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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
METHYLENE BLUE)
Purpose of primary stains: to stain all bacteria (retained by gram positive even after
decolourisation)

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

Note:
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
dye

-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
ENLARGED)

-modifications:
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

Uses:
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

Technique:
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:
Iodine
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
green
- 5 min Albert A, 1 min Albert B
- we don't wash in between because malachite green is highly water soluble