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Why do we stain?

-To increase colour contrast and therefore visibility

-done on a fixed smear
-types of staining:
1) simple stain: basic stains to see uniformly coloured bacteria
EG: Methylene Blue, Carbol Fuschin

2) Negative staining: usually used to detect capsules

EG: India Ink, Nigrosin

3) Impregnation staining: usually done to enhance visibility of thin structures

EG: Silver staining

4) Differential: used to differentiate bacteria

- Gram
- Albert/Ponder's/Neisser's
- Acid-fast

Gram Stain
Primary stains that can be used in gram stain: Crystal violet/Gentian violet/Methyl violet (NOT
Purpose of primary stains: to stain all bacteria (retained by gram positive even after

Mordants that can be used: Gram's iodine,

Gram's iodine consists of KI, and iodine solution
Purpose of a mordant: binds to the alkaline primary stains and forms complexes to be "fixed" ,
which are retained in the thicker peptidoglycan layer of gram positive bacteria

Decolourisation: absolute alcohol (95%)(15s), acetone alcohol(10s), acetone(1s)

Purpose: it removes the stain from gram negative bacteria as the dye is not tightly bound to it

Secondary stains that can be used: safranin, dilute carbol fuschin, neutral red (counterstaining)
Basic fuschin stains more STRONGLY than safranin and therefore can be used in organisms
which take up safranin poorly: Haemophilus, Legionella

Point to note: gram postive bacteria also take up counter stain but it cannot be seen because of
the darker primary stain

Gram positive cell wall contains a THICK peptidoglycan layer, and a teichoic acid layer
NAM and NAG molecules form chains and are cross linked to form a thick peptidoglycan layer
Teichoic Acid: unique to Gram Positive bacteria, maintain the structure
Gram negative cell wall has a very THIN peptidoglycan layer (1-2 layers as compared to 50-100
layers in gram positive)
Contains an outer membrane, which contains porins
Contains a lipopolysaccharide later consisting of lipid A or endotoxin and core polysaccharide

Theories of Gram Staining:

-pH theory: cytoplasm of gram postive bacteria if more acidic and therefore can retain the basic

-Cell wall theory: the thick peptidoglycan layer of the gram postive cell wall prevents the exit of
crystal violet (plus, the alcohol used in decolourisation shrinks its pores--- Dye can enter, but
dye-iodine complexes cannot exit)

-gram negative bacteria allow outflow as they have a thin peptidoglycan layer which is NOT
cross linked, LPS layer is easily disrupted( lipid is digested, and therefore pores are actually

1) Kopeloff and Beerman modification: methyl
violet is used instead of crystal violet, basic fuschin used
2) Jensen's modification: absolute alcohol is used as decolourisation, neutral red as counter
stain (used for meningococci and gonococci)
3) Preston and Morrel's modifications: iodine-acetone is used as decolouriser
4) Weigert's modification: aniline-xylol is used as a decolourisation agent

A bacterium that loses its cell wall is called a L-form. Since cell wall is lost, Gram positive also
appear negative.

Reasons why gram positive may look like gram negative:

1) over-decolourisation
2) older smears are prone to decolourisation
3) cell wall damage due to usage of antibiotics, or during division (Mycobacterium, Actinomyces)

Vice versa:
1) under decolourisation
2) growth stress

1) FIRST step in identification of bacteria
2) To start empirical treatment
3) For organisms that take too long to culture
4) Yeasts can be stained: cryptococcus, candida
5) helps choose the correct culture medium
6) rough estimation of the bacterial load

1min methyl violet + 1min Gram's iodine + 15s absolute alcohol + 30s DILUTE carbol fuschin

Albert stain:

-used to show metachromatic granules of C.diphtheria

-metachromatic granules are seen in C.xerosis as well

Contents of Albert stain:

Albert A:
Toluidine blue (more basic)
Malachite green (less basic)
Glacial acetic acid
95% ethanol

Albert B:
Potassium iodide

-based on the principle of differential staining

-both toluidine blue and malachite green are basic dyes
-cytoplasm of Corynebacterium is neutral, but the granules are highly acidic
- the pH of this stains is brought to 2.8 by glacial acetic acid
- this is comparatively more acidic than the cytoplasm, but more basic than the volutin granules
- as a result, the volutin granules take up toulidine blue, and cytoplasm takes up malachite
- 5 min Albert A, 1 min Albert B
- we don't wash in between because malachite green is highly water soluble