MOSES TABIWAN /CYTOLOGIC

1. CYTOLOGIC STAINING
TECHNIQUES
RASHID ADAMS

2. PAPANICOLAOU STAIN
 A polychrome staining reaction designed to show
the many variations of cellular morphology
demonstrating degrees of cellular maturity and
metabolic activity.
 Essentially a modification of the conventional
haematoxylin and eosin stain to be more specific
for cytoplasmic changes and to provide
cytoplasmic contrast between cells with differing
maturity and metabolic activity.
 Greatest value is the resultant cell transparency so
that overlapping cells or those in three-dimensional
configurations can be seen in detail

3.  Because of the high alcoholic content, the counterstains
provide clear visualization through areas of overlapping
cells, mucus and debris.
 The use of an alcoholic fixative instead of aqueous
solution results in a nuclear stain that demonstrate
unique chromatin patterns found in normal and
abnormal cells.
 The main advantages of this stain are:
 Definition of nuclear detail
 Cytoplasmic transparency
 Cell differentiation
 Four main steps:
 Fixation
 Nuclear staining with haematoxylin

4.  Cytoplasmic staining with counterstains orange G and
EA
 Clearing and mounting
 Interspersed with steps to add solutions that
hydrate, dehydrate, and rinse cells.
 Two ways of hydration:
 Beginning with a high concentration of alcohol and
going through graded lower levels of alcohol until the
cellular smear is immersed in water.
 Abrupt, immediate immersion of the smear from high
concentration of alcohol directly into water.
 Dehydration follows a similar pattern, either
gradual or abrupt replacement of water by alcohol

5.  The gradual displacement of alcohol by water and
of water by alcohol is thought to minimize cellular
distortion and to reduce cell loss from the slide
surface caused by convection currents of the
solutions.
 Disputed by others hence the one-step hydration
and dehydration method.
 Rinses following each staining stage must be done
in the solvent of the stain:
 Rinsing water following staining in aqueous
haematoxylin
 Rinsing in alcohol following orange G and EA.

6.  Because of variability of the cell sample both in
fixation and pH there will be variation in the normal
staining of a specific cell type.
 Papanicolaou stain should therefore be used as an
aid and not the final answer in differentiating one
cell type from another.
 Normal variations in colour of specific cell types is
important in helping the cytotechnologist in
checking a particular batch of stained slides and
adjusting the staining reaction by microscopic
checks.

7. Nuclear Stain
 Haematoxylin is the nuclear stain used in the
Papanicolaou staining method.
 One of the few natural stains used in the
laboratory.
 Name derived from old Greek words Haimato
(blood) and Xylon (wood), referring to its dark red
color in natural state, and to its method of
manufacture from wood.
 Precise chemical formula known but synthesis is
not practical.
 Dye extracted by boiling wood of South American
and West Indian logwood tree (Haematoxylon
campechianum), partly purified by recrystallization

9.  Comes as brownish tan powder, poorly soluble in
water and somewhat more soluble in ethyl alcohol
(1 gm/100ml water and 30-40 gm/100 ml alcohol).
 Haematoxylin:
 Is a colourless compound
 Easily oxidised to haematein – an orange to reddish
compound; the active dye
 Brownish powder sold commercially a crude
mixture of haematoxylin, haematein and other,
unidentified substances
 Haematein can be bought, but expensive, and
same results can be had simply by oxidizing crude
haematoxylin

10. OXIDATION OF HAEMATOXYLIN
TO HAEMATEIN

11.  Oxidation was carried out by making up
hematoxylin solution, plugging container with
cotton, and leaving it exposed to light and air for 6
weeks to several months
 However, oxidation or ripening achieved much
faster by adding variety of oxidizing agents - two
most common being sodium iodate and mercuric
oxide
 Oxidation not only begins immediately, but
apparently continues for some time, causing dye
precipitates of uncertain composition, and
eventually exhausting stain

12.  Addition of glycerin to several formulas is said to
guard against over-oxidation and improves the
keeping properties and perhaps to retard fungal
growth
 Haematein is anionic and not an efficient colorant
of cells/tissue by itself and easily removed by
solvents.
 Used alone is a poor stain, but in combination with
various metallic salts – mordants – which couple it
to the tissue, is one of the best nuclear stains
known.
 Combination of dye and mordant called dye “lake”
or coloured complexes
 Lakes with different metals have different colors

13. METAL COLOUR
Aluminum Purple to blue
Iron Blue-black
Chromium Blue-black
Copper
Blue-green to
purple
Nickel Violet shades
Tin Red
Lead Dark brown
Osmium Green brown

14.  Two methods to stain nuclei:
 Progressive Method: Nucleus stained to desired colour
intensity, leaving background unstained.
 Eliminates decolourising with HCl and subsequent
running-water bath.
 May be necessary to examine slides at several different
intervals to determine when staining is dark enough but
not too dark
 Regressive Method: Overstain with an unacidified
haematoxylin, then remove excess stain with dilute HCl
– process called differentiation
 Difference between methods largely one of
convenience:

15.  Progressive haematoxylins generally less concentrated
and work slowly to avoid overshooting the endpoint.
 Regressive haematoxylins more concentrated and many
can achieve overstaining in a matter of less than a
minute, while differentiation requires only a few
seconds.
 Also, timing not so important in regressive procedures.
As long as slide is overstained, it doesn't matter whether
it was in staining dish for 1 minute or more.
 Regressive procedures therefore faster and more
convenient than progressive ones
 Added advantage that differentiation also removes
haematoxylin from gelatin or other slide adhesive,
producing clear, transparent background.

16.  Non-gynae cell samples do not adhere to slides as well
as gynae samples so progressive method (does not
require running-water bath) recommended
 Alum haematoxylin acts like a basophilic stain with
affinity for basic nucleoproteins (pH of solution is
2.95 and with addition of 2% acetic acid pH is
2.34).
 Differentiation done in dilute acid, usually acid
alcohol because of haematoxylin's greater
solubility in alcohol.
 Stopped immediately by simply washing the slides
in water.

17. Fixed Slides
Preparing for aqueous stain
Preparing for alcohol stain
Preparing for mounting medium
Preparing for clearing
100% alcohol Mount Slides
95% Ethanol
Hydration
Haematoxylin Haematoxylin
Rinse in water Rinse in water
Blueing solution Dilute HCI
Rinse in water
Dehydration
Cytoplasmic Staining
OG
Rinse in alcohol
EA polychrone
Rinse in alcohol
Final Dehydration
Clearing

18.  Alum haematoxylin solutions impart on nucleus
light transparent blue stain which rapidly turns red
in presence of acid
 To cause colour to change from red to desired
blue, pH raised to over 8.0 (i.e. make it alkaline).
 Accomplished by rinsing in:
 Tap water if pH of the water is over 8.0
 Dilute solutions of ammonium hydroxide – pH 8.0-8.5
 Lithium carbonate pH 8.0-8.5

19.  Scott’s tap water substitute pH 8.2 - 33.5 g NaHCO3 and
20 g MgSO4 in one liter of water with thymol (to inhibit
formation of molds),
 These are referred to as “blueing agents”.
 Blueing agent not removed by water continues to
act
 Dilute HCl used in regressive method must also be
removed by thorough rinsing in water otherwise its
action will continue.
 Once the nuclei have been stained, stain is usually
“set”.
 Stain can be removed by chlorinated tap water but
not by alcohol rinses in subsequent solutions.
 If water used has high chlorine content, fading of
nuclear staining may occur.

20.  Therefore, both chlorine and pH levels of tap water
used should be tested.
 To ensure consistency of staining on a daily basis,
important to make microscopic checks after critical
steps in staining procedure e.g. after progressive
blueing and water rinses and after regressive HCl
plus running water rinses.
 Dyes should be changed after 2000 slides have
been stained or 6-8 weeks, whichever comes first.

21.  Variability of Haematoxylin
 Strength of all hematoxylins varies from day to
day, becoming a little weaker with every use
because slide racks carry a little water over with
them, causing some degree of dilution.
 Strength becomes stronger each time a new
solution is made.
 Oxidation continues slowly and irregularly from
day to day, further reducing dye strength by
producing unpredictable amounts of precipitated
dye which must be filtered out.

22.  Oxidation produces several oxidized derivatives of
hematein, from monoxy- to pentoxyhematein, each
with a different color.
 Di- and tri-oxy derivatives appear to offer optimum
colour. Tetroxyhematein is brownish and
pentoxyhematein is colorless, so obviously oxidizing
step can be carried too far and render dye unusable.
 To slow down aerobic oxidation of stock solution in
storage, some people cover surface with a layer of
oil and pipette from below surface when more stain
is needed.

23.  Original hematoxylin was a crude extract of
logwood, so may contain hundreds of unknown
substances besides hematoxylin, and some of
them and their oxidized products may be
responsible for some of the precipitates.
 Note "dye content" on label of jar containing
hematoxylin powder. A dye content of 50% means
that only half of the powder is hematoxylin, and the
other half is a mixture of unknown composition.

24. Cytoplasmic Stains
 Synthetic dyes
 Derived from coal tar products
 Chemical formulae standardised and so no
variation in batches of dye powder.
 Orange G is a monochromatic stain that gives
keratin a brilliant orange colour.
 Has relatively small molecules and so rapidly
penetrates the cytoplasm.
 EA is a polychrome composed of eosin and light
green, and in the original stain, Bismarck brown.

25.  Eosin stains the cytoplasm of mature squamous
cells, nucleoli, and cilia.
 Light green stains the cells that are metabolically
active, such as parabasal and intermediate
squamous cells and columnar.
 Bismarck brown does not add a characteristic
colour to the cytoplasm and for this reason has
been removed from many formulae.
 Superficial cells stain pink with eosin –
eosinophilic.
 Parabasal and intermediate cells stain green, blue-
green, or blue, depending on time in the EA stain –
said to be cyanophilic.

26.  Different types of EA solution:
 EA-36 – the original formula developed by Papanicolaou
using ethanol as solvent
 EA-50 – commercial preparation using methanol; has
similar formular as EA-36 except the solvent
 EA-65 – contains half the amount of light green of EA-
36 or EA-50, while eosin and Bismarck brown remain
the same; preferred by some for non-gynaecological
preparations, especially if they are thick and absorb too
much of the green colour; has been recommended for
gynae specimens to help differentiate cervical
adenocarcinomas from endometrial types
 The conterstains are removed from the cells if the
slides are allowed to remain in the alcohol rinses
for an extended length of time.

29. Clearing
 Principles
 Mount preparations to optimise microscope objective’s
performance
 Immerse fixed and stained preps in an organic solvent
that has RI close to that of fixed protein
 Final step in staining method, resulting in cellular
transparency; step between dehydration and
mounting
 To transition from stain to mounting, cyto preps
must pass through an organic solvent that will
displace any remaining alcohol between staining
and mounting

30.  Solvent should have RI similar to that of fixed
protein – 1.536 – and the mountant
 Xylene is the clearing agent used.
 Miscible with both dehydrating alcohol and mounting
media
 Transmits light rays from the microscope illumination in
the same way as the cell itself, thereby making the cells
ransparent
 Term “clearing” misleading – fixed protein naturally
transparent and colourless so does not require
clearing
 Spaces in and around the protein occupied by air
with RI of 1.00 – cells appear opaque

31.  Dry protein causes diffraction of light – light
bending as it passes through a transparent
medium – air – and meeting an edge – fixed
protein – so does not appear transparent
 Effect is scattered light or blurred images
 Replacing air with xylene (RI = 1.49) means fixed
protein and surroundings have similar RIs, light
passes through unimpeded optically and cells
become transparent
 Xylene chemically inert to biological dyes so does
not affect stains

32.  Histological grade xylene contains mixture of 3
isomers; purified xylene, expensive, 1 isomer
 Xylenes contain up to 15% benzene – a bone
marrow carcinogen, so work under fume hood to
avoid inhaling vapours
CH3
CH3
CH3
CH3
CH3
3
HC

33. Mounting the cell sample
 Mounting medium:
 Acts as a permanent bond between slide and coverslip
 Protects cellular material from air-drying and shrinkage
 Acts as effective seal against oxygen preventing fading
of stain
 Must have RI that closely matches specimen and slide
to yield best possible transparent image
 Smallest amount of liquid mounting medium that
will completely cover area under coverslip without
evaporating after drying should be used

34.  Too rapid evaporation of solvent causes air
bubbles to appear in mounting medium as it dries
 Solvent of mountant should evaporate relatively slowly
to avoid air being aspirated under cover glass before
edge seal can form around perimeter of cover glass
 Proportion of solids to volume of solution should
allow solvent to evaporate without air being drawn
under cover glass - retraction
 Mounting medium should flow easily when applied
to slide and not ENTRAIN or ENTRAP air bubbles
 Chemical composition should not cause dye to
fade or annual bands to form

35.  pH of mounting medium should be as neutral as
possible and to inhibit fading of stains
 No mounting medium possesses all these
properties and performance features

36. Alum Haematoxylins
 Many formulae for haematoxylin stains but alum
haematoxylins widely used in cytology because
they:
 Produce clear nuclear staining
 Are simple to prepare
 Give consistent reliable results
 Can be used to stain a large number of samples before
renewal
 Haematoxylin has affinity for and links up with
alums
 Aluminium binds to tissue, acting as a mordant.

37.  Aluminum lake formed with ammonium alum
(aluminum ammonium sulfate) particularly useful
for staining nuclei. Purple in acid solution, but blue
in alkaline solution.
 Aluminum potassium sulphate (potassium alum or
potash) or aluminum sodium sulphate (sodium
alum) may also be used to create the aluminum
lake.
 No appreciable difference in results found.
 Staining of nuclei by alum haematoxylin enhanced
by the addition of acetic or citric acid, which
apparently reacts with nuclear chromatin, giving it
a somewhat crisper appearance – acid is referred
to as an “accelerator”*

38.  Haematein exists in solution 3 forms:
 Free haematein – yellow
 Partially linked haematein – haematein linked 1
aluminium ion per molecule – red
 Fully linked haematein with each molecule attached to 2
aluminium ions – blue
 PRINCIPLE OF BLUEING
 Alum haematoxylin acts like a basophilic stain with
affinity for basic nucleoproteins (pH of solution is
2.95 and with addition of 2% acetic acid pH is
2.34)
 Alum usually dissociates in alkaline soln combining
with OH− of water to form insoluble AlOH3
 < pH5, H+ compete with aluminium ions for OH−

39.  Thus in presence of excess acid, AlOH3 cannot be
formed thus failure of aluminium haematoxylin dye-
lake to form, due to lack of OH− ions
 Hence, acid solutions of alum haematoxylin are
red due to partially linked haematein
 During staining alum haematoxylin stained smears
passed on to an alkaline solution to neutralize acid
and free OH- group, to form an insoluble blue
aluminium haematin-tissue lake (blueing) –
completely linked haematein
 Haematein lake thought to bind to phosphate
groups of DNA and RNA but likely that nuclear
proteins also participate to some extent in stain
uptake

40.  Once a lake is bound to tissue it is not easily
removed by neutral fluids
 Regressive haematoxylins require differentiation,
progressive haematoxylins do not – both require
blueing
 Blueing can occur over wide range of pH
 Low pHs (5-6) blue slowly over several minutes*
 Moderate pHs (8-9) blue satisfactorily within 2 minutes
 High pHs (10-11) blue rapidly within seconds#
 Differentiation omitted or incomplete in regressive
haematoxylins results in residual haematoxylin
visually obscuring chromatin and preventing
uptake of cytoplasmic counterstains

41.  Most influential factors determining type of staining
to be expected from individual hemalums are
 Amount of dye in the solution
 Amount of mordant, and ratio between dye and mordant
 pH
 Time for which the solution is applied

42. PROPERTY
PROGRESSIVE REGRESSIVE
HAEMALUM
CONCENTRATION
LESS – 1-4 G/L ≥ 5G/L
ACETIC ACID PRESENT PRESENT
RATE OF UPTAKE SLOW ABSENT
EASILY CONTROLLED? YES NO
OVERSTAINING? NO YES
DIFFERENTIATION
REQUIRED?
NO YES
BLUEING REQUIRED? YES YES

43. DIFFERENTIATION VRS BLUEING
PROPERTY DIFFERENTIATION BLUEING
PURPOSE DIFFERENTIALLY EXTRACT
EXCESS HAEMATOXYLIN
FROM CHROMATIN AND
CYTOPLASM; QUANTITATIVE
CONVERT SOLUBLE RED
COLOUR TO INSOLUBLE
BLUE COLOUR; QUALITATIVE
FUNCTION ATTACKS CELL/MORDANT
BOND
OXIDISES AL-HAEMATEIN
TYPE OF STAIN REGRESSIVE
HAEMATOXYLINS
PROGRESSIVE &
REGRESSIVE
HAEMATOXYLINS
WORKING pH ABOUT 2.5 5-11
COMMON E.G. 0.5% HCl IN 70% ALCOHOL SCOTT’S TAPWATER SUBST
TIMING DIPS/SECONDS MINUTES
TIMING ACCURACY CRITICAL NOT SO CRITICAL
TIME TOO BRIEF HYPERCHROMASIA PURPLE COLOUR
TIME TOO LONG HYPOCHROMASIA DECOLOURISATION IF pH ≥
11

44.  Harris Haematoxylin
 Chemically ripened – sodium or potassium iodate
 Used as progressive stain in cytology (regressive
in histology)
 Uneven staining is likely to occur during
differentiation of nuclear stain in HCl
 Hyperchromatic and hypochromatic staining of nuclei
may occur in batch staining due to variability of
thickness of smear
 Needs frequent filtering to remove metallic scum
 Should be stored in dark to prevent over-oxidation
 Mayer haematoxylin
 Chemically ripened – sodium iodate

45.  Can be used both as a regressive and progressive
stain
 Used as a progressive stain with short immersion
time and low haematoxylin concentration,
selectively stains chromatin allowing for more
control over nuclear density without any
appreciable coloration of the cytoplasm.
 Gill haematoxylin
 Half-oxidised haematoxylin – No. 1 - 2g; No. 2 - 4g
 Stains chromatin at a controllable rate without risk
of over-staining
 Highly selective so differentiation not necessary
and subsequent cytoplasmic staining is enhanced

46.  Carazzi haematoxylin
 Chemically ripened using potassium iodate
 Used progressively or regressively
 Gives selective nuclear staining with minimal
cytoplasmic coloration.
 Erlich haematoxylin
 Naturally ripened
 Stains mucin and not recommended for cytology.

47. Orange G
 Strongly acidic dye
 Stains keratin within the cytoplasm and granules in
eosinophilic, superficial cells
 Reacts with the basic groups of protein
 Only slightly soluble in ethanol; 50 times more
soluble in water
 In 95% ethanol, OG stains cytoplasm sufficiently
lightly to maintain transparency of cytoplasm.
 Stains rapidly and intensely when glacial acetic
acid is added to formula

48.  Binds more strongly with tungstate-treated
cytoplasm, so phosphotungstic acid added to
enhance depth of colour – acts as a mordant

49. EA
 Light Green SF Yellowish
 An acid dye and binds well to basic protein side
chains
 Most light-sensitive of the dyes in the
Papanicolaou stain
 Eosin
 Is tetrabromofluorescein
 When all four bromine atoms are present is
deepest red
 Tends to stain more yellowish at lower bromine
content

50.  Eosin is formed by a reaction between bromine
and fluorescein.
 There are two eosin variants typically used: eosin
Y which is slightly yellowish and eosin B which is
slightly bluish. Eosin Y is most popular.

51. PRINCIPLE OF PAP STAIN
 After nuclear staining with alum haematoxylin
small anions of orange G enter all unstained
components
 Dye binds to proteins only from a solution with pH
less than 3
 Single ions and dimers of eosin and large ions or
aggregates of light green compete with each other
for cytoplasmic binding sites but do not displace
orange G from keratin*
 Single eosin ions enter erythrocytes and produce
an orange-pink colour

52.  Eosin dimers enter cytoplasm of most cells and are
bound by proteins there to produce a red-pink
colour
 Light green is bound by mucus which acquires
bluish green colour of dye
 Optimum pH for competition between eosin and
light green is 6.5
 pH of each dye solution after preparation
determined by concentration of phosphotungstic
acid added to the solution
 Large anions of PTA appear to assist in exclusion
of light green from most cells – acts as dye
excluder and allows differential staining by light
green and eosin*

53. SUMMARY
 When stains are chemically competent –
appropriately prepared and dye is not exhausted –
are applied in prescribed sequence for the right
times and appropriate rinses undertaken:
 Haematoxylin stains chromatin
 Orange G colours keratin
 Eosin stains superficial squamous cells, cilia, nucleoli,
and erythrocytes
 Light green colours everything else
 If haematoxylin overstains, it blocks the uptake of
cytoplasmic dyes

54.  If orange G stains excessively, it cannot be
displaced by eosin
 If staining time in EA is too brief, eosin will not be
displaced by light green

55. ROMANOWSKY STAINS
 Polychrome stain containing two or three thiazine
dyes (methylene blue and its oxidation products)
and eosine.
 Originally designed to incorporate cytoplasmic
(pink) staining with nuclear (blue) staining and
fixation as a single step for smears
 Primary use of Romanovsky-type stains is for
cytoplasmic detail, such as intracytoplasmic
mucins, fat droplets and neurosecretory granules
 Advantage over Pap stain

56.  Solvent is usually methanol and glycerine
 Glycerine helps to stabilize stock solution but can
be replaced by diethylamine hydrochloride, which
does not increase viscosity
 Air dried Romanowsky-stained preparations are an
integral part of diagnostic assessment of many
non-gynaecological specimens
 Adapted for automatic staining
 Stock solution can be stored but working solution
should be freshly made – 10ml of stock to 90 ml of
phosphate buffered distilled water (pH 6.8)
immediately before use
 pH sensitive therefore buffer and rinse water critical

57.  All Romanowsky stains have tendency to
precipitation and should be filtered before use
 Ensure that ethanol used for fixation after air-
drying is acetone-free

58.  The eosin and methylene blue azure dyes in the
Romanowsky methods stain at neutral pH
 Allow differential staining of epithelial cells and
blood cells
 Mucin, myxoid material and cartilage stain in a
characteristic manner which allow easy recognition
of these components
 Romanowsky stains can be prepared in the lab
from the separate ingredients but commercially
available ready-mixed preparations are more
reliable
 Dye should be dissolved in acetone-free methanol
for use.

59.  The alcoholic solution must be diluted with water or
buffer immediately before use, to liberate the
colorant ions in an active form.
 The pH of the diluted stain and of the water for
rinsing is critical factor – 6.8
 Methylene Blue
 A cationic dye soluble in both water and alcohol.
 With time and exposure to air and alkali is
demethylated to azure B and azure A, and other
thiazine dyes – a change called polychroming.
 Deliberately oxidised for use in Romanowsky
stains to give a mixture of dyes known as
polychrome methylene blue.

60.  Main source of variation among different batches is the
rather unpredictable assortment of dyes produced by
oxidation.
 Variants of the Romanowski group differ in the degree
of oxidation (polychroming) of the methylene blue stain
prior to the precipitation
 When solutions of methylene blue and eosin are
mixed a precipitate is formed, which is soluble in
ethanol and methanol
 Dried precipitate known as "methylene azure“
 Leishman's Stain
 Wrights Stain (America)
 Giemsa and May-Grünwald stains in Germany and
Europe

61.  Properties of the Romanowsky stains attributable
only to azure B and eosin
 Based on electrostatic interaction between dye and
target molecules
 Staining solution contains positively charged basic
dyes (methylene blue and related azures) and
negatively charged acidic dye (eosin)
 Presence of other thiazine dyes, especially
methylene violet, has no adverse effects at low
concentrations
 Azure B in diluted stain is present as dimers – two
cations of dye held together by van der Walls
forces, producing a charge of +2

62.  Initial coloration involves acidic and basic dyeing.
 Acidic dye, eosin colours erythrocytes and
eosinophil granules
 Basic dye, Azure B, rapidly gives rise to blue-
stained chromatin and ribosome-rich cytoplasm,
neutrophil specific granules, platelets and also to
violet basophil granules
 Azure B in certain structures then combines with
eosin to produce a purple Azure B-eosin complex
 Selectivity of complex formation controlled by rate
of entry of eosin into Azure B stained structures
 Only faster staining structures permit formation of purple
complex in the standard method

63.  Purple colouration of nuclei, peculiar to these
stains – the Romanowsky-Giemsa effect – thus
due to:
 Binding of cationic dimers to nuclear DNA
 Binding of eosin anions to the already bound azure B
cations
 Ionic and non-ionic forces involved in binding of
dyes:
 Negatively charged phosphate groups of DNA and RNA
attract the azure B cations, and the attachment of the
dye is reinforced by van der Waals and hydrophobic
forces between aromatic rings of dye and purine and
pyrimidine rings of DNA
 Both types of force also involved in binding of eosin to
bound azure B

66.  Diff-Quick Stain
 Rapid manual Romanowsky technique
 Commercial kit stain
 Based on a modification of the Wright-Giemsa
stain
 Three-solution, three-step, fast
 Provides good cytoplasmic detail
 intracytoplasmic mucins, fat droplets and
neurosecretory granules are well visualised
 Extracellular substances appear metachromatic
 Free mucin, colloid, ground substance, etc, are also
easily stained
 Microbiologic agents, such as bacteria and fungi, also
appear easier

67.  Comparable to May-Grunwald-Giemsa and Wright-
Giemsa, but much quicker.
 Allows air-dried smears to be examined quickly –
advantageous for:
 Rapid initial assessment of fine needle biopsy samples
 Rapid assessment of fine needle biopsy/aspiration
samples to ensure that representative sample is
obtained
 Fixation is with methanol containing 1.8mg/l
triarylmethane dye
 Diff Quick solution I – eosinophilic – is buffered
eosin Y with sodium azide
 Diff Quick solution II – basophilic – is a buffered
soln of thiazine dyes, methylene blue and azure A

68.  Azure A undergoes slow constant oxidation to
azure B
 PROCEDURE
 Air-dry the smear
 Fix in “Diff Quick” Fixative (or methanol) for 30
secs/drain
 Stain with “Diff Quick” solution II for 30 secs/drain
 Counterstain (optional) with “Diff Quick” solution I
for 30 secs/drain
 Rinse in tap water to remove excess stain
 Rapidly dehydrate in absolute alcohol
 Clear and mount

69.  TECHNICAL NOTE:
 (step 5) - exposure to alcohol should be as brief as
possible to prevent excessive decolourisation

70. RINSES
 Rinses constitute 80% or more of all the solutions in
most stains
 Purposes of rinses vary
 Effect transition from organic solvents to aqueous
solutions and vice versa – dehydration and hydration
 Stop action of previous solution – post-haematoxylin
water rinses
 Differentially extract excess haematoxylin – differentiation
 Convert haematoxylin from red to blue – blueing
 Promote redistribution of dye within cells/tissue –
unfiformity

71.  Allow expression of differential staining
 Dehydrate – absolute alcohol
 Clear – xylene
 Amount of stain that remains within cells
represents the difference between what staining
solutions put in and the rinses take out
 TECHNICAL NOTE
 Post-eosin rinses perform most effectively when
solution is clean
 There is maximum difference in concentration gradient
between dyes in cells and the rinsing solution
 Stained smear immersed in clean alcohol, dyes diffuse
effectively into surrounding solvent

72.  Rinse becomes laden with dye and as concentration of
dye increases, concentration gradient reduces and dye
diffusion slows
 When concentration of dye in cells equals that in rinse,
concentration gradient is zero, and net dye diffusion
becomes zero
 For effective rinsing:
 Keep rinses deep for maximum dilution – slides must be
deeply covered by rinsing fluid
 Use in sets of 3
 Dip racks in at least recommended number of times
 Change rinse as needed – when third rinse becomes
coloured*

73. COVER GLASS
 Plus mounting medium – constitute “front lens” of
every microscope objective
 Their combined thickness and optical properties
influence the quality of image formation by objective and
oculars
 Specification of Standard Microscope Cover Glass
 Refractive Index – amount of bending of light as it
passes through one transparent medium into
another
 Dispersion – measure of chromatic aberration
 Homogeneity – RI varies little throughout glass

74. SPECIFICATIONS OF STANDARD MICROSCOPE COVER GLASS
SPECIFICATION ROYAL MICROSCOPICAL
SOCIETY
AMERICAN SOCIETY FOR
TESTING AND MATERIALS
STANDARD SPECIFICATION E211
THICKNESS 0.18 mm ± 0.003mm NO. 0 0.085-0.13 mm
NO. 1 0.13-0.17 mm
NO. 1.5 0.17-0.19 mm
NO. 2 0.19-0.25 mm
R.I. 1.524 ± 0.0007 TYPE 1: CRITICAL MICROSCOPY:
1.523 ± 0.0005
TYPE 2 ROUTINE USE 1.52 ±0.02
DISPERSION 52.0 ± 2.75
HOMOGENEITY R.I. CONSISTENTLY
WITHIN ± 0.0007
SURFACE
QUALITY
MACHINE POLISHED
PARALLEL AND FLAT ON
BOTH FACES, TO “PLATE-
GLASS” QUALITY

75.  No. 1.5 thickness cover glass often recommended
as thickness range within 0.17 on objectives
 Correct if specimen is in direct contact with underside of
cover glass, without intervening mountant
 Specimen must be mounted in contact with
underside of the cover glass, otherwise layer of
mountant interposed – equivalent to increasing
cover glass thickness
 Layer of specimen must be thinner than 0.03 mm
(3µm) otherwise lower surface will lie beyond
tolerance on cover glass thickness
 Therefore, for routine use, No. 1 cover glass
should be recommended

76. DESTAINING CELL SAMPLE
 Following steps are taken:
 Removal of Coverslip
• Accomplished by :
• Soaking slide in xylene until coverslip falls off
• Placing the slide in a freezer up to 1 hour – not for newly
stained and coverslipped slides
• Placing slide on a warming plate at 60oC for 3 to 4 hours
• Slide is put through 2 changes of xylene to remove
all traces of mounting medium.
• Time required depends on
• Mounting medium used
• Interval from staining and coverslipped

77. • Thickness of the cell sample
• Thickness of the mountant used
• Destaining proceeds in reverse direction to
staining process omitting the stains in the steps
• Carrying slide through the alcohols to water
removes the counterstains
• Nuclear stain is removed by soaking the slide in
dilute HCl, aqueous or alcoholic for 5 to 10
minutes, the slide examined under the microscope
and if any nuclear stain still remains it is re-
immersed in the acid solution.
• Acid is then removed by placing slide in running
water for 10 to 15 minutes or in several changes of
water

78.  TECHNICAL NOTE:
 Destaining process times for counterstains are not
critical and it is better to prolong the process to be
sure all original stain is removed
 Slide can remain in acid for as long as 1 hour
during removal of nuclear stain without ill effect.
• After removal of nuclear stain, acid completely
removed by putting slide in a blueing solution.
• Slide is rinsed in 2 changes of tap water if Scott’s
tap water substitute or lithium carbonate is used.
• Slide is ready for restaining and may be put
directly into haematoxylin.
• Filter staining solutions after re-staining as cells
may more readily float off slide into staining
solutions

79. RESTAINING THE CELL SAMPLE
 The cells of de-stained preparations may appear
more sensitive in their uptake of haematoxylin
 If batch re-staining is being undertaken it may be
necessary to test one slide so that the time spent
in the haematoxylin does not exceed the time
necessary to stain the nucleus
 The type of fixation originally used to fix the cells
may affect de-staining and re-staining procedures
– remains an unknown parameter

80. FREEZER METHOD FOR REMOVING
COVERSLIP
 Place slide flat in freezer compartment with
coverslip side down for 10-60 minutes – time
depends on how long ago slide was coverslipped;
inverse relationship
 Remove slide – area between coverslip and slide
should appear frosty with a separation of coverslip
from slide along edges. If these changes are not
seen repeat step 1.
 Place a blade under coverslip at area of separation
and move it along the outer edge of the slide.

81.  Coverslip should pop or fall away from the slide
easily.
 PROTECTIVE GLOVES AND GOGGLES
SHOULD BE WORN DURING THE FINAL STEP