Alkaline phosphatase activity : Reddish brown
α-Naphthyl Acetate Method for Nonspecific Esterase
This method employs α-naphthyl acetate as the substrate,
the enzyme releases α-naphthol during the hydrolysis
of the substrate. The α-naphthol is then coupled with a
suitable diazonium salt to produce an insoluble azo dye at
Sodium dihydrogen orthophosphate 2.75 g
d. Pararosanilin HCl - Stock solution
Pararosanilin hydrochloride 2 g
Preparation of Incubating Medium
A volume of 0.4 mL of solution C and D is mixed together
and allowed to stand before adding to incubating medium.
a. Incubate smears at 37°C for 20 minutes.
c. Counterstain in 2% methyl green (chloroform extracted).
e. Dehydrate, clear and mount.
Diaminobenzidine Method for Peroxidase
Preparation of Incubating Solution
3:3 - diaminobenzidine tetrahydrochloride 5 mg
a. Rinse fixed smears in distilled water.
b. Transfer to incubating solution for 5 minutes at room
c. Rinse in 3 changes of distilled water.
d. Dehydrate clear and mount in DPX.
Peroxidase—fine brownish granules.
40% Formaldehyde (HCHO): 10 mL
To 30 mL of 30% ethanol, add 0.9 g of Benzidine, mix well
and then add 3 mL of zinc sulfate (3.8%). A precipitate is
a. Fix the smear with fixative for one minute.
b. Wash gently with tap water and soak out the excess
c. Take 5 mL of staining solution and 3 drops of 3%
hydrogen peroxide, mix well and cover the slide for
d. Wash the slide in running tap water for 10 seconds.
e. Counterstain with dituted Giemsa’s stain for 2 minutes.
f. Wash with tap water then dried and mounted in DPX.
Peroxidase positive seen as green granules.
It is a useful preliminary method to indicate two major
lipid classes. For detailed morphology oil red O method is
The working solution is prepared an hour in advance by
mixing three parts of a stock solution of oil red O (saturated
in 99% isopropanol) with two parts of distilled water and
Fix the smears in formalin vapor for 5 minutes and wash in
running tap water for 10 minutes.
b. Stain for 15 minutes in Oil Red O.
c. Differentiate in 60% isopropanol until a delipidized
control section appears colorless.
d. Wash in water and counterstain nuclei with Mayer’s
f. Rinse in distilled water and mount in glycerin jelly.
Unsaturated hydrophobic lipids and mineral
This is an immunohistochemical technique, aimed at
the specific histological localization of particular tissue
antigens by immunological method. These techniques
are being increasingly used for diagnostic histology. The
phosphatase enzymes are nowadays commonly used for
histochemical detection of various antigenic markers.
Direct method: The primary antibody conjugated with
with specific substrate. Tissues/smears are examined under
light microscope to detect the substrate color at the antigenic
Indirect method: It is more sensitive and commonly used.
The specific primary antibody is applied directly to the
tissues/smears. This is followed by the second antibody
(antispecies specific IgG) conjugated with enzyme. The
color of the reaction is developed by using specific substrate
and examined under light microscope.
Peroxidase conjugated with antirabbit immunoglobulins
(Igs), PBS (pH 7.2/0.2M), DAB (3.3 Diamino benzidine-4
HCl), normal swine serum, hydrogen peroxidase, antigen
specific antibody raised in rabbit.
1. Dewaxed paraffin sections are hydrated in usual
manner. Where prefixed smears are used, these are
washed with buffered distilled water.
3% solution of hydrogen peroxide in distilled water for
10–30 minutes or with acid alcohol for 15 minutes.
For Cryostat, use acid alcohol or phenylhydrazine
3. Wash twice with phosphate buffer saline *(pH 7.2, 0.2M).
4. Expose sections/smears to normal swine serum diluted
C, 5–10 minutes. Excess NSS/NGS
is removed, without washing prior to stage 4.
5. Sections/smears are treated with optimally diluted
primary rabbit antiserum at 22oC, 15–30 minutes or
24–48 hours at 4oC with highly diluted antiserum.
6. Treat sections/smears with horseradish peroxidase
labeled swine/antirabbit IgG 1:20-1:100 for 15–30
7. Wash twice with phosphate buffer saline* (pH 7.2). The
end product is revealed with a freshly made solution
of 0.05%, 3, 3-diaminobenzidine tetrahydrochloride
(DAB) in 0.01% H2O2 in wash buffer.
DPX for the DAB or PDP reactions (brown to dark brown).
Aqueous mountants, e.g. glycerin gelatine, are used for the
(Sodium dihydrogen orthophosphate)
NaH2PO4. 2H2O—31.2 g for 1 liter.
Na2HPO4. 2H2O—31.6 g for 1 liter.
Solution A 70 mL + Solution B 180 mL. Make the volume
up to 1 liter and dissolve 5.7 g NaCl in a liter and filter
It is a histochemical or cytochemical technique for in
situ detection and localization of specific intracellular
antigens. Specific antibodies conjugated with fluorescent
dyes, such as fluorescein or rhodamine, are used to trace
the specific antigenic areas on the tissue smear or section.
This can be visualized under the fluorescent microscope,
as bright purple green/red color fluorescence.
In this method, conjugated antiserum is added directly to
the tissue sections or viable cell suspension.
It is more sensitive and commonly used. The unlabeled,
specific antibody is applied directly to the tissue smears/
sections, followed by a second antibody treatment, i.e.
antispecies specific Ig conjugated with fluorescein or
rhodamine and examined under UV-microscope. Due to
the use of second antibody, the sensitivity and specificity
of the reaction is highly improved.
FITC/antihuman Igs conjugate, phosphate buffer saline
(PBS), specific antibody, glycerol buffer, fluorescent
microscope. Smear or section of tissue.
1. Reasonably diluted antibody put on the antigen slide
fixed in methanol for half an hour at room temperature
2. Wash the slide twice in PBS pH 7.2. All washes are
carried out on a magnetic stirrer.
3. Incubate slides for 30 minutes with 1:20 diluted
FITC (Fluoroscein isothiocynate) conjugated with
Igs in PBS/pH 7.2* containing 0.01%. Evans blue
as counterstain at room temperature in a moisture
4. Wash the slide twice in PBS pH 7.2.
5. Mount the slide with 90% glycerol buffer pH 8.6.
6. Examine the slide under UV-microscope. The antigen
positive areas of the cell will show purple green
fluorescence, whereas the negative area would appear
A programable cytocentrifuge from WESCOR is available,
which can be used to prepare slide from any body fluid.
With the help of cytocentrifuge sample cells, one can
safely and quickly deposit a monolayer of cells on to a
microscope slide for staining or any other processing.
This can be used on any of the body fluids, such as CSF,
urine, synovial fluid, aspirates, washes, etc. and can be
programed as per the requirements.
* PBS (Phosphate buffer saline) pH 7.2/0.2 M
* PBS preparation described earlier.
In the following pages maximum stress is laid on diagnostic
Schizomycetes (Bacteria and related forms)
These members form elongated cells and have a tendency
to branch, produces spores, not all are pathogenic to man
This represents the true bacteria forms, classifiable as
bacilli, cocci, or vibrios. Their staining reaction can either
be gram-positive or gram-negative. Some are motile and
possess peritrichous flagella. They multiply by binary
fission. Widely distributed they can be saprophytes,
parasites and many are pathogenic to human beings.
¾ Pseudomonadaceae → Spirillaceae
¾ Enterobacteriaceae → Bacteroidaceae
¾ Corynebacteriaceae → Bacillaceae
¾ Lactobacillaceae → Neisseriaceae
¾ Micrococcaceae → Brucellaceae.
These are slender, spiral shaped cells, aflagellate but move
by flexing or whirling and spinning. Stainable by special
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