September 28, 2021: Pharmaceutical Interview Questions and Answers

#AllAboutGram’sStaining #Gram’sStainingPrinciple #Gram’sStainingSteps #Gram’sStainingReagents

1. What is Gram-stain?

The Gram stain is an important technique for identification of bacteria. It divides bacteria into two groups, Gram-positives and Gram-negatives.

2. How the Gram-staining technique is useful?

Gram-staining technique that allows to visualize the morphological types of bacteria using a compound light microscope under magnification of 100x.

3. Explain the principle of the Gram-staining technique?

Step 1: Crystal violet (primary stain) aqueous solutions consist of +ve and –ve ions. These ions penetrate through the cell wall and cell membrane of all types of bacteria. The +ve ion stains the bacterial cells and stains the cells purple.

Step 2: Iodide (the mordant) interacts with crystal violate and forms complexes.

Step 3: Decolorizer (made of acetone and alcohol 95%) interacts with the lipids of the cell membrane. A Gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed.

On the other hand, a Gram-positive cell becomes dehydrated because of property of decolorizer. The complexes trapped within the Gram-positive cell due to the multilayered nature of the peptidoglycan.

Step 4: When decolorization is added, the Gram-positive cell remains purple, and the Gram-negative cell loses its purple color.

Step 5: A Safranin, the counterstain is used to provide color to Gram-negative bacteria a pink/ red color.

Gram ve and ve

4. What is the procedure of Gram-staining technique?

a. Take a clean, grease free slide.

b. Prepare the smear of suspension on the clean slide with a loopful of sample.

c. Air-dry or heat-fix smear of cells for around 1 minute with crystal violet staining reagent. (Note: Too heavy or too light cell concentration will affect the Gram Stain results.)

d. Rinse the slide with gentle stream of tap water for 2-3 seconds.

e. Flood the gram’s iodine for 1 minute and wash with gentle stream using tap water for 2-3 seconds.

f. Flood slide with 95% alcohol or acetone for about 15-20 seconds until decolorizing agent running from the slide shows clear liquid.

g. Add counterstain, safranin and wait for 1 minute

h. Wash slide in a gentile stream of tap water until it appear colorless.

i. Blot dry with absorbent paper.

j. Observe under oil immersion using a Brightfield microscope.

k. Results:

Gram-negative bacteria will stain pink or red

Gram-positive bacteria will stain blue or purple

5. What is the procedure of Gram-staining technique?

a. Primary Stain: Crystal Violet Staining Reagent.

Solution A for crystal violet staining reagent

Crystal violet (90% dye content) – 1g

2g Ethanol, 95% (v/v) – 10 ml

Solution B for crystal violet staining reagent

Ammonium oxalate, 0.4 g

Distilled water, 40 ml

Mix solution A and solution B to obtain crystal violet staining reagent. Store for 1 day and then filter using filter paper.

b. Mordant: Gram’s Iodine

Iodine, 0.5 g

Potassium iodide, 1.0 g

Distilled water, 150 ml

Triturate iodine and potassium iodide in a mortar and add water slowly with continuous trituration until the iodine is dissolved. Store in amber bottles.

c. Decolorizing Agent

Ethanol, 95% (vol/vol)

Alternate decolorizing agent – 1:1 acetone and ethanol mixture.

d. Counterstain: Safranin

Use 1.25g Safranin O and mix it with 50 ml 95% Ethanol (Solution A). Take this Solution A 5 ml and mix it with 45 ml distilled water

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