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- Gram Stain -

Purpose, Procedure and Preparation

Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria.





Gram positive bacteria will typically have a stronger affinity for crystal violet on applying gram's iodine than the gram negative cell wall.

Being a mordant, gram's iodine forms a complex with crystal violet in the stain that has attached more tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria. 

Whereas the gram positive bacteria stain violet as a result of the presence of a thick peptidoglycan layer in the walls of their cell, the gram negative bacteria stain red, due to the thinner peptidoglycan layer in their cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).


Technique


The staining involves 3 major steps/processes that include; 

o   Staining with crystal violet (a water soluble dye), 

o   De-colorization (using ethanol/acetone), 

o   Counterstaining (using Safranin), 

Due to the differences in the thickness of the peptidoglycan layer on the cell walls of these bacteria, gram positive bacteria will retain the crystal violet stain after the de-colorization process using ethyl alcohol/acetone. 

After staining the sample with crystal violet, ethyl alcohol is used to decolorize the sample. It achieves its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it. In doing so, large crystal violet cannot penetrate the tightened layer of peptidoglycan, and hence it is trapped in the cell wall of gram positive bacteria.

On the other hand, the outer membrane of the gram negative cells cannot retain the crystal violet iodine complex and hence the color is lost. 

Safranin is a lighter stain as compared to crystal violet and thus it does disrupt the purple coloration in the gram positive cells.

Theory


In an aqueous solution, crystal violet dissociates into ions of CV+ and CV-. These ions penetrate the walls and membranes of both gram positive and negative cells.

CV+ will interact with the negatively charged components of the bacterial cells, and take up the purple coloration. On adding iodine, iodine cations (I- or I3-) interact with CV+, which results in the formation of larger complexes of CVI within the cytoplasm and the outer layers of the cell.  

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

This results in the loss of the outer membrane, which in turn leaves the peptidoglycan layer exposed. For the gram negative cells, ethanol causes the walls to be leaky and hence they cannot hold the large complexes of CV-L during de-colorization.   

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

Some generas, such as ArthrobacterActinomyces and Corynebacterium have a cell wall that is particularly sensitive to breakage during cell division. 

This results in gram negative staining of the gram positive cells.

On the other hand in cultures of Clostridium and Bacillus, the reduced thickness of peptidoglycan during growth coincides with an increased number of cells that in turn stain gram negative.

Preparations


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

 - Solution A 

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

o   20ml of 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 crystal violet staining reagent and store for 24 hours.  

2.  Mordant (grams iodine) 

o   gram of iodine, 

o   grams of potassium iodide, 

o   300ml of distilled water, 

Using a mortar, iodine and potassium iodide are ground, while slowly adding water with continued grinding until all the iodine has completely dissolved. (Store this in an amber bottle) 

3.  Decolorizing agent 

o   Ethanol, 95 percent (vol/vol) 

However, acetone or 1:1 acetone with ethanol,  

o   50ml acetone 

o   50ml ethanol (95%) 

4.   Counterstain (safranin) 

 - Working solution: 

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

o   90ml of distilled water,

Procedure


Smear preparation 

It is important to note that the thickness of the sample smear on the slide is an important consideration during the preparation of the sample. The smear should not be too think or too thin. 

Label the slide; 

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

This should then be left to air dry before heat fixing by carefully passing the slide through the Bunsen burner (avoid burning the sample). 

Actinomycetes- same as bacteria, but by trying to get a portion of the colony on the slide while it is still intact, this can be achieved by using a scalpel,

Staining Procedure


o   Flood the slide with crystal violet staining reagent for about 1 minute , 

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

o   Wash the slide for the again in a gentle, indirect stream of tap water for about 2 seconds, 

o   Flood the slide with the decolorizing agent then wait for 15 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent  running from the slides runs clear, 

o   Flood the slide using counterstain safranin( and wait for about a minute (30 seconds to 1 minute) 

o   Wash the slide using a gentle and indirect stream of tap water to a point where the color appears in the effluent and then blot dry the absorbent paper, 

o   Add a drop of immersion oil on the stained sample and observe under the microscope


Gram staining helps to characterize bacteria as gram positive or gram negative allowing microscopist enthusiasts/professionals to verify a bacterial cell's wall and membrane which in turn influences various facets of its pathogenicity and level of virulence.


Further reading:  Cell Division and Cytopathology

Return from Gram Stain to Cell Staining in Microscopy

Return from Gram Stain to Microscopy Information Home


References 

Visca, P, Petrucca, A., De Mori, P., Festa, A., Evangelo, B., Antinori, A., and Petrosillo, N., (2001) Community –Acquired Acinetobacter radioresistens Bacteremia in an HIV-Positive Patients. Emerging Infectious Disease 7 (6):1032 – 1035. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631918/pdf/11747736.pdf

Anderson, G.K. Beveridge, T. J., and H. C. Clark. 1983. Chemical Mechanism of the gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. J. Bacteriol. 156 (2):837-845. 

McClelland, R., 2001. Gram’s stain the key to microbiology. Medical Laboratory Observer [serial on the internet]. April 2001 [cited August 6, 2005]

Available from: http://www.mlo-online.com/ce/pdfs/apr01.pdf. 

Links 

http://www.microbelibrary.org/component/resource/gram-stain/2886-gram-stain-protocols 

faculty.stcc.edu/rapp/BIOT251/gram_stain_procedure.htm 

http://serc.carleton.edu/microbelife/research_methods/microscopy/viable_quant.html


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