Preparation of Gram Stain and Normal Saline
1. Gram's Staining
(a) Crystal Violet solution
Crystal Violet 20.0 g
Ammonium Oxalate 9.0 g
Ethanol or Methanol 95 ml
Distilled Water (D/W) to make 1 litre
Preparation: In a clean piece of paper, 20 gm of crystal violet is weighed and transferred to a clean brown bottle. Then, 95 ml of ethanol is added and mixed until the dye is completely dissolved. To the mixture, 9 gm of ammonium oxalate dissolved in 200 ml of D/W is added. Finally the volume is made 1 litre by adding D/W.
(b) Lugol’s Iodine
Potassium Iodide 20.0 gIodine 10.0 g
Distilled Water 1000 ml
Preparation: To 250 ml of D/W, 20 gm of potassium iodide is dissolved. Then 10 gm of iodine is mixed to it until it is dissolved completely. Finally the volume is made 1 litre by adding D/W.
(c) Acetone-Alcohol Decolourizer
Acetone 500 mlEthanol (Absolute) 475 ml
Distilled Water 25 ml
Preparation: To 25 ml D/W, 475 ml of absolute alcohol is added, mixed and transferred into a clean bottle. Then immediately, 500 ml acetone is added to the bottle and mixed well.
(d) Safranin (Counter Stain)
Safranin 10.0 gDistilled Water 1000 ml
Preparation: In a clean piece of paper, 10 gm of safranin is weighed and transferred to a clean bottle. Then 1 litre D/W is added to the bottle and mixed well until safranin dissolved completely.
2. Normal saline
Sodium Cholride 0.85 gDistilled Water 100 ml
Preparation: The sodium chloride is weighed and transferred to a leak-proof bottle premarked to hold 100 ml. Distilled water is added to the 100 ml mark, and mixed until the salt is fully dissolved. The bottle is labelled and stored at room temperature.