Gram stain is most likely one that the most commonly used staining steps used in the ar of microbiology. It is among the differential stains that are offered to characterize bacteria in one of two groups: either gram optimistic bacteria or gram an adverse bacteria.

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Gram positive bacteria will generally have a more powerful affinity for crystal violet on using gram"s iodine than the gram an adverse cell wall.

Being a mordant, gram"s iodine develops a facility with decision violet in the stain that has attached an ext tightly come the cell wall of gram hopeful bacteria 보다 that that the gram an unfavorable bacteria. 

Whereas the gram hopeful bacteria stain violet as a result that the presence the a special peptidoglycan class in the walls of their cell, the gram an adverse bacteria stain red, because of the thinner peptidoglycan great in your cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).

Technique

The staining entails 3 major steps/processes that include:

o Staining v crystal violet (a water dissolve dye) 

o De-colorization (using ethanol/acetone) 

o Counterstaining (using Safranin)

Due come the differences in the thickness that the peptidoglycan class on the cell walls of these bacteria, gram optimistic bacteria will retain the decision violet stain after the de-colorization procedure using ethyl alcohol/acetone. 

After staining the sample v crystal violet, ethyl alcohol is offered to decolorize the sample. That achieves its purpose by dehydrating the peptidoglycan class by tightening and shrinking it. In law so, big crystal violet cannot pass through the tightened class of peptidoglycan, and also hence it is trapped in the cell wall surface of gram optimistic bacteria.

On the various other hand, the outer membrane the the gram negative cells can not retain the crystal violet iodine facility and thus the shade is lost. 

Safranin is a lighter stain as contrasted to decision violet and thus the does disrupt the violet coloration in the gram hopeful cells.

Theory

In an aqueous solution, crystal violet dissociates right into ions that CV+ and also CV-. These ions permeate the walls and membranes of both gram positive and an adverse cells.

CV+ will communicate with the negatively charged components of the bacterial cells, and take up the violet coloration. On including iodine, iodine cations (I- or I3-) interact with CV+, which outcomes in the development of bigger complexes the CVI in ~ the cytoplasm and the external layers that the cell.  

On adding the decolorizing agent (ethanol), it interacts through the membrane lipids of both the gram positive and gram negative positive and also gram negative.

This results in the ns of the external membrane, which consequently leaves the peptidoglycan great exposed. Because that the gram negative cells, ethanol causes the wall surfaces to it is in leaky and hence castle cannot host the big complexes that CV-L throughout de-colorization.  

In several of the staining processes using gram stain, a pattern of gram-variables are obtained, which is a mix of pink and also purple.

Some generas, together as Arthrobacter, Actinomyces and Corynebacterium have a cell wall surface that is specifically sensitive to breakage during cell division.

This outcomes in gram negative staining that the gram optimistic cells.

On the other hand in societies of Clostridium and also Bacillus, the reduced thickness of peptidoglycan throughout growth synchronizes with an increased variety of cells that subsequently stain gram negative.

Preparations

1. The major stain (crystal violet reagents for staining) 

 - Solution A 

o 2 grams of decision violet (certified 90 percent of the dye content) 

o 20ml that ethanol (95percent vol/vol) 

- Solution B

o 0.8 grams of ammonium oxalate, 

o 80ml of distilled water, 

Mix A and B so as to obtain decision violet staining reagent and store for 24 hours.  

2. Mordant (grams iodine) 

o gram the iodine, 

o grams of potassium iodide, 

o 300ml that distilled water, 

Using a mortar, iodine and potassium iodide room ground, if slowly adding water with ongoing grinding till all the iodine has fully dissolved. (Store this in an amber bottle) 

3. Decolorizing agent 

o Ethanol, 95 percent (vol/vol) 

However, acetone or 1:1 acetone through ethanol,  

o 50ml acetone 

o 50ml ethanol (95%) 

4.

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Counterstain (safranin) 

 - Working solution: 

o 10ml of the stock equipment (2.5 grams Safranin O and 100ml the 95 percent ethanol)

o 90ml that distilled water

Procedure

Smear preparation 

It is crucial to keep in mind that the thickness the the sample smear on the on slide is critical consideration throughout the ready of the sample. The smear must not be too think or also thin. 

Label the slide. 

Bacteria - smear the sample ~ above the slide using an inoculating needle. This can also be excellent by introducing a autumn of saline on the slide followed by the sample and then mixing.

This should then be left to air dry prior to heat resolving by closely passing the slide through the Bunsen burner (avoid burning the sample). 

Actinomycetes - same as bacteria, however by trying to obtain a part of the colony on the on slide while that is quiet intact, this can be completed by making use of a scalpel.

Staining Procedure

o overwhelming the slide through crystal violet staining reagent for around 1 minute , 

o to wash the slide using a gentle, indirect stream of madness water for about 2 seconds, flood the slide with a mordant (Gram’s iodine) then wait for a minute, 

o wash the slide because that the again in a gentle, indirect present of madness water for around 2 seconds, 

o overwhelming the slide through the decolorizing agent then wait because that 15 seconds. This can also be done by adding a autumn by drop come the slide till the decolorizing agent to run from the slides operation clear, 

o flood the slide making use of counterstain safranin( and wait for around a minute (30 secs to 1 minute) 

o wash the slide using a gentle and also indirect currently of madness water to a point where the color shows up in the effluent and then blot dried the absorbent paper, 

o add a fall of immersion oil ~ above the stained sample and also observe under the microscope