Although living microorganisms can be directly examined with the light microscope, they often must be fixed and stained to increase visibility of specific morphological features, and preserve them for future study.

1. STAINING TECHNIQUES
Presented By:
Manu Bhardwaj
M. Sc. Biotechnology (I Yr.)
Department Of Advanced Science & Technology
NIET, Nims University, Jaipur, Rajasthan, India

2. STAINING…?
Although living microorganisms can be directly
examined with the light microscope, they often
must be fixed and stained to increase
visibility of specific morphological
features, and preserve them for future
study.

3. STAIN TECHNIQUES
 SIMPLE STAIN TECHNIQUES
 NEGATIVE STAIN TECHNIQUES
 GRAM STAIN TECHNIQUES
 H & E STAIN TECHNIQUES

4. SIMPLE STAIN TECHNIQUES
Place small amount of bacteria in a droplet of water on a glass slide. Air
dried.
Pass slide through a flame in a process called heat fixing. Which fixes
the cells of slide, kill most of the micro-organism and prepare them for
staining.
Slide is flooded with a basic dye such as Crystal violet or
Methylene blue for a minute or so.
The positive charged dye is attached to the Bacterial cell which
has negative charge and thus staining takes place.
This technique is effective for vegetative cells. The stain does not easily
penetrate spores.

5. SIMPLE STAIN TECHNIQUES

6. NEGATIVE STAIN TECHNIQUES
 Opposite to the simple staining technique.
 Bacteria are mixed on a glass slide with an acidic dye such as
Congo red or black stain Nigrosin.
 The mixture s smeared across the faces of slide and allow to air
dry because the stain carries negative charge. It is repelled by the
bacteria which also possess negative charge.
 The stain gathers around the cell since a chemical reaction has
been take place.
 Because the heat fixing is avoided thus the cells appear less
shrivelled or distorted.
 They often appear larger than stained cells and more natural.

7. NEGATIVE STAIN TECHNIQUES

8. GRAM STAIN TECHNIQUES
 Developed by Cristian Gram in 1884.
 Bacteria can be grouped in to two grouped either
Gram +ve (stained Purple) or Gram –ve (stained
pink).

9. GRAM STAIN TECHNIQUES
 Prepare bacterial smear on the clean slide.
 Pass the slide through over the flame 2–3
times. (Heat fixing)
 Apply Crystal Violet (Primary stain) on
smear for 1 minutes & rinse with water.
 Apply Gram’s iodine (Mordant) for 1 minute
& wash with water.
 Then wash with 95% alcohol
(Decolouriser) for 10-20 seconds & rinse
with water.
 Apply Safranin (Seconday stain) for 1
minute & wash with water.
 Air dry, Blot dry & Observe under Microscope.

10. H & E STAIN TECHNIQUES
H & E is a charge-based stain Techniques.
Hematoxylin stains acidic molecules shades of blue.
Eosin stains basic materials shades of red, pink and orange.
H & E stains are universally used for routine histological
examination of tissue sections.

11. H & E STAIN TECHNIQUES
 Immerse section in Hematoxylin for 1 minute.
• Rinse with tap water.
• Exchange tap water until the water is clear.
 Immerse sections in EOSIN stain for 1-2 minutes.
Rinse with tap water.
• Exchange tap water until the water is clear.
 Dehydrate in ascending alcohol solutions (50%, 70%, 80%, 95%, 100%).
 Clear with xylene .
 Mount cover slip onto a labeled glass slide with DPX.