staiing Flashcards
coloring agent used for general purposes
Dye
- – dye used for biological purposes (specific)
Stain
derived from coal tar
rendered synthetically to become a stain
aniline dyes,
o Colorless organic compound that can bind to the nitro
group of chromophores
Benzene
Chemical groups with conjugated double bonds,
imparts color to the colorless benzene.
Chromophore
Groups that intensify the color of chromophore by
conveying the property of ionization to the chromogen
and enabling it to form salts and binding to the
biological substance. This makes up the stain.
Auxochrome
as “an organic compound
containing both chromophores and auxochrome link to the
benzene.
stain
Natural stain
Example: hematoxylin, carmine
synthetic
safranin, methylene blue, crystal violet, aniline etc
stains bacteria directly
Aniline dyes
indirect - stains only the background
India ink and Nigrosin dye
stains based on staining activity
Based on staining activity
o Nuclear – staining the nucleus
▪ Acidic in nature
▪ Ex. Hematoxylin, Carmine
o Cytoplasmic – staining the cytoplasm
▪ Basic in nature
▪ Aniline blue, eosin
o Histologic – staining particular tissues
▪ Ex. Safranin
e
o – negatively-charged chromophore, anionic;
more affinity to positively-charged cellular components
such as protein
Acidic
– positively-charged chromophore, cationic,
more affinity for negatively-charged cellular
constituents such as the DNA and RNA
Basic
basic dye example
Ex. Crystal violet, methylene blue, hematoxylin,
gentian violet
neutral example
Ex. Giemsa stain, Lishman stain, Wright stain,
Romanowsky stain (used in hema)
Protein will be coagulated and the bacterial cell will
adhere to the slide
Heat fixation
Protects cellular structure and morphology of
larger and more delicate organisms
Chemical fixation
Done through application of methanol or any
alcohol (dipping it for 5 to 6 times); it has to be
dried first before staining
o Chemical fixation
▪ direct stains the bacteria
o Chemical fixation
Uses a single stain, will create uniformity of color in all
materials found in the smear
Simple staining
dye used for simple staining
usually basic dye
Impart different colors to different bacteria, utilizes two or
more stains
Differential staining
o Most widely used differential staining procedure
- Gram staining
staining, called safranin,added
Karl Weigert
what is the most critical step ion gram stain
Decolorization
, Mycoplasma, Rickettsiae, Chlamydiae
do not take up the dyes used in Gram stain or are too small
to be seen with light microscopy
stain weakly with gram stain (they are gram
resistant
Mycobacteria
To be visible on a slide, organisms that stain by the Gram
method must be present in concentrations of about
104 to 105 organisms per ml of uncentrifuged fluid
All cocci are gram positive except
Neisseria, Moraxella,
Veillonella
example of acid fast bacteria
Mycobacterium and Nocardia
o Mycobacterium tuberculosis – causes tuberculosis
o Mycobacterium leprae – causes leprosy; weakly acid
fast bacteria (0.5% – 1% decolorizing solution instead
of 3%
counter stain for ziehl neelsen
methylene blue, malachite green
acid fast result
: Acid fast bacilli – red
: Non-acid fast bacilli - blue
decolorizer of cold method or kinyoun
Reagents required:
▪ Carbol Fuchsin stain (filtered)
▪ Acid alcohol 3% v/v (or 20% sulfuric acid)
▪ Malachite green 5 g/l (0.5 w/v) or Methylene blue 5g/l
in hot method it is
heated to enable dye to
penetrate the waxy
mycobacterial cell wal
Phenol-carbol fuchsin stain
Stain is not heated but the
penetration is achieved by
increasing concentration of
basic fuchsin and phenol
and incorporating a ‘wetting
agent’ chemical
Cold method (Kinyoun
Method)
Primary fluorochrome and their AFB fluoresces
Auramine O Green
Auramine O-Rhodamine B Yellow/orange
Acridine Orange = Yellow/orange
When no AFB are seen after examining 300 fields, report
the smear as:
No AFB seen/300 visual fields
Acid-fast organisms other than Mycobacterium
- Nocardia spp. – partial acid fast
- Rhodococcus spp. – partial acid fast
- Legionella micdadei – partial acid fast in tissue
- Cyst of Cryptospordium – acid fast
- Cyst of Isospora – acid fast
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