and free from grease.
• With the loop pour the sample and suspend it on the slide.
• Dry the slide with air and then provide heat to the slide. This heat is given to the sample to fix the sample on the slide so that it doesn’t get washed off too easily. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly.
• Now after the heat finally the dye (crystal violet) is poured onto the slide and minimum for 30 seconds and maximum for 1 minute it is kept on the slide and then it is washed with water. This time is given so that the bond is formed between the dye and the cell membrane of the bacteria. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly. 
• The mordant or it can be said the iodine solution is applied and it is also kept for a maximum 1 minute and then the slide is washed with normal water. It gets trapped in the cell after making a bond with the crystal dye.
• Now it’s the turn for acetone or alcohol to be used on the slide and then after 10-20 seconds, the slide is washed again with water. These two agents work as decolorizers.
• The last step is the application of safranin for 1 minute and then again it is rinsed with water.
• The slide undergoes blot and air dry and now it is ready for observation under the microscope.

Purpose

There are many purposes and uses of this technique that are explained briefly

The main purpose of this technique is the identification of the different types of bacteria i.e., the difference between gram-positive and gram-negative bacteria. It is a laboratory technique that can only be performed in labs. The main thing which is responsible to give color to the bacteria is their cell wall composition and thickness. There are a lot of bacterial families and species which can not get differentiated by this technique. For e.g., archaea, mainly archaebacteria. Some of the bacteria are gram-variable which means they can stain anyone either positive or negative while some are doesn’t stain any dye, they neither come under positive nor under negative. Such bacteria which doesn’t come under either the negative or positive category such bacteria are not identified by this technique. There are other techniques that are used for such bacteria. For such bacteria genetic sequencing, molecular techniques are used for their identification in detail. In medical research, this technique is very helpful. When the infection is there inpatient then staining is done on the fluid of his body. This technique is fast result giving so it is used more often than culturing in the case of infection. It is also used in the treatment of the patient. It is used in some special cases of infection. For e.g., in septic arthritis synovial fluid is used for gram staining, and in meningitis cerebrospinal fluid is used for diagnosis. In case of infection in the throat, lungs, skin wounds, or genitals this technique is used. It is also used to check infection in some body fluids such as urine and blood.

Frequently Asked Questions

Question 1: What is the most important and crucial step in staining?

Answer:

As the main purpose of this staining is to differentiate between positive and negative bacteria. Decolorization is very important step in this technique because it rinses off the violet crystal dye and then help in differentiating between the bacteria.

Question 2: What is the composition of gram stain in this technique of gram staining?

Answer: 

There are four components in this stain that are-

Decolorizer (acetone/ethanol), Mordant (Iodine), Primary stain (Crystal violet), Counterstain (Safranin). Every component of this stain has its own function like the primary stain is used to color the bacteria, a mordant is used for fixing the dye with the cell wall of bacteria, decolorizer is used for washing off the extra dye on the cells so that differentiation can be done, counterstain is used to give color for identification.

Question 3: What is the process of retaining crystal violet dye in gram bacteria?

Answer: 

The actual binding of peptidoglycan doesn’t happen there is a series of steps that happens to get the dye trapped in the cell membrane. Firstly, the complex is formed between mordant and the crystal violet dye by which the size of the molecules is changed so that escaping from cell membrane because impossible. Secondly decolorizer shrinks and dehydrates the cell which decreases the size of the cells through which the dye gets retained in the cells and give color to the bacteria.

Question 4: In a cell what are the things that can be known by gram staining.

Answer:

The structure of its cell wall and cell shape can be determined by this technique.

Question 5: Give the uses of staining.

Answer:

There are a lot of uses of staining in the medical and research field. In the field of research, it is very useful for the scientist to determine different bacteria and helpful in the basic step of identification of bacteria. It is used in different projects for study and research whereas in the medical field it is used for the detection of infection and also used in detecting the infection in body fluids like urine and in blood.