Gram's Staining
It is a type of differential staining where two or more stains are used to differentiate between types of bacteria. This staining technique the year 1884. was discovered by Hans Christian Gram in.
Importance:
°It is important test for rapid diagnosis of infectious agents.
°It differentiates types of bacteria whether Gram positive or Gram negative.
It helps in study the morphology of bacteria.
°It helps in find the evidences of capsule, spores, pus cell, epithelial cells, Yeast cell etc.
°To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of their cell walls.
°It is important test for rapid diagnosis of infectious agents.
°It differentiates types of bacteria whether Gram positive or Gram negative.
It helps in study the morphology of bacteria.
°It helps in find the evidences of capsule, spores, pus cell, epithelial cells, Yeast cell etc.
°To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of their cell walls.
Steps:
1.Crystal violet:
It is the primary stain which is used as a simple stain because it dyes the cell wall of bacteria cell.
2.Gram's Iodine:
It is acts as mordant which helps to fix the primary dye to the cell llew
It is the primary stain which is used as a simple stain because it dyes the cell wall of bacteria cell.
2.Gram's Iodine:
It is acts as mordant which helps to fix the primary dye to the cell llew
3. Decolourizer:
It is used to remove the stain of primary dye (crystal violet) from the Gram negative bacterial cell wall. Decolourizer is composed of an organic solvents like acetone, ethanol or in combinations.
It is used to remove the stain of primary dye (crystal violet) from the Gram negative bacterial cell wall. Decolourizer is composed of an organic solvents like acetone, ethanol or in combinations.
4.Safranin:
Finally, a counter stain is applied to stain those cells that have lost their primary stain (especially Gram negative bacteria) as a result of decolourization.
Finally, a counter stain is applied to stain those cells that have lost their primary stain (especially Gram negative bacteria) as a result of decolourization.
The procedure is based on the ability of microorganisms to retain colour of the stains used during the gram stain reaction. Gram-negative bacteria are decolourized by the alcohol, losing the colour of the primary stain, purple. Gram-positive bacteria are not decolourized by alcohol and will remain as purple. After decolourization step, a counterstain is used to impart a pink colour to the decolourized gram-negative organisms.
Gram positive bacteria: IDENTIFICATION OF BACTERIA USING STAINING TECHNIQUES Stain: Gram positive bacteria: IDENTIFICATION OF BACTERIA USING STAINING TECHNIQUES Stain:
A stain is a substance that adheres to a cell which gives cell colour because bacteria are slightly negative charge at pH 7.0. The presence of colour gives the cells significant contrast so that they are prominently visible. Different stains have different affinities for different organisms. Hence, the staining technique enhances the contrast in the microscopic image. Generally, crystal violet, Gram's lodine, acetone and safranin are used as stains.
Simple Staining
Simple staining is carried out to visualize bacteria and to compare morphological shapes and arrangements of bacterial cell. In this technique the bacterial smear is stained with single basic dye such as crystal violet, safranin, methylene blue etc.
Principle:
These single basic dyes will give up a hydroxyl ion or accept a hydrogen ion that leaves the stain positively charged. Since, the surface of bacteria is negatively charged so these charges are strongly binds to the cell surface (cell wall). against a light background.
Steps
1. Clean and dry microscope slides thoroughly.
2 Flame the surface in which the smear is to be spread.
3. Flame the inoculating loop.
4. Transfer a loop full of tap water to the flamed slide surface.
5. Reflame the locop making sure the entire length of the wire that will enter the tube has been heated to redness.
6 Remove the tube cap with the fingers of the hand holding the loop.
7. Flame the tube mouth.
8 Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear .
9. Reflame the inoculating loop to redness including the entire length that entered the tube.
10. Allow the smear to dry thoroughly.
11. Fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.
12. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered with the stain for the time designated below.
Methylene blue - 1 minute
Crystal violet-30 seconds
Carbol fuchsin- 20 seconds
During the staining the slide may be placed on the rack or held in the fingers.
13. At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.
14. Wipe the back of the slide and blot the stained surface with a paper towel. 15. Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.
16. Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply oil directly to the smear, and focus the smear under oil with the 100X objective
2 Flame the surface in which the smear is to be spread.
3. Flame the inoculating loop.
4. Transfer a loop full of tap water to the flamed slide surface.
5. Reflame the locop making sure the entire length of the wire that will enter the tube has been heated to redness.
6 Remove the tube cap with the fingers of the hand holding the loop.
7. Flame the tube mouth.
8 Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear .
9. Reflame the inoculating loop to redness including the entire length that entered the tube.
10. Allow the smear to dry thoroughly.
11. Fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.
12. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered with the stain for the time designated below.
Methylene blue - 1 minute
Crystal violet-30 seconds
Carbol fuchsin- 20 seconds
During the staining the slide may be placed on the rack or held in the fingers.
13. At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.
14. Wipe the back of the slide and blot the stained surface with a paper towel. 15. Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.
16. Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply oil directly to the smear, and focus the smear under oil with the 100X objective
Observation:
Spherical shaped bacteria coccus is turn blue colour whereas rod shaped acteria, bacilli turn red colour.
Use:
To study morphology and arrangement of bacteria cell.
Stain dark purple due to retaining the primary dye called Crystal violet in the cell wall. Example: Staphylococcus aureus.
Gram negative bacteria:
Stain red or pink due to retaining the counter staining dye called Safranin.
Example :Escherichia coli
Example :Escherichia coli
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