Laboratory‎ > ‎ Gram Stain


The purpose of this experiment was to learn the gram staining method and to observe the characteristics of gram negative and gram positive microorganisms.


The gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between gram-positive and gram negative bacteria. Bacteria that stain purple with the gram staining procedure are termed gram-positivethose that stain pink are said to be gram-negative.
The terms positive and negative have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. Gram-positive and gram negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. The gram-positive cell wall appears thick and consists of numerous interconnecting layers of peptidoglycan. Also interwoven in the cell wall of gram-positive bacteria are teichoic acids. Generally, 60% -90% of the gram-positive cell wall is peptidoglycan. The gram-negative cell wall, on the other hand, contains a much thinner, single layer of peptidoglycan only two or three layers thick. This is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, and lipoprotein. Only 10% – 20% of the gram-negative cell wall is peptidoglycan.

The gram staining procedure involves four basic steps:

1. The bacteria are first stained with the basic dye crystal violet. Both gram positive and gram-negative bacteria become directly stained and appear purple after this step.

2. The bacteria are then treated with gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both gram-positive and gram-negative bacteria remain purple after this step.

3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex while gram-negative are decolorized.

4. Finally, the counterstain safranin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, that are now colorless, become directly stained by the safranin. Thus, gram-positive appear purple, and gram-negative appear pink.


– Microscope                                                              – Bunsen burner

– Inoculating loop                                                        – Test tube rack

– Nutrient agar of Escherichia coli                              – Pisette

– Nutrient agar of Bacillus subtilus                             – Distilled water

– Crystal violet                                                            – Alcohol

– Iodine                                                                      – Safranine

– Oil immersion                                                           – Microscope slide

– Holder                                                                     – A piece of napkin


In here, firstly, immersed to alcohol microscope slide was taken and was held to flame very short time, then plates of Escherichia coli and Bacillus subtilis were taken and in the meantime inoculating loop was held to flame as far as redness. Afterwards; one loop E. coli and B. subtilis were taken and spread out on microscope slide and a drop of water on a slide was added, meanwhile loop was held to re-flame, then microscope slide was waited in air for drying. After that; microscope slide of bacteria were passed through the flame of the bunsen burner 3 or 4 times to heat-fixed and thus heat-fixed smear of bacteria was prepared. Afterwards; crystal violet was added to microscope slide and was waited for one minute. Gently it was washed with distilled water. Then, gram’s iodine solution was added and was waited for one minute and then gently it was washed with distilled water. Then, on microscope slide, stained bacteria were decolorized immediately with alcohol for 30 seconds and again bacteria were washed with distilled water. Then, safranine was added on microscope slide, bacteria were stained with safranine and waited for one minute and rinsed with distilled water. After that; excess water was taken with a piece of napkin, then it was dried in air. Next, a drop of oil immersion was added and finally, slide was placed on light microscope and bacteria were observed under oil-immersion objective (100X) and colour, shape, size of bacteria was recorded.


                    Bacillus subtilis                                            Escherichia coli

Colour : purple or dark pink                                        Colour : red

Shape  : rod                                                                Shape  : rod

Size      : long                                                               Size      : short


Because of these results, B. subtilis is gram positive bacteria and E. coli is gram negative bacteria


In this experiment, gram staining technique was tried to apply. Firstly; Bacillus subtilis and Escherichia coli cultures were used. These were put on the microscope slide. The microscope slide was sterilized previously. Then, microscope slide was passed through flame, that is; heat fixation was taken place, because of this B. subtilis and E. coli cultures were to fix on the microscope slide and to prevent their motile, thus microorganisms were non-motile. After that; crystal violet stain was applied above microorganisms. We applied it because this stain provides to be observed readily. Then iodine solution was applied on microorganisms. Iodine solution incorporates with crystal violet and crystal violet iodine complex (CV-I) occurs within the cell, and cells stain violet colour. Then microorganisms were decolourized with alcohol. These cells have cell wall and the cell wall contain lipid. This lipid layer is affected by alcohol. In addition; if cell is gram positive, cell wall dehydrates and shrinkage of pores occurs on cell wall, permeability of cell decreases and cells appear violet colour. If cell is gram negative, extend of pores occurs and permeability of cell increases and cells appear colourless. After then; when safranine is added, gram positive bacteria appear violet colour, that is; bacteria do not affect from safranine, gram negative bacteria appear red colour with effect of safranine. 

In our experiment, Bacillus subtilis was observed as purple or dark pink colour, therefore it was gram positive. Escherichia coli was observed as red colour, therefore it was gram negative. As a result; experiment results were compared with original results and original results came seasonably with our experiment results.

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