Gram Staining | Principle | Procedure and Results

Gram Staining

Introduction of Gram Staining

What is Gram Staining: Dr. Christian Gram in 1884 introduces Gram staining technique and classified bacteria on its Gram characters which might be Gram-negative or Gram-positive. In the microbiology field, extensive staining techniques are used. In accumulation, these staining techniques also help in the determination of cell size, structure, and configuration. It�s a sort of identification test.

Gram Staining

Gram Staining Principle

Here are the following main principles of gram staining techniques.

  1. Cell wall structure identifies either cell is Gram-positive or negative in nature.
  2. During the procedure when we stained by primary stain and secure it by a mordant.
  3. At that time, some bacteria have the ability to hold or retain the primary stain by counterattacking decolorization.
  4. While some bacteria decolorized by the use of a decolorizer. The bacteria which retain its primary stain are called Gram-positive and the bacteria which will be decolorized called Gram-Negative.
  5. Crystal violet (CV), when dissolved in water, make CV+ and Cl� ions in its solutions. These ions infiltrate by a cell wall and cell membrane. This happens in both Gram-negative and Gram-positive cells. The CV+ ion interrelates with (-) negative charge bacterial cells components and gives a purple stain to the cell.
  6. Iodine (I), is practice as a mordant that intermingles with CV+ cells and produces large multiplexes of violet crystal.
  7. When alcohol or acetone as decolorizer is added it reacts with cell membrane lipids.
  8. Gram-negative cells have very thin 1-2 layers of peptidoglycan and have a lipopolysaccharide layer which is dissolved by adding Alcohol.
  9. Due to this ability gram-negative cells unable to recollect the complexes and were decolorize when the complex is washed away completely.
  10. In divergence, Gram-positive celled organisms by Ethanol treatment renovates desiccated. Due to this cell wall pores closed. and due to this stain cannot exist in the cell.
  11. After this decolorization procedure, Gram-positive celled retained its purple and in contrast, the Gram-negative celled lost its purple color.Gram Staining

Gram Staining Reagents

Gram Staining Kit

  1. Primary Stain: Crystal Violet

Solution A :

Ethyl Alcohol= 20 ml

Crystal Violet = 2 gm

Solution B :

Distilled Water = 80 ml

Ammonium Oxalate = 0.8 gm

After this mix A and B solution. �Keep this solution for 24 hours and then filter it. Place in the yellowish-brown painted bottle.

  1. Mordant: Gram�s Iodine Solution

Potassium Iodide = 2 gm

Iodine = 1 gm

Distilled water = to 100 ml

Mix them all and stockpile in a yellowish-brown colored bottle.

  1. Decolorizer: 95% Ethanol or 1:1 Acetone with Ethanol

Ethanol (95%) = 50ml

Acetone = 50 ml

  1. Counterstain: Safranin

Safranin O = 0.34 gm

Distilled water = 90ml

Absolute alcohol = 10ml

Mix them all and filter it and store it in a yellowish-brown colored bottle.Gram Staining

Gram Staining Protocol

Smear preparation:

  1. First of all, take a lubricant permitted dry slide.
  2. Take the inoculating loop and disinfect this inoculating loop by the heating it on the Bunsen burner flame.
  3. Later on the transference of a loopful culture with the help of antiseptic loop and make a slur at the epicenter.
  4. Smear neither reedy or nor very profuse.
  5. Consent the smear to parched in the Air. Fix the gasping smear on the slide by transient the slide 3-4 times through the flame quickly with the smear side facing up.

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Gram Staining

  1. Dwelling the slides on the special rods which are used for staining.
  2. Refuge the smear with the stain of crystal violet and consent for 1 minute.
  3. Rinse prudently below tap water.
  4. Inundation the smear with solution Iodine and consent it for one minute.
  5. Trench off the iodine gram solution.
  6. Rinse the slide again in tap water.
  7. Deluge the slide by a decolorizing agent.
  8. Wait for 20-30 seconds.
  9. This process is also performed by accumulating drop by drop on the slide.
  10. Continue this process until the decolorizing agent seriatim from slides.
  11. Moderately wash the slide by water and trench it wholly.
  12. Counterstain is added with Safranin.
  13. After this wait for almost 30 seconds to 1 minute.
  14. Rinse the slide in the ancillary tap water stream.
  15. Continue this process till no color seems in the seepage
  16. Dry the slide by blotting paper.
  17. After this observe the slide beneath the Microscope.

Gram Staining

Gram Staining Results

The staining outcomes are as follows :

Gram-Positive Cell: Dark Purple color �seemed

Gram-Negative: Pale to dark Red color seemed

Yeasts cell: Dark purple color

Epithelial cells: Pale red color

Gram Staining

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Gram Stain Result Interpretation

Gram Staining

Gram Positive and Gram Negative Bacteria List


Gram-Positive Bacteria

Bacillus Species Clostridium Species Corynebacterium Species

Bacillus anthracis

Bacillus cereus

Bacillus subtilis

Clostridium difficile

Clostridium perfringens

Clostridium tetani

Corynebacterium diphtheria

Corynebacterium jeikeium

Corynebacterium urealyticum

Gram-Negative Bacteria

Brucella Species

Haemophilus Species:


Neisseria species:


Brucella abortus

Brucella canis

Brucella melitensis

Brucella suis

Haemophilus influenzae

Haemophilus ducreyi

Haemophilus avium

Neisseria gonorrhoeae

Neisseria meningitidis