SIR MADAN LAL INSTITUTE OF
NURSING AND PARAMEDICAL
COLLEGE
TOPIC – Staining Techniques.
From – Miss Shweta yadav
M.Sc Nursing .
Dept of Obs & Gyne
Introduction
Staining is an auxiliary techniques used in
microscopy .
Stains and yes are frequently used in biology and
medicine to highlight structure in biological tissues
Stains may be used to define and extreme bulk
tissues, cell populations or organelles with
individual cells.
Bacteria are microscopic organisms they are
mostly colorless to visualize them to study their
structure ,shape and other structural
characteristics
Types of stains
ACIDIC- Negatively charged and radicals imparts
color in eosin ,acid fuchsine , malachite green , indian
ink .
BASIC – positively charged basic redicals combines
with nigatively charged particles and gives color .
E.g – HEAMATOXILLIN , METHYLENE BLUE
,CRYSTAL VIOLET.
NEUTRAL – both positively and negatively chargd
imparts different colors to different components .
E.g – GEIMSA’S AND LEISHMAN ‘S STAIN .
STAINING TECHNIQUES
POSITIVE STAINING –
Where the actual cells are themselves colored and
appear in a clear background .
Simple staining ; - a stain which provides color
contrast but gives same color to all bacteria an cells .
E.g – Malachite Green and Crystal Violet.
Differential staining – a stain which imparts
different colors to different stain which contains
more than one stain .
E.g- GRAM’S STAIN ,Acid fast Staining and
Endospore staining .
Negative staining
Where the cells remains clear ( uncolored ) & the
background is colored to create a contrast to
aid in the better visualization of the image .
Nigrosin .
Indian ink .
Bacterial smear preparation
SMEAR – Is a distribution of bacterial cells for
the purpose of veiwing them under the
microscope .
METHOD –
Aseptically a small sample of the culture is
spread over a slide surface .
This is then allowed to air dry .
The next step of heat fixation to help the cells
adhere to the surface .
The smear is now ready for staining .
Smearing techniques
Heat fixation –
a. Pass air dried smears through a flame two or
three times .do not over heat .
b. Allow slide to cool before staining .
Methanol fixation –
a. Place air dried smears in a coplin jar with
methanol for one minute . Alternatively ,flood
smear with methanol for one minute .
b. Drain slides nd allow to dry before staining .
Simple staining
Loeffler’s Methylene blue ;-
It is generally the most useful ,its shows the characterstics
morphology of polumorphs ,lymphocytes and other cells
more clearly than do stronger stains such as gram stain or
dilute carbol fushsin .
Polychrome Methylene blue –
This is made by allowing loefflers methylene blue to
‘ripen’ slowly .
The slow oxidation of the methylene blue forms a violet
compound that gives the stain its poluchrome properties .
The ripening takes 12 months or more to complete ,or it
may be ripened quickly bt the addition of 1.0 potassium .
In contrast to the blue staining of most structures by methylene blue
,the violet components stain acidic cell structures red- purple .
Dilute carbol fuchin – made by diluting zeihl- neelsen ‘ stain with
10-20 times its volume of water .
Over staining must be avoided ,as this is an intense stain , and
prolonged applications colors the cell protoplasm in addition to
nuclie and bacteria .
Requirements –
loefflers methylene blue .
Dilute carbol fushsin .
Distilled water .
Compound microscope .
Cader wood oil
Fixed smear .
Procedure
Make a thin smear on a slide .
Heat fixes the smear by passing the slide 2-3 times
gently over the bunsen flame wu=ith the smear side up
.
Pour loeffer’s methylene blue over the smear and allow
it to stand for 3 minutes .
Wash the stained smear with water and air dry it.
Observe the smear first under low power (10x)
objective ,and then under oil immersion (100x)
objectives .
Observe the presence of organisms and also the cellular
content of sample .
