The Gram Stain
Gram staining can be used to identify, classify and study two different types of bacteria; the Gram-positive bacteria and the Gram-negative bacteria. The first stain comprises of the crystal violet and gram iodine complex. After which, decolouriser (containing of 95% of ethanol) is being added to the samples. Following that, a second stain called the red Safranin dye is being added. Gram-positive bacteria have a thick layer of peptidoglycan which causes the purple coloured dye complex to remain within the cell. The retention of the first stain in the Gram-positive bacteria causes the bacteria to be stained purple. On the contrary, after the addition of decolouriser to the Gram-negative bacteria, the lipid contents of the cell are being dissolved and the contents of the cell including the purple coloured dye complex leaves the cell wall. This causes the Gram-negative bacteria to remain colourless after the addition of the first stain and the decolouriser and retains the red colour when the second dye is being added. The differences in the dye retention help to distinguish the Gram-negative and the Gram-positive bacteria where blue-purple coloured are the Gram-positive and the pink-red are the Gram-negative bacteria.
2)Aim and Objectives
To determine and classify Escherichia coli and Bacillus subtillis into Gram-positive and Gram-negative bacteria. To be able to distinguish the structural differences and the staining colour differences between Gram-negative bacteria and Gram-positive bacteria.
Differential stain is staining process that makes use of more than one type of stain. The Gram Stain is an example of differential stain. Gram stain makes use of crystal violet and gram iodine to form purple dye complex which is being used as the first stain. A counter stain, Red Safranin dye, is used as the second dye. These two dyes and the addition of decolouriser (contains 95% of ethanol) between the addition of two dyes help to distinguish between the Gram-positive bacteria and the Gram-negative bacteria. Upon the addition of the first stain and the decolouriser, the Gram-positive bacteria would be coloured purple under the microscope while the Gram-negative bacteria would remain colourless. The counter staining causes the Gram-negative bacteria to be stained red and would appear red when viewed under the microscope.
The different staining of the bacteria is due to the structural and chemical differences in the two different types of bacteria. Gram-positive bacteria have a much thicker layer of peptidoglycan in their cell walls as compared to the Gram-negative bacteria. The thick layer of peptidoglycan in Gram-positive bacteria traps the purple dye even after the addition of decolouriser. This causes the Gram-positive bacteria to remain purple coloured. However, when the decolouriser is added to Gram-negative bacteria, the alcohol dissolves the lipids in the cell membrane and causes the contents in the cell and the purple dye to escape outside the cell.
Glass slides were labelled and a circle at the center was drawn on the other side of the slide. Sterile water was placed on the center of the circle using an inoculation loop. After which, another inoculation loop was used to take up a portion a specific colony of E. coli from the growing colonies of E. coli on the agar plate. Heat fixation of the smears were then carried out until all the sterile water had evaporated. The glass slide was then placed on a metal rack beside the stains and the decolouriser for gram staining to be carried out. The glass slide containing E. coli was first stained with excess crystal violet dye for approximately a minute. After which, the excess dye was being washed off with distilled water. Similarly, excess Gram iodine was also stained on the E. coli for a minute and was being washed off using distilled water. Following that, the decolouriser was gently squirted in small amounts on the top part of the slide away from the smear. The end of gram staining was when Red Safranin dye was added on top of the smear for a minute. After which the excess dye was being washed off using distilled water and the slide was dried using lens cleaning paper. The dried slide was then placed under the microscope at 10X,40X objective lens and oil was added before the smear was viewed under the 100X objective lens. At 100X objective lens, the structural characteristics and the colour of the cell was examined. Following which B. subtilis went through the same process.
5)Results and Discussion
The figure below shows E. coli and B. subtilis after staining viewed under a microscope.
Figure 1: Escherichia coli Figure 2: Bacillus subtilis
Shape and cell arrangement
Figure 1 shows E. coli which had been stained red through the Safranin dye and the cell is rod shaped. They also seem to be more clustered together as compared to the B. subtilis. The staining of red proves that E. coli is Gram-negative. However, figure 2 shows B. subtilis which contains some parts of the smear stained violet and some parts of the smear stained red. This indicates that some of the bacteria are Gram-positive and some of the bacteria are Gram-negative although they were obtained from the same colony. Theoretically, B. subtilis are supposed to be Gram-positive. P.J. Piggot, 2009 However, the result of this experiment shows that B. subtilis is Gram-variable. A possible reason to back this up would be the using of old specimens for the experiment. This would have caused some of the bacteria cell walls to have damaged. Damaged cell walls would have led to some of the peptidoglycan and the contents of the cell to leave the cell. Even if the contents remain in the cell, the addition of ethanol would have dissolved the lipid content of the cell and the thinning of peptidoglycan alone would have caused some of the bacteria to appear as Gram-negative. Another possible reason could be the excess addition of ethanol onto the smear. The ethanol added may be too much which causes the cell to be damaged or direct squirting of ethanol onto parts of the smear could have resulted in damage of some of the bacteria cell walls. This would have caused some of the bacteria to be stained red.
Gram staining can be used to distinguish Gram-positive and Gram-negative bacteria. However, it must be ensured that fresh colonies are being cultured to obtain more accurate results. Also, through the staining process, the cell shape, arrangement and even cell size can be measured between the Gram-negative and Gram-positive bacteria and even between each of the sub groups of Gram-positive and Gram-negative bacteria itself.
1)Why is Gram stain considered a differential stain?
The Gram stain classifies two types of bacteria into Gram-negative and Gram-positive bacteria. The gram stain comprises of 2 stains; first the addition of crystal violet and the gram iodine complex stain and following that it would be the safranin stain. A decolouriser is being added between the first and the second stain.
2)How do Gram-positive and Gram-negative bacteria differ in cellular structure, and how does this contribute to their differential staining properties?
The Gram-positive bacteria have a much thicker peptidoglycan cell wall than the Gram-negative bacteria. Gram-positive with a thicker peptidoglycan cell wall, it can retain the purple dye and the contents of the cell even after the addition of the decolouriser. However, the thin layer of peptidoglycan in the Gram-negative causes the escape of the purple dye and appears colourless after the addition of the first stain and the decolouriser.
3)Why are some cells Gram-variable? How does the age of a culture affect the Gram stain reaction?
Older specimen of Gram-positive bacteria can cause some of the cells to be damaged. This causes the bacteria which was Gram-positive when it was young to become Gram-negative or Gram-variable cells. As the bacteria culture ages, the Gram-positive bacteria which might have had thick peptidoglycan cell walls may have a reduction in peptidoglycan and may not be able to retain the purple dye as much as when it was young.
4) Which step in the Gram stain procedure is most prone to error? If not done correctly, how might that step affect the gram reaction result?
The addition of decolouriser (95% of ethanol) is the step that may cause the greatest amount of error. Decolourisation is the step after the addition of the first stain to help to differentiate between the Gram-negative and the Gram-positive cells. Excess of decolouriser may cause the Gram-positive cells to lose its purple complex dye stain and it may appear as a Gram-negative cell due to the red coloured appearance. Shortage of decolouriser may cause Gram-negative bacteria to retain the purple dye and may be misinterpreted as a Gram-positive bacteria due to its purple coloured appearance.
5)List the reagent of the Gram stain technique in order and their general role in the staining process.
1. Crystal violet dye – acts as the primary stain to help to classify the Gram-positive and the Gram-negative cells
2. Grams iodine – A mordant and fixes itself with the primary stain to form a purple complex
3. Alcohol – A decolourizer that dissolves the lipid layer in the Gram-negative cells
4. Safranin – acts as a counterstain to stain the Gram-negative bacteria which are colourless after the addition of the decolouriser
6)If you are performing Gram stain technique on Pseudomonas aeruginosa and forgot to wash with decolouriser, what gram reaction and colour of the cells would you observe under the microscope? Explain.
Pseudomonas aeruginosa would appear as blue-purple colour. Theoretically, P. aeruginosa may be a Gram-negative organism. Marcus,2017 However, without the addition of decolouriser, the lipids in the cell membrane cannot be dissolved to allow the purple dye to leave the cell. This would cause the cell to not be counterstained as the red stain cannot be seen amidst of the purple dye.
7)There is another method to identify Gram reactions of bacteria cell types. Describe this method. (Hint: use reagent to break down the cell wall).
Another method would be the use of dilute alkali solutions like Potassium Hydroxide(KOH). A drop of 3% of KOH is to placed on a glass slide. After which, remove a small amount of bacteria from the same colony using the inoculation loop from the incubated agar plate. Place the bacteria into the KOH and keep mixing the solution for 1 minute to notice and formation of string when the loop is being lifted.
Figure 3: Show an example of formation of string
After 1 minute if the mixture forms a string when lifted it is Gram-negative. If it does not, the bacteria is Gram-positive.
8)With the help of a table, list down the characteristic differences between the Gram-positive and Gram-negative bacteria cells.
Bacteria remains purple in colour even after the decolourizer is being used
Bacteria becomes colourless after decolourizer is being used
Thick peptidoglycan cell wall
Thin peptidoglycan cell wall
Has a final stain colour of blue-purple
Has a final stain colour of pink-red
Outer membrane present
Outer membrane absent
Very little pathogenic bacteria belong here
Most pathogenic bacteria belong here
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