What is staining and classifications of staining, the steps of the staining or procedure.

1. Bsc nursing 1 year
Microbiology
Unit :-2 (staining)
BY:- PRAGYA TIWARI

2. Staining :-
 Since, bacteria are two small and we can
not seen with naked eyes, therefore to
visualize them, either dyes or stain.
 Staining a bacterial cell is relatively fast,
and simple.
 Staining not only makes bacteria more
easily seen, but it allows their morphology
(shape and size) to be visualized more
easily.

4. Stain :-
 Stain are dyes or reagent used for differentiate colouring of microorganisms.
 To observe their structure with much variety under microscope.
Staining :- By applying penetrative dyes or chemical.
Purpose of staining:-
 To view the organism with much clarity.
 To differentiate one organisms from another.
 To determine peculiar structure eg. Spore, nucleus etc.

5. Types of staining:-
Staining
technique are
broadly divided
into two
group:-
1. Simple
staining
2. Differential
staining.

6. 1. Simple staining:-
In this procedure reagent methylene blue crystal violet or carbon Fuchsin (nigrosin) is used
and all cells and their structure sat in in thr same manner this procedure are two types. :-
 Positive staining:- In positive staining the stain (methylene blue) is basic cationic have
positive charge and attach to the surface of the object that is negatively charged is
called positive staining.
 Negative staining:- In negative staining the stain nigrosin is acidic and anionic having
negative charge and it is repelled by the object bacteria. That’s why the staining, stain
only background but not the bacteria is called negative staining.

8. 2.Differential staining:-
 Differential staining is a procedure that takes advantage of differences in the
physical and chemical properties of different groups of bacteria.
 Using multiple stains can better differentiate between different microorganisms
or structures/cellular components of a single organism.
 In this procedure, more than one staining reagent are used and specific object
(Specific microorganisms) exhibit different staining reaction.
 To most widly used differential staining procedure are :-
1. Gram staining.
2. Acid fast staining.

9. 2(a). Gram staining:-
 Gram Staining is the common, important, and most used differential staining
techniques in microbiologyand bacteriology, which was introduced by Danish
Bacteriologist Hans Christian Gram in 1884.
 It is important in identification and differentiation of bacteria.
 The gram stain differentiate bacteria in to two broad group:-
1. Gram positive bacteria.
2. Gram negative bacteria.

10. Reagents Used in Gram Staining:-
1. Crystal Violet, the primary stain
2. Iodine, the mordant (The mordant is Gram’s Iodine. This binds to the crystal
violet making a large complex that adheres to the cell membrane.)
3. A decolorizer made of acetone and alcohol (95%)
4. Safranin, the counterstain

11. Procedure of gram staining:-
1.
1. Take a clean, grease free slide.
2. Prepare the smear of suspension on the clean slide with a loopful of sample.
3. Air dry and heat fix
4. Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
5. Flood the gram’s iodine for 1 minute and wash with water.
6. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
7. Add safranin for about 1 minute and wash with water.
8. Air dry, Blot dry and Observe under Microscope.

13. Gram positive and gram negative
bacteria:-
1. Gram positive bacteria are those which resist the
decolonization and retain the primary stain appearing violet.
Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium,
Corynebacterium, Enterococcus, Gardnerella, Lactobacillus,
Listeria, Mycoplasma, Nocardia, Staphylococcus,
Streptococcus, Streptomyces ,etc.
2. Gram negative bacteria are Decolourized by organic
substance (acetone alcohol) and therefore take the counter
stain appearing red.
Gram Negative Bacteria: Escherichia coli (E. Coli),
Salmonella, Shigella.

16. 2(b). Acid fast staining:-
 The main aim of this staining is to
differentiate bacteria into acid fast
group and non-acid fast groups.
 This method is used for those
microorganisms which are not
staining by simple or Gram
staining method, particularly the
member of genus Mycobacterium,
are resistant and can only be
visualized by acid-fast staining.

17. Procedure of acid fast staining:-
 Prepare bacterial smear on clean and grease free slide, using sterile technique.
 Allow smear to air dry and then heat fix.
 Cover the smear with carbol fuchsin stain.
 Heat the stain until vapour just begins to rise (i.e. About 60 C). Do not overheat.
Allow the heated stain to remain on the slide for 5 minutes.
 Wash off the stain with clean water.
 Note: When the tap water is not clean, wash the smear with filtered water or
clean boiled rainwater.

18. Continue..
 Cover the smear with acid alcohol for 5 minutes or until the smear is sufficiently
decolorized, i.e. Pale pink.
 Caution: Acid alcohol is fiammable, therefore use it with care well away from an open
fiame.
 Wash well with clean water.
 Cover the smear with malachite green stain for 1–2 minutes, using the longer time
when the smear is thin.
 Wash off the stain with clean water.
 Wipe the back of the slide clean, and place it in a draining rack for the smear to air-
dry (do not blot dry).
 Examine the smear Under microscopic.

20. Acid fast and non acid fast :-
 Acid fast: Bright red to intensive
purple, Red, straight or slightly
curved rods, occurring singly or in
small groups, may appear beaded
 Non-acid fast: Blue color; In
addition, background material
should stain blue.