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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)
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
-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.
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:
Albert B:
Iodine
Potassium iodide