Differential Staining
👉In this staining process used more than one dyes .
👉It is differentiate different types of microorganisms.
👉It is mainly two types .
A- Gram Staining B- Acid-fast Staining
A-Gram Staining 👇
👉This technique was discovered by Han's Christian Gram in 1884.
👉This staining technique helps to differentiate between Gram positive bacteria & Gram negative bacteria.
Principle:-
Same as previous methods
Procedure:-
👉First of all clean slide is taken , wash it with detergents , air dry it and sterilize .
👉 Making smear of bacterial Culture with the help of sterile inoculating loop.
👉 Then smear allowed to air dry , heat fixation by burner flames.
👉 Smear Stained with 1°dye (Crystal voilet dye) for 30sec.
👉 Allow to dry.
👉 then smear slide washes with dist.water for 2-3sec.
👉 Then smear stained by Gram iodine for 1minute.
👉Again smear slide washes with distt.water 2-3sec.
👉After this process smear slide treated with decolourizing agent ( alcohol,acetone) for 10-30seconds.
👉 Again Smear slide washes with distt. Water 2-3sec.
👉Then apply 2°dye(Safranin dye) on smear slide for 30-60sec.
👉Smear slide washes with distt. water 2-3sec.
👉 Next smear covered by coverslip.
👉 Finally bacterial smear observed under microscopy.
Results:-
👉Gram positive bacteria gives -(blue-purple colour)
👉Gram negative bacteria gives -(red-pink colour)
B- Acid-fast Staining 👇
👉This staining technique also k/a Ziehl-Neelsen Staining method.
👉Because, this technique discovered by Ziehl & modified by Neelsen.
👉 This staining method used for those microbes which are not stained by simple staining & gram staining.
👉This staining method helps to differentiate between Acid-fast bacteria & Non Acid-fast bacteria.
👉In this method Carbol-fuschin dye is used.
Principle:-
(same as previous methods)
Procedure:-
👉First of all takes clean slide , wash it with detergents then air dry it & sterilize.
👉Making smear of bacterial Culture with the help of sterile inoculating loop.
👉 Then smear allowed to air dry , heat fixation by burner flames.
👉Slide stained by Z-N Carbol fuschin dye. 👉 After this slide placed on the water bath for boiling (3-5min.).
👉 Continuously add ZN-CF dye to avoid the drying of smear.
👉Next, smear slide treated with decolourizing agent (alcohol,acetone).
👉then smear slide washes with distt. water (2-3sec.).
👉Next, flood the smear slide with Counter -stain dye(Methylene blue) for 2-3min.
👉Once again smear slide washes with distt.water 2-3sec.
👉then allow to air dry it.
👉 Next, smear slide cover by Coverslip.
👉Finally bacterial smear observed under microscopy.
Results:-
👉 Acid-fast positive bacteria gives-Red colour.
👉Acid fast negative bacteria gives-Blue colour.
That's all about differential staining .
I hope you understand differential staining.🗯️🗯️🗯️🗯️🗯️💥👋🏻👋🏻
