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.
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.
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