Gram Stain

What sample is needed for a Gram Stain?

1. The Gram stain can be performed 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 a Gram stain?

1. The 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 fluid.
3. To diagnose bacterial meningitis.
4. It can stain yeast, and this needs to be reported.

What is the history of the Gram stain?

1. Bacteria are colorless and usually invisible under light microscopy, so a stain was developed to visualize them.
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, and urine.
4. Bacteria that absorb crystal violet and retain it appear blue; these are called Gram-positive organisms.
5.  If the crystal violet is washed off with alcohol, it will absorb the counterstain safranin, which stains gram-negative organisms.

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 both 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 are needed for protein synthesis.
6. Gram-positive organisms are hydrophilic, and this property prevents bacteria in the intestine from being affected by 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?

What solutions are needed for the Gram stain?

1. Crytal violet = 0.5% in distle water.
2. Gram’s iodine:
   1. iodine = 1 gram
   2. Potassium iodide = 2 grams
   3. Distle water = 300 mL
3. Ethyl alcohol 75%.
4. Dilute carbol fuschsin:
   1. Zeihl-Nelsen carbolfichsin = one part
   2. Distle water = 9 parts
5. Acetone:
   1. Acetone, 1 part with 2 parts ethyl alcohol, is a much more rapid decolorizer than 70% ethyl alcohol.
6. Neutral red:
   1. Neutral red 1 gram, 1% acetic acid 2 mL, and distilled water 1000 mL, may be used as a counterstain.
   2. It is useful for staining intracellular organisms and does not mask weakly gram-positive organisms.
7. Safranin:
   1. Safranin 0.5% in distle wateris another counterstain.
   2. But it has no obvious advantages.

What are the steps in the Gram stain?

1. Fix the slide by heating.
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 membrane of Gram-negative bacteria, after which 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 dye extraction (still 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, or carbolfuchsin.
   1. Counterstaining with dilute carbol fuchsin is done for one minute
   2. The counterstain cannot enter Gram-positive bacteria, so they appear purple.
   3. In gram-negative bacteria, safranin can enter and give a pink color.
6. Rinse in water and dry.