Gram Stain

What sample is needed for Gram Stain?

1. Gram stain can be done on sputum, pus, tissue, and urine.
2. The sample can be obtained from the infected ulcer or wound.
3. The CSF may be stained.

What are the indications for Gram stain?

1. Gram stain differentiates between gram-positive and gram-negative organisms.
2. To diagnose the presence of bacteria in sputum, pus, or any other tissue or fluids.
3. To diagnose bacterial meningitis.
4. It can stain yeast, and this needs to be reported.

What is the history of Gram stain?

1. Bacteria are colorless and usually invisible to light microscopy, so a staining color was developed to show those bacteria.
2. The name comes from its inventor, Hans Christian Gram. He published a gram stain method in 1884.
   1. He was searching for the organism and was diagnosed with pneumonia.
3. This is a special stain for diagnosing gram-positive or gram-negative organisms in various samples, such as sputum, pus, urine, etc.
4. The bacteria that absorb the crystal violet and hold on to it will appear blue, called gram-positive organisms.
5.  If the crystal violet is washed off with alcohol, it will absorb the counterstain safranin, a gram-negative organism.

What is the structure of the bacteria?

1. Bacteria are prokaryotes (single-cell organisms).
2. The layer outside the cytoplasm is called the peptidoglycan layer and is present in gram-positive and gram-negative bacteria.
3.  Gram-positive bacteria have a thick wall and extensive cross-linking of the amino acid chains. In contrast, gram-negative bacteria have a very thin wall and a simple cross-linking pattern.
4. Bacteria consist of circular DNA molecules (Continuous coding of the gene).
   1. DNA is tightly coiled.
5. Only ribosomes are seen, which is needed for protein synthesis.
6. Gram-positive organisms are hydrophilic, and this property prevents bacteria in the intestine from bile.
7. Gram-negative bacteria’s outer cell walls are also hydrophilic, but the lipid component molecules give hydrophobic properties.

1. Gram-positive = Blue.
2. Gram-negative = Red.
3. The gram-negative bacterial wall consists of three layers.

4. Gram-positive bacteria consist of Two layers.

How will you differentiate Gram-positive and Gram-negative bacteria?

How will you do Gram staining?

It consists of the following steps:

1. Fix the slide by heat.
2. Primary stain: Stain the slide smear with gentian violet or crystal violet. It will penetrate the cell membrane.
3. Mordant: wash the violet stain and flood the smear with the Iodine solution. This acts as a mordant and forms a complex with Crystal violet.
4. Decolorizer: Wash off the smear and flood it with alcohol (95 %) or an Acetone-alcohol mixture.
   1. This will remove the outer cell membrane in gram-negative bacteria, where the complex will be washed off.
   2.  Gram-positive bacteria’s cell membrane remains intact, and the stain will not be washed off after alcohol treatment.
   3. Decolorization with acetone or alcohol will lead to the following:
      1. Gram-positive bacteria block the dye extraction (still, this step is unclear).
      2. This step will decolorize gram-negative bacteria and not gram-positive bacteria.
5. Secondary stain: Counterstain the smear with safranin O, a red dye.
   1. The counterstain cannot enter gram-positive bacteria, so the bacteria are purple.
   2. In gram-negative bacteria, safranin can enter and give a pink color.