Fixation of Tissues - 2

Northern Borders University
Faculty of Applied Medical Sciences

Department of Medical Laboratory Technology
Histopathology Technique-I
2nd Semester
2023 – 2024
1. Fixation of tissues
Introduction
 Histology:
 Science of examination of normal tissues
 Histopathology:
 Examination of tissues for presence /
absence of changes in structure due to disease
process.
12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 3

Introduction
 Preparation of tissues for microscopic examination,

through a series of processes namely fixation,
dehydration, clearing, embedding, cutting, staining
and examination under microscope.
 Tissues decompose when removed from the body or
it is cut off from the blood supply - Autolysis and
Putrefaction.

Introduction
 Definition of fixation
 Complex series of chemical events which differs for
different groups of substances found in tissues .
 Or
 A histological technique used in preserving biological tissue
specimens for later cytologic, histologic, or pathologic
examination through the microscope.
 Definition of Fixative:
 A fixative may be defined as a substance which prevent post
mortem changes and preserves the morphological and
chemical characteristics of cells and tissues..

Fixation of tissues
 As soon as cells or tissues are removed from
the body they begin to die and undergo post-
mortem changes.
 These changes may be autolytic or
putrefactive .
 Autolysis: Is a self-destructive process due to
the release of autolytic enzymes (lysosomes)
from the dead cells.
 Putrefaction: Occurs due to the action of
bacteria that invade the tissue.
Principle in the action of fixative
 Fixatives act by denaturing or precipitating

proteins which then form a meshwork due to cross
linking of proteins.
 This meshwork tends to hold the other cell
constituents in vivo relation to each other and
insoluble proteins provide mechanical strength for
subsequent procedures.

Pre-fixation procedures & precautions
 Dissecting table made up of hard wood with a working

surface area of 45 X 35 cm
 Wood- actual cutting surface
 Rubber pads of various dimensions- suitable
 Instrument- thin bladed knives 20-30 cm in length, scissors,
probes, scalpels, stainless-steel rule, forceps, sponges
 First-aid box & eye-wash bottle
 Cutting board & instruments should be cleaned before
procedures
 Washing of the gloved hands before dissection
 Fixation of tissue- as soon as possible after death or
removal from the body.

Pre-fixation procedures & precautions
 Screw capped specimen

jars containing
appropriate fixatives
 Amount of fluid in the
jars- 15-20 times
 Early dispatch of the
specimens to
histopathology
laboratory

Washing
 Tissues should be
washed in
physiological saline
 Excessive blood &
mucous should not
be there.

Selection of tissue
 Tissues selected for sectioning should be sufficiently thin to be adequately fixed
throughout a reasonable time
 Plastic tissue cassettes are suitable for holding and identifying tissue samples in
processing, embedding, and sectioning procedures. The cassettes fit securely in
microtome.
 Best thickness- 3-5mm.

All fixatives have advantages and disadvantages, including
 Molecular loss
 Tissue swelling or shrinkage
 Variations in staining quality
 The ability to perform accurate biochemical
analysis.

Purpose of Fixation
 Preservation of cells & tissue constituents in a condition identical to
that existing during life.
 To prevent autolysis, bacterial decomposition & putrefaction of the
cell. To coagulate the tissue as to prevent loss of easily diffusible
substances.
 To fortify the tissue against deleterious effects of further stages.
 To facilitate differential staining with dyes & other reagents.
 It should penetrate evenly and rapidly.
 It should harden the tissues
 Increase the optical differentiation of cells & tissues
 Should not cause shrinkage or swelling of the cells
 Must not react with the receptor sites & thus must not interefere with
the staining procedure
 It must be cheap and easily available.
 Good fixative is most important factors in the production of
satisfactory results in histopathology
Following factors are important
 Fresh tissue
 Proper penetration of tissue by fixatives
 Correct choice of fixatives
 No fixative will penetrate a piece of tissue
thicker than 1 cm.

Types of methods of fixation
1. Perfusion/ in vivo fixation:

 Fixation of whole animals by perfusing the
animal’s vascular system with a fixative.
1. Immersion/ in vitro fixation
2. Perfusion-immersion
3. Fixation using vapors method.

For dealing with specimen thicker, following
methods are recommended
1. Solid organ:
 Cut slices as necessary as
but not thicker than 5 mm.
2. Hollow organ:
 Either open or fill with
fixative or pack lightly with
wool soaked in fixative.
3. Large specimen:
 It requires dissection, inject
fixative along the vessels or
bronchi as in case of lung
so that it reaches all parts
of the organs.
Methods of specimen fixation
Solid organ Hollow organ

Properties of an ideal fixative
 Preserves tissue in their natural state and fix all tissue components.
 Make the cellular components insoluble to reagent used in tissue
processing
 Cause minimum physical and chemical alteration of the tissue and its
components
 Should penetrate a tissue quickly
 Neither shrinks nor swells
 Makes the specimen hard enough
 Raises refractive indices
 Provides full range of staining methods with great selectivity
 No rigid upper limit of fixing time
 No tendency to deteriorate
 Non-toxic, stable & safe to handle
 Non-inflammable & non-irritant
 As such no ideal fixative
 Choice depends on the cell or tissue constituent to be demonstrated
Mechanism / Action of Fixatives
 Most fixatives act by denaturing or precipitating

proteins which then form a sponge or meshwork,
tending to hold the other constituents
 At the molecular level, fixative have the property
of coagulating proteins in the tissue, through the
formation of crosslink's between protein molecules
thereby keeping their relation to each other.

General classification of fixatives
1. Aldehydes (cross linking agents):

 Formaldehyde and Gluteraldehyde
 Act by creating covalent chemical bonds between
proteins of tissues- Oxidizing agents- joins with
various side chains of protein molecules & other
biomolecules- allow formation of cross link-
stabilizes tissue structure.

2. Protein denaturing agents:

 Acetic acid, methyl alcohol, ethyl alcohol
 Reduce the solubility of protein without
combining with it & disrupts the
hydrophobic bonds which is needed for its
tertiary structure to form.

3. Unknown mechanism:
 Mercuric chloride (B5 fixatives)
 It increases the staining brightness & give
good nuclear detail. Good for reticulo-
endothelial tissue & haemopoetic tissue.

4. Picrates:
 Picric acid binds with histone & basic proteins to
form crystalline picrates with amino acid &
precipitates protein.
 5. Oxidizing agents:
 Osmium tetroxide, potassium permanganate,
potassium dichromate.

1. Physical methods
 Such as heating, microwaving, and freeze-drying

are independent processes and not used
commonly in the routine practice of
Histopathology, except for the use of dry heat
fixation of microorganisms prior to Gram
staining.

1. Physical methods
1. Heat fixation:
 The simplest form of fixation is heat. Boiling or
poaching an egg precipitates the proteins and, on
cutting, the yolk and egg white can be identified
separately.
2. Microwave fixation:
 Microwave heating speeds fixation and can reduce
times for fixation of some gross specimens and
histological sections from more than 12 hours to
less than 20 minutes.

1. Physical methods
3. Freeze-drying and freeze substitution:

 Is a useful technique for studying soluble
materials and small molecules; tissues are
cut into thin sections, immersed in liquid
nitrogen, and the water is removed in a
vacuum chamber at -40°C.
 The tissue can be post-fixed with
formaldehyde.

2. Chemical fixation
 Most methods of fixation used in processing of tissue

for histopathological diagnoses rely on chemical
fixation carried out by liquid fixatives.
 Chemical fixation utilizes organic or non-organic
solutions to maintain adequate morphological
preservation.
 Chemical fixatives can be considered as members of
three major categories:
1. Coagulant
2. Crosslinking
3. Compound fixatives.

2. Chemical fixation
 1. Coagulant fixatives:
 Both organic and non-organic solutions may coagulate proteins, making them
insoluble.
 Coagulating such proteins maintains tissue histomorphology at the light
microscopic level.
 Dehydrant coagulant fixatives
 The most commonly used coagulating fixatives are alcohols (e.g. ethanol,
methanol) and acetone. Methanol is closer to the structure of water than
ethanol.
 Dehydrant fixatives act to remove free and bound water, causing a change to
the tertiary structure of proteins so that they precipitate, leaving the nucleic
acids relatively unchanged.
 Ultrastructure is destroyed by any of these four dehydrants due to the
extraction of lipids, and each may cause excessive shrinking of tissue
components after more than 3–4 hours of fixation.
 Each of these fixatives can be modified by adding other chemicals to produce
specific effects.

1. Coagulant fixatives
 Examples of dehydrant coagulant fixatives

1. Absolute Ethanol
2. Absolute Methanol
3. Ethanol, 95%
4. Acetone
 Methanol is useful for touch preparations and smears,
especially blood smears.
 Other types of coagulant fixative
 Acidic coagulants such as picric acid and trichloroacetic
acid change the charges on the ionizable side chains
 Acetic acid coagulates nucleic acids but does not fix or
precipitate proteins; it is therefore added to other fixatives
to prevent the loss of nucleic acids.
2. Non-coagulant cross-linking fixatives
 Several chemicals were selected as fixatives

secondary to their potential actions of forming
cross-links within and between proteins and
nucleic acids as well as between nucleic acids and
proteins.
 Examples of chemical fixatives include:
 Formaldehyde, glutaraldehyde, and other
aldehydes, e.g. chloral hydrate and glyoxal, metal
salts such as mercuric and zinc chloride, and other
metallic compounds such as osmium tetroxide.

1. Simple Fixatives
 1. Formaldehyde fixation:
 Formaldehyde in its 10% neutral buffered form (NBF) is the most common fixative used
in diagnostic pathology.
 Pure formaldehyde is a vapor that, when completely dissolved in water, forms a solution
containing 37–40% formaldehyde; this aqueous solution is known as ‘100 % formalin’.
 The time to saturation of human tissues with active groups by formalin is about 24 hours,
but cross-linking may continue for many weeks.
 When formaldehyde dissolves in an un-buffered aqueous solution, it forms an acid
solution (pH 5.0 - 5.5) because 5 - 10% of commercially available formaldehyde is formic
acid.
 Stabilizer – 10 - 14% methanol
 On storage becomes acidic by formation of formic acid.
 Turbid on keeping - paraformaldehyde.
 Yellow - contaminated with ferric iron from metal containers (Positive Prussian blue
reaction).
 Fixes protein, lipids well preserved.
 Favors staining of acidic structures like nuclei with basic dyes
 Diminishes effect of acid dyes on basic structures like cytoplasm.

1. Formaldehyde fixation
Advantages of Formaldehyde
 Formaldehyde primarily preserves peptide protein bonds
and the general structure of cellular organelles.
 Easy to prepare
 Relatively stable
 It allows subsequent use of most staining procedures.
 Frozen sections can be prepared with ease.
 Fixes lipids for frozen sections and tissue enzymes.
 Penetrates tissues well.
 Beneficial hardening with little shrinkage
 Natural tissue colors are retained.
 Does not require washing before processing.
 Best fixative for nervous system.

 Disadvantage of Formaldehyde
 Using acid formalin for fixation is the formation of a
brown-black pigment with degraded hemoglobulin.
 Unpleasant vapour irritant to the nose, the eyes and
mucous membranes
 Irritation to respiratory epithelium
 Formalin dermatitis
 Formation of precipitate of paraformaldehyde which
can be prevented by adding 11- 16 % methanol.
 It causes shrinkage of collagen.
 It suspected to contain cancer producing agents.

 Formalin pigment
 Brown, granular material, extracellular, birefringent
 Progressive in deposition
 Often seen after several days
 Action of acid formalin on blood
 Avoided by using buffered formalin.
 Removed by treatment with saturated alcoholic solution of
picric acid for 20mins
 For routine histopathology, 10% neutral buffered formalin
(NBF) is frequently used for initial fixation and for the first
station on tissue processors.

2. Glutaraldehyde fixation
 Glutaraldehyde is a bifunctional aldehyde that

probably combines with the same reactive groups
as does formaldehyde.
 In aqueous solutions glutaraldehyde polymerizes,
forming cyclic and oligomeric compounds.
 It is oxidized to glutaric acid.
 To aid in stability, it requires storage at 4°C and at
a pH of around 5.
 Used for Electron Microscopy with osmium
tetroxide.

2. Glutaraldehyde fixation
 Advantage:
 Most efficient cross linking agent for collagen
 More rapid fixation than formalin.
 Disadvantage:
 False positivity with Periodic Acid Schiff’s (PAS).

3. Osmium tetroxide fixation
 Pale yellow.
 Toxic solid
 Soluble in water as well as non-polar solvents and can
react with hydrophilic and hydrophobic sites
including the side chains of proteins, potentially
causing crosslinking.
 Excellent preservation of detail of single cells
 Use as a secondary fixative for electron microscope
examinations
 Used to stain lipids (myelin) in frozen sections.
 Storage in dark, cool place.

3. Osmium tetroxide fixation
 Disadvantages:
 Uneven penetration for pieces more than 2-3mm
 Vapor is irritating, causes conjunctivitis
 Causes tissue swelling which is reversed during
dehydration steps.
 Swelling can be minimized by adding calcium or
sodium chloride to osmium-containing fixatives.

4. Mercuric chloride
 White crystalline substance.

 Powerful protein precipitant, fixes both nucleus &
cytoplasm well favoring its staining.
 Enhancing the staining properties of tissues,
particularly for trichrome stains.
 Conjunction with other fixatives.
 Hardens tissue
 Radio-opaque.
4. Mercuric chloride
 Disadvantages:
 These fixatives penetrate slowly, so specimens must be thin
 Mercury and acid formaldehyde hematein pigments may deposit in tissue after
fixation.
 Formation of deposits of intensely black precipitates of mercuric pigment in
the tissues.
 Should be dissolved in distilled water to prevent the precipitation of mercury
salts.
 These precipitates can be readily removed by a Lugol’s iodine step in the
staining procedure, followed by bleaching of the section in sodium
hypochlorite solution (Hypo).
 Mercury-based fixatives are toxic and should be handled with care.
 Corrosive to metals, they should not be allowed to come into contact with
metal.
 Pollution to environment, mercury-containing chemicals are an environmental
disposal problem.

2. Compound fixatives
 Other agents may be added to formaldehyde to produce specific effects that are
not possible with formaldehyde alone.
 The dehydrant ethanol, for example, can be added to formaldehyde to produce
alcoholic formalin.
 This combination preserves molecules such as glycogen and results in less
shrinkage and hardening than pure dehydrants.
 Compound fixatives are useful for specific tissues, e.g. alcoholic formalin for
fixation of some fatty tissues, such as breast to aid in identifying lymph nodes
embedded in fat.
 Some combined fixatives including alcoholic formalin are good at preserving
antigen immunorecognition.

 Types of Mercuric chloride containing fixatives
1. Zenker's Solution:
 M.C. Fixatives rapidly penetrates tissues and
permit excellent staining of nuclei and connective
tissues.
 Disadvantage :
 Causes hardening of tissues
 Fixed tissue should be washed overnight in
running tap water before processing.

2. Helly’s solution:
 By adding formalin to Zenker’s solutions the beneficial
effects of both fixatives are combined, minimizing their
disadvantages.
 Staining of nucleus, cytoplasm and connective tissue is
good with Helly’s fluid.
 Fixation time 12-24 hours.
3. Schaudinn’s solution
4. Ohlmacher’s solution
5. Carnoy-Lebrun solution
6. B5 fixative.

Dichromate-containing fixatives:
 Fixatives containing chromate at a pH of 3.5–5.0 make proteins
insoluble without coagulation.
 More acidic pH – fixes nucleus & cytoplasm
 Chromate is make unsaturated but not saturated lipids insoluble upon
prolonged (>48 hours) fixation and hence mitochondria are well
preserved by dichromate fixatives.
 Dichromate-containing fixatives have primarily been used to prepare
neuroendocrine tissues for staining, especially normal adrenal medulla
and related tumors (e.g. phaeochromocytomas).
 Wash in running water after to prevent formation of insoluble
precipitate.
 Prolonged exposure causes tissue to become brittle.
 Use as mordant.

 Types of dichromate-containing fixatives:

1. Chromic acid
2. Miller’s or Möller’s solution
3. Möller’s or Regaud’s solution
4. Orth’s solution.

 Picric acid -containing fixatives
 Many picric acid fixatives require a saturated
aqueous solution of picric acid.
 Aqueous picric acid 2.1% will produce a
saturated solution and 5% picric acid a saturated
solution in absolute ethanol.
1. Bouin’s fluid
2. Hollande’s solution
3. Fixatives for DNA, RNA, and protein analysis.

3. Histochemical fixatives
 These are used to demonstrate the chemical constituents

of the cell.
 Preserve the constituent to be demonstrated & its
morphological relationships.
 Without affecting the reactive groups & reagent to be
used in its visualization.
 Best – cryostat cut sections of rapidly frozen tissue.
 Cold acetone – where enzymes are to be studied especially
phosphatases
 Vapour fixatives like formaldehyde, acetaldehyde,
gluteraldehyde, acrolein to fix cryostat cut sections of
fresh tissue & blocks of frozen dried tissue.

The most commonly fixatives used in histopathology lab
1. 10% Formalin
2. Carson’s modified Millonig’s phosphate:
 Is buffered with sodium monobasic phosphate and sodium
hydroxide.
3. 10% Formal calcium:
 Recommended for the preservation of lipids especially
phospholipids.
4. 10% Formal saline
5. Formalin, buffered saline
6. 10% Zinc formalin (unbuffered):
 It gives improved results with Immunohistochemistry
(IHC).

Factors affecting the quality of fixation
1. Buffers & hydrogen ion concentration (pH)

2. Duration of fixation and size of specimens
3. Temperature
4. Concentration
5. Osmolality of fixatives and ionic composition
6. Additives
7. Volume ratio
8. Choice of fixatives
9. Penetration
10. Tissue storage

1. Buffers & hydrogen ion concentration (pH)
 The most reliable way of achieving optimal formalin

fixation is through its buffering at pH 7.2–7.4 (i.e. neutral
buffered formalin).
 The choice of specific buffer depends on the type of
fixative and analyte.
Commonly used buffers are:
 Phosphate, Cacodylate, Bicarbonate, Tris, and Acetate.
 It is necessary to use low salt-buffered formalin in the
new complex tissue processors in order to keep the
machine ‘clean’, and reduce problems in its operation.

2. Duration of fixation and size of specimens
 Blocks should be small or thin (3 – 5 mm) to ensure adequate penetration.
 The depth (d) reached by a fixative is directly proportional to the square root of
duration of fixation (t) and expressed this relation as:
d = k √t
 The constant (k) is the coefficient of diffusability, which is specific to each
fixative.
 NBF, will penetrate tissue to the depth of approximately 1 mm in one hour
 Gross specimens should not rest on the bottom of a container of fixative: they
should be separated from the bottom by wadded fixative-soaked paper or cloth,
so allowing penetration of fixative or processing fluids from all directions.
 When surgical specimens are to be processed to paraffin blocks, the time of
penetration by fixative is more critical.
 Proteins inactivate fixatives, especially those in blood or bloody fluids.
 Bloody gross specimens should therefore be washed with saline prior to being
put into fixative.
 The fixative volume should be at least 15 times the volume of the tissue
specimen for optimal, rapid fixation i.e 1:15.

3. Temperature of fixation
 Most tissues fixed at room temperature.
 The diffusion of molecules increases with rising
temperature due to their more rapid movement and
vibration; i.e. the rate of penetration of a tissue by
formaldehyde is faster at higher temperatures.
 Microwaves therefore have been used to speed
formaldehyde fixation by both increasing the temperature
and molecular movements.
 Increased vapor levels are a safety problem.
 Most chemical reactions occur more rapidly at higher
temperatures and therefore formaldehyde reacts more
rapidly with proteins.
 Electron microscopy & histochemistry at 0 - 4˚C.
4. Concentration of fixative
 Different concentrations have different effects on
morphology.
 Effectiveness and solubility primarily determine the
appropriate concentration of fixatives.
 Concentrations of formalin above 10% tend to cause
increased hardening and shrinkage.
 Higher concentrations result in formalin being present in
its polymeric form, which can be deposited as white
precipitate.
 Ethanol concentrations below 70% do not remove free
water from tissues efficiently.
 Effects subsequent staining.

5. Osmolality
 6. Osmolality of fixatives and ionic composition

 The osmolality of the buffer and fixative is important.
 Hypertonic solutions lead to shrinkage.
 Hypotonic solutions cause swelling of cells & poor fixation.
 The best morphological results are obtained with solutions that are
slightly hypertonic (400–450 mOsm)
 The osmolality for 10% NBF is about 1500 mOsm.
 The ionic composition of fluids should be as isotonic as possible to the
tissues.

6. Additives
 The addition of electrolytes and non-electrolytes to fixatives improves
the morphology of the fixed tissue.
 These additives include calcium chloride, potassium thiocyanate,
ammonium sulfate, and potassium dihydrogen phosphate.
 The electrolytes may react either directly with proteins causing
denaturation, or independently with the fixatives and cellular
constituents.
 Fixatives buffered with electrolytes such as phosphates may cause
problems with some processors due to precipitation of the salts.
 The addition of non-electrolyte substances such as sucrose, dextran, and
detergent improve fixation.
Secondary fixation
 Tissues fixed with two fixatives in

succession.
 Improved preservation & staining.
 Tissues fixed in buffered formalin –
fixed with mercuric chloride.
 Tissues fixed with gluteraldehyde is
post fixed with osmium tetroxide for
electron microscopy.

Post chromatization
 Treatment of tissues with 3% potassium

dichromate following normal fixation.
 Before processing – tissue in dichromate
solution for 6- 8days
 After processing – for 12- 24 hrs
 Both followed by washing in running water
 Employed to mordant tissues.
 Mitochondria & myelin demonstrated.
 Improved preservation and staining of
elements.
THANK YOU

Fixation of Tissues - 2

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Northern Borders University

Faculty of Applied Medical Sciences

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 3

 Preparation of tissues for microscopic examination,

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 4

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 5

 Fixatives act by denaturing or precipitating

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 7

 Dissecting table made up of hard wood with a working

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 8

 Screw capped specimen

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 9

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 10

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 11

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 12

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 14

1. Perfusion/ in vivo fixation:

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 15

Solid organ Hollow organ

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 17

 Most fixatives act by denaturing or precipitating

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 19

1. Aldehydes (cross linking agents):

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 20

2. Protein denaturing agents:

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 21

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 22

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 23

 Such as heating, microwaving, and freeze-drying

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 24

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 25

3. Freeze-drying and freeze substitution:

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 26

 Most methods of fixation used in processing of tissue

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 27

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 28

 Examples of dehydrant coagulant fixatives

 Several chemicals were selected as fixatives

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 30

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 31

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 32

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 33

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 34

 Glutaraldehyde is a bifunctional aldehyde that

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 35

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 36

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 37

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 38

 White crystalline substance.

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 40

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 41

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 42

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 43

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 44

 Types of dichromate-containing fixatives:

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 45

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 46

 These are used to demonstrate the chemical constituents

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 47

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 48

1. Buffers & hydrogen ion concentration (pH)

12/2/2023 NBU, AMS, Dr.EIM & DMO, Histo 49