MICROSCOPY
acid, nitric acid and Sudan IV are
Light Microscopy
 Bright field Microscopy
 Dark field Microscopy
 Fluorescence Microscopy
 Phase-Contrast Microscopy
 Differential Microscopy
 Confocal Microscopy
 Polarizing Microscopy
 Electron Microscopy
 Transmission Electron Microscopy
 Scanning Electron Microscopy
Histochemistry is defined as the application to
histological preparations of physical or chemical
methods of analysis to identify the chemical
substances present in their normal sites in tissues.
FOUR BASIC REQUIREMENTS FOR A
HISTOCHEMICAL
① The substances being studied must not be
diffuse out their original sites.
② The product of the reaction should be
insoluble and stained or electron-scattering
③ The method employed should be specific for
the substance or chemical groups being
studies.
④ The procedure must not denature or block
reactive groups.
ACTIVE PRINCIPLES IN PLANTS
 ALKALOIDS
o Place a drop of iodo-potasssium
iodide (IKI) on the freshly-cut
sections mounted on a slide.
Heat the slide slightly if needed.
o RESULTS: chocolate brown
precipitate with alkaloids
o NOTE: Other reagents like picric
utilized to serve as confirmatory
tests for the presence of
alkaloids.
 GLUCOSIDES
o Treat fresh sections with 10%
nitric acid. A dark orange color
is indicative of the presence of
arbutin.
o Immerse sections in 1% picric
acid for 30 minutes, then wash
with water and treat with a drop
of 1% aqueous sodium carbonate
on a slide.
o RESULTS: A red color means
amygdalins’ is present.
 TANINS
o Treat fresh sections with 10%
aqueous ferric chloride plus a
little sodium carbonate.
o RESULTS: The positive color
reaction for tannin is blue green.
 SAPONINS
o Place fresh sections on a drop of
concentrated sulfuric acid on a
slide.
o RESULTS: Color reaction initially
is from yellow, then red reaction
within 30 minutes and finally, a
violet or blue green reaction
indicating the presence of certain
saponin.
o ANOTHER PROCEDURE!!!
o Place a fresh section in saturated
solution of calcium chloride, then
place 10% aqueous potassium
dichromate. At first, the
compound is found broken down
and the barium unites with
chromium chromate, which is
yellow in color.
o RESULTS: Cells with tannins will
yield brownish to red color
reactions.
  FORMIC ACID
o Place fresh sections in a few
drops of mercuric chloride
solution heated in water bath for
one hour, then wash with water
acidified.
STEPS IN TISSUE PROCESSING
 Specimen Accessioning – number
 Gross examination – size, weight, age
 Fixation – preservation
 Tissue Processing
 Sectioning
 Frozen Sections
 Staining
FIXATION
 The death of a cell leads rapidly to a
breakdown of cell constituents resulting
from internal enzymes and from damage
caused by external agents such as bacteria
and enzymes. The cell contents begin to
diffuse out of position as well as to change
chemically.
 Aim of tissue preparation: preservation of
the cell in a state as close to the living
condition as possible.
 Therefore, the selected tissue must be
placed in the selected fixative as soon as
possible after death. There must be no
delay.
 All solutions must be ready before the tissue
is removed from the state.

 H & E staining Characteristics:

 Cover slipping  Rapid penetration

 Ability to change the soluble cell contents
to insoluble substances so that they will
Histolog Light Electron remain in their original position.
ical Microscope Microscope  Protect tissues against shrinkage and
Techniq distortion during subsequent treatment.
ues  Mordant cell constituents enabling them to
Fixation Buffered Double Fixation: be reactive to certain stains
isotonic solution Buffered  Changing the refractive index of the
of 37% glutaraldehyde + cell/organelles so that they can be seen in
formaldehyde Buffered osmium the light microscope
tetroxide
Purpose:
Dehydrat Water + Dioxane↑or
ion Alcohol↑ (EtOH) Acetone↑ The purpose of fixation is to preserve tissues
Embeddi Paraffin, Resin Epoxy, Resin permanently in as life-like a state as possible.
ng Fixation should be carried out as soon as possible
Staining Staining H&E Phosphotungstic after removal of the tissues (in the case of surgical
(Hematoxylina acid [Negative pathology) or soon after death (with autopsy) to
nd Eosin) stain – Viruses, prevent autolysis.
Staining Nerves,
Fixative Agents:
Papanicolaou Polysaccharides]
staining [Pap Osmium tetroxide  Special fixative agents – multiple
smear] Sudan [Lipids] composition
staining [Lipids]  Primary fixative agents
 Coagulant fixatives were said to
Mounting Canada Balsam, Glycerol result in a permeable meshwork of
protein strands
 Non-coagulant fixatives which are
additive in nature, formed extensive
 cross-links producing a less a. Fix 12 to 24 hours in the dark. (depending
permeable gel. on the size of the tissue)
b. Wash in running tap water for 24 hours
SPECIAL FIXATIVE AGENTS: c. Transfer to 50 % ethanol for 2 hours.
Then transfer to 70% ethanol.
 Bouin’s Fluid
d. To remove mercuric chloride.
Composition:
Place the tissue in 70% ethanol to which
1. Picric acid, saturated aqueous solution enough iodine solution has been added to
(1.2gm/100cc) give a deep brown color. Leave in iodine-
70% ethanol until the color fails to disappear
2. Formalin (40% formaldehyde) (up to 6
3. Glacial acetic acid hours).

Preparation: Iodine Solution

a. Fix 12 to 24 hours depending on tissue 1. Potassium iodide 3 gm___
size. 2. 95% Ethanol 100cc___
b. Place directly in 70% ethanol with a small 3. Dissolve and add Iodine 2 gm
amount of lithium carbonate for at least 24
hours. Change the ethanol several times during These are excellent fixatives. Helly's is
this period. As much of the picric acid as especially good for cytoplasmic granules (secretion,
possible should be removed at this time neurosecretion). Helly's does not harden tissues as
because it tends to slow down paraffin much as Zenker's, but Zenker's penetrates faster.
penetration during embedding. Small Stock solution keeps indefinitely; keep it away from
amounts of lithium carbonate added to the strong sunlight.
ethanol help to remove the picric acid. All
picric acid must be removed before  Carnoy's Fluid
staining is begun. Composition
1. Glacial acetic acid 10 cc
Bouin's fluid works well with most 2. Absolute ethanol 60 cc
mammalian and invertebrate tissues. It is 3. Chloroform 30 cc
good for nuclei, but fixes cytoplasmic Preparation:
structures only indifferently. It is an excellent a. Fix 20 minutes to 3 hours (depending on
field fixative. The solution keeps indefinitely the size).
and tissues can be left in it for weeks. Tissue should be kept small for this fixative
no more than 5mm across.
 Zenker’s or Helly’s Fluid b. Transfer to 95% ethanol (1hour) and then
to 70% ethanol for temporary storage or
Composition
dehydrate immediately and embed in paraffin.
1. Potassium dichromate 2.5 gm
2. Mercuric chloride 5.0 gm Carnoy's works very rapidly and is an
3. Sodium sulfate 1.0 gm excellent cytoplasmic fixative. It is used
4. Distilled water 100.0 cc extensively for histochemical work. The
fixative keeps indefinitely.
Preparation:
Zenker's Fluid Mahdissan's Fluid is a variant of Carnoy's
Just before use, add 1 part formalin (40% Fluid.
formaldehyde) to 10 parts stock solution; - insect tissues.
use immediately. Composition
1. Absolute ethanol 60 cc
Helly's Fluid 2. Chloroform 30 cc
1. Immediately before use, add 1 part 3. Formalin (40% formaldehyde) 10 cc
formalin 4. Glacial acetic acid 5 cc
2. (40%formaldehyde) to 10 parts stock
solution; use immediately.  Baker's Formal-Calcium Fluid
Preparation:
 Composition
1. Formalin (40% formaldehyde) 10 cc
2. Calcium Chloride (anhydrous) 1 gm
3. Distilled water 90 cc
4. Calcium carbonate excess (Keep excess
carbonate in the bottom of the bottle).
Preparation
a. Fix 2 to 3 days. Store in fluid if necessary.
b. If lipid preservation is not important,
transfer the tissue directly to 70 % ethanol
and treat it in the same way as other tissues
to be embedded in paraffin. Tissues may be
stored in 70% ethanol.
When lipids are fixed special methods are
required for sectioning and staining. Avoid
fat solvents (xylene and benzene) because
Baker's fluid is neutral, it is also used for
preservation of calcareous structures.
However it should not be used for
localization of phosphatases. Penetration is
very slow; therefore it is important to cut the
tissues into small pieces. Formalin fluids are
excellent for preservation of an entire
organism if it is not to be used for general
dissection. The fluid keeps indefinitely.
 Regaud’s Solution
Composition
1. Potassium dichromate 2.4 gm
2. Distilled water 80.0 cc
3. Formalin 20.0 cc___
Preparation
a. Fix 2 days, change the fluid every
day.___
b. 3% potassium dichromate, 4 to 8 days,
changes the fluid every day.
c. Running water, 24 hours.___
d. Transfer to 50 % ethanol for 2
hours,___e. Transfer to 70% ethanol.
Regaud's solution is used for
mitochondria. Mix the solution immediately
before use. Fix the tissues in the dark.
 FAA Solution
Composition
1. 95% Ethanol 50 cc
2. Glacial acetic acid 5 cc
3. Formalin 10 cc
4. Distilled water 35 cc
Preparation
a. Fix thin leaf pieces 12 hours, small twigs
24 hours.___
b. Wash in 2 to 3 changes of 50 % ethanol,
24 hours each.___
c. Store temporarily in 70 percent ethanol.
Long term storage (1 month or more) should
be in 1 percent glycerine in 70 % ethanol.
 Craf Solution
Stock Composition
1. Chromium trioxide 1.0 gm
2. Distilled water 96.0 cc
3. Glacial acetic acid 4.0 cc
Immediately before use, mix equal parts
(1:1) of stock solution and 25 percent
formalin.
Preparation
a. Fix small, soft tissues (onion root tip) for
24 hours, larger tissues for 3 days to 1
week.
b. Was in running water 6 hours to
overnight.
c. Dehydrate in the following successive
solutions: 15, 30, and 50 % ethanol; 1/2 hour (root
tip) to 4 hours (larger tissue) each.
d. Store in 70 % ethanol until ready to
process.
Factors influencing chemical fixation
 Temperature – increasing the temperature
of fixation will increase the rate of diffusion
of the fixative into the tissue and speed up
the rate of chemical reaction between the
fixative and tissue elements.
 Time – the optimal time for fixation will vary
between fixatives
 Penetration rate – the penetration rate of a
fixing agent depends on its diffusion
characteristics and varies from agent to
agent.
 Specimen dimensions - should not be
more than 4 mm thick. Ideally a 3 mm thick
slice should provide excellent fixation and
processing.
 Volume ratio – fixative to tissue ratio of
20:1 is considered the lowest acceptable
ratio but I would advocate a target ratio of
50:1.
 pH and buffers – most popular
formaldehyde solution in use today is
therefore buffered to pH 6.8 – 7.2 for this
reason. For electron microscopy pH is
more important and should match
physiological pH.
 Osmolality - the osmotic effects exerted by
the fixative are again more important at the
ultrastructural level than at the level of the
light microscope because it is the
phospholipid membranes that are easily
damaged by excessively hypotonic or
hypertonic solutions, but osmolality does
have some relevance in routine
histopathology.