The document discusses several staining techniques used to identify different characteristics of bacteria under a microscope. The Gram stain distinguishes between Gram-positive and Gram-negative bacteria and was an important early technique. The acid-fast stain identifies bacteria with waxy cell walls like Mycobacterium tuberculosis. The endospore stain reveals if a bacteria can form dormant endospores. Capsular staining highlights the capsules of virulent bacteria that are difficult to see with regular stains. Each technique has a specific multi-step procedure to prepare and differentially stain samples for examination.

2. CONTENT:
Introduction
Gram stain
Acid fast stain
Endospore stain
Capsuler stain

3. The gram stain developed by Christian Gram in the 1800’s was the
first differential staining technique in use and is still an important
tool for distinguish between two main type of bacteria Gram-ve
Gram+ve.
PROCEDURE:-

4.  Place slide with heat fixed smear on staining tray.
 Gently flood smear with crystal violet and let stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
 Gently flood the smear with Gram’s iodine and let stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The
smear will appear as a purple circle on the slide.
 Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop
by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
 Immediately rinse with water.
 Gently flood with safranin to counter-stain and let stand for 45 seconds.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
 Blot dry the slide with bibulous paper.
 View the smear using a light-microscope under oil-immersion.


5. Gram+ve cell will appear purple and Gram-ve cell appear pink

6. Acid Fast staining
Acid Fast staining is also known as Ziehl Neelsen stain is used to
identify specialized bacteria that have waxy mycolic acid in there
cell wall.
PROCEDURE
1 A slide shaped piece of blotting paper is placed over the bacterial
smear.
2 Apply bright pink dye (Ziehl’s carbolfuchsin) on blotting paper
and place over a water bath for 3-5 minutes and rinse the slide.
3 Decolorize with acid alcohal
4 Lastly purple counter stain(crystal violet) is applied and slide is
rinsed.
5 Acid-fast(waxy)bacterial cell are bright pink and non acid fast
bacterial cell are purplish blue.

7. ENDOSPORE STAINING
The endospore stain is used to identify bacteria that can
produce endospore or not.
The endospore stain is used to identify bacteria that can
produce endospore (small,dormant structure alike to
bacterial seeds).
Forming endospore is very advantages to the bacteria
allow to survive under difficult circumstances,
including dessication , starvation high heat , exposure
to chemicals and radiation.
This stain is designed to distinguish between vegetative
cell of bacteria from spore.

8. PROCEDURE:
1 Place bacterial smear on water bath and stain with
malachite green.
2 Heat on water bath for 5 minutes.
3 Rinse the slide and a pink counter stain (SAFRANIN) is
applied for one minute.
4 After final rinse when viewing slide under oil
immersion with a compound light microscope ,
vegetative cells will appear pink and endospore are
stained a bluish green.

10. CAPSULAR STAINING
Bacterial capsular are composed of high molecular
weight polysccharides and are associated with virulent
and bioflim formation. Capsule are not stain well with
crystal violet, methylene blue or other simple stain.
Here two method of capsular staining are present
following :-
1 Manavel’s staining
2 Hiss method of capsular staining