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 MVJ Medical College & Research Hospital
Department of Anatomy, Pathology & Microbiology
Hoskote, Bengaluru
Karnataka

CME & WORKSHOP ON

_L_g__g__Laborato
Stainin Techniues

Date:
13th & 14th Aug ust-201 5

WORK MANUAL

Prepared by:

Dr. Shameem Shariff

Organizing Chairman,
CME and Workshop; and
Professor and Head
Department of Pathology
MVJMC & RH

Microbiology Section :
Contributed by Department of Microbiology, MVJMC & RH
Patron-in-Chief : Dr. M. J. Mohan
Secretary & Correspondent
Venkatesha Education Society
& Chairman
MVJMC & RH

Patrons : Dr. P. Mohan Rao

Executive Director
MVJMC & RH

Air Vice Marshal (Retd.) Dr. T.S. Raghuraman

Principal, MVJMC & RH

Mrs. Dharani Mohan

Chief Executive Officer
MVJMC & RH

Organizing Chairman : Dr. Shameem Shariff

Professor and Head
Department of Pathology
MVJMC & RH

Organizing Committee: Dr. Sangeeta. M

HOD, Department of Anatomy

Dr. Rama. N.K

HOD, Department of Microbiology

Dr. Raja Parthiban

Professor of Pathology
 Special Histochemical Stains

Diagnosis of any lesion at histology rests primarily on the routine Haematoxylin-Eosin

stain and a good deal of common sense. The eyes cannot see what the mind does not
know! is not always true and at times the unmasking of hidden substances which can be
brought to light with the help of special stains is needed in making a diagnosis.

WHismchemist
, is defi ' tification, localization and quantification
w in cells
and tissues of specific substances, re'__‘gactiveLoups and enL_L__42me catalzed substances
WMF—hu‘ch
are igh lgihte'ggdaor coloured as a result of the histochemical stains.

Principles: All histochemical stains are based on one of the following principles:

1. Simple ionic interaction between groups of opposite charges.

2. A colored product eg. Reaction of aldehydes with Schiff’s reagent.

3. Detection of an enzyme by providing a substrate that is converted by the

enzyme to a colored product.

4. Precipitation of salts of a metal eg precipitation of silver from methenamine

silver solution.

The various histochemical staining techniques used in histopathology are:

1. Routine Histochemistry

2. Enzyme Histochemistry

3. lmmunohistochemistry

Routine Histochemistry:

l. Staining of Mucosubstances and Glycogen.

The mucosubstances may occur either in epithelium or in connective tissue and

therefore, in either instance, may be called by the term "epithelial mucosubstances" or
"connective tissue mucosubstances".

Types of epithelial mucins:

Mucopolysaccharides form the bulk of the substances known as mucins secreted by

cells. Mucins are composed of hexosamine units such as glucosamine and
galactosamine. These are the predominant epithelial mucins. Other examples are
mucoproteins found in thyroid colloid, basement membranes, amyloid and beta cells of
the pituitary gland.
r‘
hpids exist as cerebrosides and gangliosides in the nervous tissue.

Types Of conn ective tissue mucins:

Chondroitin sulphate A found in cartilage

Chondroitin sulphate B found in skin, heart valves and umbilical cord
Chondroitin sulphate C found in cartilage, aorta and skin
Hyaluronosulphate found in cornea
Keratan sulphate found in cartilage, nucleus pulposus
Hyaluronic acid found in synovium, skin, aorta, cartilage, bone.

These mucosubstances irrespective of their origin may be either neutral in type being
identifiable at pH 5 and over, or acid in type identifiable usually at pH 2.8 and under.
Neutral mucins: consists of hexosamine and hexose units and do not have free acid
groups, identifiable at pH 5 and over. These are found in lining epithelium of stomach,
brunner’s glands of duodenum and in prostatic epithelium. They are PAS positive and
Alcian Blue negative.
The acid mucosubstances are composed of 3 estimable fractions - the carboxylated
mucosubstances, the weakly sulfated mucosubstances and the strongly sulfated
mucosubstances. Acid mucins consists of hexosamine and glucoronic acid, iduronic acid
or sialic acid, identifiable at pH 2.8 and under. They are Alcian Blue positive.

Strongly sulphated acid mucins: Comprise of chiefly connective tissue mucosubstances

like chondroitin sulphate, heparan sulphate and Keratan sulphate. They are found in
bronchial mucus glands (sulphomucins) and minor fraction in intestinal goblet cells. So
designated because of their ability to stain with alcian blue at pH levels of 0.5 and
below. They are largely PAS negative.

Weakly sulphated acid mucins: These are sialomucins and are usually epithelial in
origin and present in a wide range of cell types and mucous glands eg bronchial
submucous glands and in colonic goblet cells. They are PAS positive and stain with
alcian blue at pH 1.0 and above.

Sulphated Sialomucosubstances are also found in prostatic carcinoma and malignant

synovioma.

Carboxylated mucosubstances:

Sialomucosubstances (Non sulphated): These sialomiucins contain a sialic acid moiety

which is an acetylated derivative of neuraminic acid. It reacts with alcian blue at pH of
2.5 and above.

Enzyme labile are found in goblet cells of peripheral airways of lungs, intestine. mucous
cells of submandibular salivary gland.
 Enzyme resistant found in gastric epithelium and mucous gland of major bronchi

Hyaluronic acid containing mucosubstances (Non sulphated):

tI Enzyme labile containing Uronic acid are found in connective tissue, synovial fluid of
joints and pleural mesotheliomas.

ii) Enzyme resistant (nonsulphated sialidase and hyaluronidase resistant) are found in
gastric pyloric glands.

The single universal stain for mucosubstances irrespective of whether these

mucosubstances are neutral or acidic is the MUCICARMINE STAIN. THE PERIODIC ACID
SCHIFF REACTION (PAS) tests neutral mucosubstances, chiefly, and the ALCIAL BLUE
for the acid mucosubstances at pH 2.8. The alcian blue may be used at different
molarities too with the addition of varying quantities of magnesium chloride, to
estimate the amount of carboxylated, weakly sulfated and strongly sulfated acid
mucosubstances.

WTM
Periodic Acid Schiffs reaction (PAS) (Schift’s Leucofuchsin

[abductionandmdzcations:'"' In 1946, McManus first applied the Periodic Acid Schiff’s

(PAS) reaction in histology. This technique is CI~LM_J~eosel
interlinked to carbohdrat
. . v
his’tochemlstry, and may be used to demonstrate glycogerf neutral mucosubstances,
basement membran/e, pigments like lipofuclfs'in, lipids such as cerebrosides, fungi like
candida albicanS/macrophages in Wipple's disease, hyaline proteinaceous renal cysts,
intracytoplasmic globules in hepatocytes in or 1 anti- trypsin deficiency, tMaMroh'
plakce_nta and beta cells in the pituitary. It is also useful in lysosomal storage disorders
like Neiman,Gn-\d“———"5Plckiseaseauche"r's disease and in glycogen demonstration in
glycogen storage disorders. It is used to differentiate between myeloblasts and
lymcp____hoblasts,“in;the diagnosis ofALL (block staining) and AML-M6.

The substances positive to PAS are:

Polysaccharides: Glycogen, cellulose, starches. Many leucocytes contain glycogen,

capsules of fungi (candida albicans, histoplasma capsulatum, Cryptococcus,
blastomycosis), actinomycosis and bacteria.

Glycoproteins: Mucins, mucoid secretions of the intestinal tracts, uterine glands, ducts,
tracheobronchia tree, hormones (TSH), megakaryocytes etc.

Glycolipids: Gangliosides, mainly grey matter composed of fatty acids ie. cerebrosides,
globoid cells of Krabbe's disease.

Non carbohydrate containing substances: unsaturatedl/pil‘d's, phospholipids, phospho-

inositides.

Certain pigments and substances: Ceroid, lipofucsin, pigment in melanosis coli, Dubin
]ohnson pigment (related to lipofuscin).
 Plasmogens: They are acetyl phospholipids eg Russell bodies

Miscellaneous: amyloid, cartilage matrix, colloid, corpora amylacea, ocular ‘lens

material.

W: Principle.-

< The principle is to release the dialdehydes from carbohydrate by oxidation with
K/
“15‘3" periodic acid and the subsequent combination of such aldehydes with Schiff’ reagent to
give a substitute dye which is red in color (this dye is not Basic Fuchsin) but localized to
*F—.——5
I
atfw—healdehfle
site of t id‘s—rere'ase.

xidation:
w” ‘45}?
goafl'i/ Periodic acid is the most common substance to be used for oxidation. One ofits superior
properties is thagwffiat
it will not further oxidize the resultin ld__eh4d_g Thus standardization
is easier. It is used in any strength between 0.5 to 2.5% (preferably 1%). However, four
important rules must be observed when periodic acid is used.-

1'. Oxidation time must be limited to a maximum of 10 minutes.

ii. Oxid'ation should not be carried out at more than 200C.
iii. The Periodic acid solution should have a pH of 3-5.
iv. Prepared solutions must be kept at 4°C in a refrigerator.

Schiff’s reagent

Basic Fuchsin (pronounced as fook-sin) when treated with sulfurous acid results in a
CéO'v/ colourless product called Schiff’s reagent. Wsic Fuchsin is not a ure de and consists of
Mfg) >.~fpv_d__’_.__mag_4.Darar°salmmeandentail”rincipally. Pararosalinine is usually present in
w?” combination with an acetate or a chloride and is consequently unstable. It is for this
‘ reason that when Basic Fuchsin is treated with sulphurous acid, it produces Schiff’
MOD» ‘ reagent. This substance in comPJ’JWbinatio
' aldeh de molecules roduces a
reddish
w purple complex. This color is not, therefore, due to Basic Fuchsin and may vary
_____\
in intensity depending on the various aldehydes present in the tissues examined. The
two most important rules for.preservation of Schiff’ reagent are:
M’
9‘4 i. A deep brown black bottle to prevent oxidation taking place. (lfthe solution

My” ea ,
'1“
should ever turn pink, discard it).
ii. A 40C temperature.
QL 3’0 9/” Sulphorous acid [Sulfite] rinse
to“,

‘i
This step is optional. The purpose ofthe rinse is to remove excess leucofuchsin, which
may become recolorized and give false positive staining of some structures. The
sulphite rinse must be prepared fresh each day.
 Formula:

Schfif reagent

Basic fuchsin .................. 1gm

N. Hydrochloric acid ............ ZOmJI
Sodium metabisulfite ................1 gm W
Activated charcoal ..........2 gm '
Distilled water

Boil the distilled water. Allow to cool to 80°C, add basic fuchsin. Filter at 50°C and add
hydrochloric acid. At 25°C, add sodium metabisulfite. Store in dark for 48 hours. Add
activated charcoal. Shake for 1 minute. Filter. This filtrate should be clear.

Testing for activity ofSchfif’ reagent

Pour few drops of Schiff’ reagent into 10 m1 of concentrated formalin. If the reagent is
active, the solution turns reddish pink rapidly. If the blue color develops after some
time, the solution is breaking down.

The Periodic Acid 1 % solution

Control: Use skin, aorta or normal liver for positive PAS staining.
Fixative: Standard paraffin section fixed in 10% neutral buffered formalin.
Technique: On paraffin sections 4-5 um

Procedure:

1. Bring sections to waters—’7

2. Rinse in distilled water.
3. Oxidise with 1% periodiqacid for 5 minutes—2”. z? 56 wink/5M4” PM“
4. Rinse in distilled water.
5. Use Schiff's reagent for 10 minutes.
6. Sulphite rinse (Optional)
7. Wash in tap water for 10 minutes; this intensifies the color reaction.
8. Stain the nuclei with Alum haematoxylin for 5 minute.
<3“ ‘9.Wash in tap wateWr—fi—fir-Niw £0.13 w: (7'.
5M n ‘
10. Bring to mountant.

Results: ,
PAS positive material ..................... Magenta p/ink
to red
Nuclei ........................................... Blue
 Note: The temperature for oxidants to act should not exceed 25°C as it is like
oxidize other substances besides aldehydes.

Safety: Basic fuchsin (in Schiff 's reagent) is a known carcinogen. It is advisab . .
gloves, goggles, particle mask and lab coat, while preparing the solution. Avoid contact
and inhalation to hydrochloric acid as it is a strong irritant to skin, eyes and respiratory
system.
NR

( Connective tissue stains

$va
LOO 0The main function of connective tissue is to connect together and provide support to
ther tissues of the body. It is derived from Latin word meaning to 'to bind’. Connective
/lotissue usually consists of a cellular portion in a surrounding framework of a non-cellular
substance. The cell types of connective tissue include fibroblasts, mast cells, histiocytes,
adipose tissue, reticular cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, and
blood forming cells etc. The intercellular substance is usually composed of both
amorphous (nonsulphated and sulphated mucopolysaccharides) and formed elements
(collagen, reticular fibres and elastic fibres).

The collagen fibres are the most common type of intercellular substance and found in
abundance n most of the tissues of the body. They can occur individually as in loose
/W
13/"
M areolar tissue, arranged in an open weave pattern or as a large bundles of fibres
Wclumped to form a structure of tensile strength eg. Tendons viewed under polarized
light shows collagen fibres to be birefringent. Connective tissues are divided into the
following types:

02
Connective Tissue proper — includes loose or areolar, dense and reticular WSW.

Cartilage-hyaline, elastic and fibrocartilage

Bone — spongy or cancellous bone and dense or cortical tissue

Various types of collagen fibres are identified:

Type I collagen, the most common form encountered in the human can be found in ,6 \
bone, fibrous connective tissue, ligaments, skin and tendons. The fibrils are thick, zHV/D
closely packed (75mm in diameter) and strongly birefringent but not argyrophi'll'cgfillawb

Type ll collagen is present in hyaline and elastic cartilage mainly as very thin, loose
fibrils dispersed through the ground substance as a copius meshwqu of pforteoglycans.
Fibrils are birefringent and stain pink to red with Sirius red. 9, Wabflgvv by“ i z

szeJll-co.llagen occurs in conjunction with other types of collagen fibres (eg. Type I)
and IS Mill—Wma'or
com onent of reticulin. It is found as a loose network of thin, striated
fibrils yysurroundedbcarboraterlcmterfibrillary material. Fibrils are argyrophilic
and weakly birefringent.
 (a:
31C]
‘<
01

03
JESM

Type EV collagen is a non-fibrillary form which occurs in basement membranes. It is

weakly birefringent and associated with significant amounts of carbohydrate which is
responsible for the strong PAS positivity.

Type V collagen, also non-fibrillary is found mainly in b\_’_’_____‘__al.lood

vessels and fet
membranes although it does occur in small quantities in other tissues. It is found

structure.

Van Gieson Stain

Van Gieson Stain is used to differentiate between collagen and smooth muscle in tumors
fl,_~,,

\(‘QJ/ and VAWO

demonstrate th. inc ease of c_g____ollaen
¥
in diseases. This method combines two or a

des and rely on differential binding by tissue components. The

r“ V‘ y/ more _____'____L_____anionic
/ifferentiation is determined by a combination of differences in the relative size of the
' dye molecules, differences in the physical structure of the tissue, and differences in the
. . . . . ' Q2" _ c
by amino and composmon of tlssue Elements. _7 C(9m120m OZ WW. w. (“Wm {(17, «A a)
who ‘ r ’ Cb
$09) Principles ofthe Procedure: MY 2'“ ‘W‘é‘fij‘l éwDLf/k/ W M
aJ/QVan Gieson's Stain is a mixture of Picric Acid and Acid Fuchsin. It is the simplest method
.& effigy of differential staining of Collagen and other Connective Tissue. When usmg combined
\ lo W’ solutions of picric acid and acid fuchsin, the small molecules of picric acid penetrate all
../

by} of the tissues rapidly, but are only firmly retained in the close textured, red blood cells
b/g/I?‘ and muscle. The larger molecules of Ponceau S displace picricacid molecules from
RM We ,collagen fibres, which have larger pores, and allow the larger molecules to enter

R Applications .-

1. To differentiate between leiomyoma from fibroma and shwannoma,

leiomyosarcoma from fibrosarcoma.
Peutz legher's polyp to demonstrate the central core smooth muscle
To demonstrate fibrous stroma of endometrial polyp.
To show (delineate) the extent of fibrosis in a given tissue.
To differentiate amyloid from collagen
Specific for collagen '
lt can be used as counterstain with Von Kossa stain(mineralized bone black and
$9 19 3!”

osteoid red) and Verhoeffs stain (elastic tissue black and background yellow to
red)

Disadvantages: Immature (young) collagen does not stain with van Gieson.

Control: Artery or skin

Formula:

Acid- fuchsin 1% aqueous solution — 2.5 ml 0 ( \

AS/J

Picric acid saturated aqueous solution — 97.5 ml lOG/VW

 Procedure.-

Bring sections to water.

Rinse in distilled water.
Stain with Weigert's Haemotoxylin (freshly prepared) — for 10 minutes
Wash in distilled water.
Stain in van Gieson's solution for 1-3 minutes
Dehydrate in 95% alcohol
Do not use water
Clear and mount.
PGNP‘SI‘PWE‘JE“

Results :
Collagen .................................red
Muscle and other non-collagenous tissue ............ yellow
Nuclei .............. Dark Blue

Elastic Tissue
Elastic fibres are strongly eosinophilic and when arranged compactly as in the arterial
elastic laminae are easily identified due to their refractivity. They differ from collagen
by their insolubility in organic and inorganic solvents (collagen is soluble in 2% acetic
acid).

Indications:

1. Malignant hypertension: To demonstrate hyalinization of blood vessels and

reduplication of lamina elastic interna.
2. Malignant nephrosclerosis: The arcuate arteries and other renal blood vessels show
reduplication.
3. Arteriovenous malformations : to demonstrate elastic fibres in the arterial segments.
' 4. Aneurysms: To differentiate true and false aneurysms.
5. Breast lesions: Periductal elastosis in infiltrating carcinoma, central ealstosis in radial
scar..
6. Skin lesions: to demonstrate the elastic degeneration of the skin e. Solar elastosis,
pseudoxanthoma elasticum, elastofibroma.
6. Marfan’s syndrome: To demonstrate the depletion and fragmentation of the elastic
lamina and vascularity ofthe media and adventitia.
7. Giant cell arteritis: to show the disruption of elastic lamina.
In almost all its applications to the staining of vessels as well as elsewhere, the elastic
stain is best combined with the van Gieson stain.

Several methods are used for the demonstration of elastic fibres. Some ofthe popular
ones are: 3
Verhoeffs method
‘

Orcein
Weigert's Resorcin Fuchsin
Gomori's Aldehyde Fuchsin
 0

f” W 0/,
(a/’\Q"

.e"l‘;he principles behind these stai'Cfizflys

are not well uestood. The __Whoeffs rocedure is
overstaining
combinv_____________________fl'ationofiod1nefr1c.Chlordiehaem--"”'___yatox,__£ollo_welm'd
with a by
‘

ferric Chloride differentiation. The technique works after any fixation, it is the easiest to
prepare, quick to perform and is most consistent, giving—wan intense black staining of the
coarse elastic fibres, although the inner fibres are less well demonstrated. The results |
are permanent and show little fading even after several years. ") 2, MI» W0
«I V M
In the Weighert’s Resorcin-Fuchsin method, the principle of staining involved is that in
the presence of Ferric salts, which act as oxidizers, the elastic fibres stain with Basic
Fuchsin to give a brown to purple color. The results obtained with this method are good
but the preparation of the stain is tricky and time consuming and at times may give
variable results or even fail to act. The ferrous salts contained in the Ferric Chloride may
also interfere with the staining.

Verhoeff‘s
Jr“ - Van Gieson Stain for elastic tissue .-

Elastic Tissue stain

Dissolve 1g of HwLaematoxh’n i122 ml of absolute alcohol on an open dish on a hot plate.

Cool, filter and add 8ml ofa 10% aqueous solution of Ferric Chloride and 8 ml of Iodine
5-
solution (2g of Iodine plus 4g of Potassium Iodide dissolved in 100ml ofdistilled water).

For better results, make up fresh solutions just before use.

fix/Q 3&7“ x4,
Ferric Chloride Solution
Ferric Chloride .............................. 2g
Distilled water ............................... 100fiml lei/“k.
Dc
Sodium Thiosulphate solution
Sodium Thiosulphate .............................. 5g
Distilled water ............................... 100'ml
Method

Bring sections from alcohol to distilled water.

Expose to Verhoeff‘s elastic tissue for 15 minutes.
.Wash in .dlStlkdflle
water. . ' I f . /\9
Differentiate 1n 2% Ferric Chloride — only a ew—n'm‘mtes, check under C :0 34A,)
PENN!"

microscope and if differentiated too far, restain.

Place in 5% Sodium Thiosulphate for 1 minutes (r____e§_excess_1mn_)emov'.
Wash in tap water for 5 minutes. ‘
Counterstain with van Gieson stain for 1-2 minutes.
. . . O Flt- E
M“®fi
Differentiate 1n 95 /0 alcohol (Do not use waterL/j d/o NJ
\ 01 tf‘
V} 134
W

M3
Clear and mount.
PPNQW

JV‘ WJ/
Results: :o
Elastic fibres ............................. Blue black to black I? Mr

Collagen................................. Red
Other tissue elements Cytoplasm & muscle) ..................yellow.
Reticulin Stain

Reticulin is a procollagen. It is finer than collagen, stains black with reticulin stain an .
unstained with collagen stain. Collagen fibres on the other hand are coarse, doubly
refractile, stain red with a collagen stain like van Gieson; and yellow, lavender or brown
on silver impregnation. Reticulum and collagen are basically similar and though there
may be chemical differences in the amino acid content of collagen and reticulin, most of
the observed differences in the physical arrangements of molecules and the presence of
additional bindings or cementing substances in collagen such as a mucopolysaccharides
resembling hyaluronic acid.

Principle of staining: The staining procedure for reticulin is an impregnation method.

The aldehyde groups of the carbohydrate of r‘Meuculmfibresreduc"e the colorless silver 7_
CMA—“flomplex
to a dark brown oxide of silver whic is precipitated in particulate form on
reticulin fibres.

The methods of Foot, Bielschowsky-Maresch, Perdrau Da Fano, Wilder, Gorden and

Sweet's , Gomori and Lillie, all use Silver oxide for hydroxide in ammonical solution.

e.g. AgN03 + NaOH = AgOH + NaNO3

The del Rio-Hortego, Foot and Laidlaw variants use ammonical solution of silver
carbonate.

2AgNO3 + L1'2C03 = AngOs + 2 LiN03

The precipitated Silver Carbonate is dissolved with ammonia water to given Ammonium
Silver Carbonate.

Ag2C03 + 4NH3 = [Ag(NH3)2 ]2 CO3

So, Ammonium silver carbonate (Laidlaw's method) or Ammonium Silver Oxide or

hydroxide (other method eg. Foot's] are reduced to a dark brown silver oxide by
reticulin fibres and subsequently reduced to black metallic silver by formalin.
l

Reticulin Stain — Bielschowsky's Method — Foot's modification l

Applications:

1. In kidney lesions-diabetic glomerulosclerosis display laminated argyrophilia of the K-

W lesions; Chronic lobular glomerulonephritis shows a tangle of reticulin; amyloid is
coloured with a diffuse pale grey with a silver reticulin stain.

2. ln liver, reticulin is helpful in early cirrhosis and detects creeping in fibrosis

 3.. 1n bone marrow: reticulin detects fibrosis in myelofibrosis. metastatic carcinoma
‘

shows reticulin free areas.

4. In vascular tumors: In diagnosing hemangiopericytoma which shows pericytes

external to the basement membrane in contrast to hemangioendothelioma; to outline
vascular pattern in angiosarcoma.

5. In lymph nodes, some early lymphomas are differentiated by production of

abundant reticulin with loss of nodal architecture as compared to normal lymph node
architecture or follicular hyperplasia. This application of reticulin stain in lymph nodes
is rarely used these days as immunomarkers have replaced the utility value of reticulin
in lymph node pathology. Angioimmunoblastic lymphomas and T- cell lymphomas are
typically rich in high endothelial venules outlined by reticulin.

6. In ovarian tumors, granulosa cell tumors show group of cells surrounded by reticulin
while in thecomas, individual cells are surrounded by reticulin fibres.

7. Tumors of nervous system arising from mesodermal tissues show abundant reticulin
eg. Gliomas, meningeal tumors, sarcomas. Astrocytomas are characterized by the
pattern of angiogenesis.

9. In paraganglioma, the reticulin stain reveals the typical cell nests or organoid (Zell
ballen) pattern.

10. In endometrial stromal sarcoma, the individual cells are surrounded by reticulin as
also an enhanced vascular pattern.

A chicken wire pattern of vascularity is characteristic of myxoid liposarcoma and

oligodendroglioma.

11. Absence of reticulin fibres may be helpful in the diagnosis of epithelial neoplasia as
well as Ewing's sarcoma of bone.

Solutions :ffoot' Silver Oxide Solution

“ 0491/
Add 20 drops 9% sodium Hydroxide to 20 ml 10% Silver Nitrate. Dissolve the brown
precipitate by adding strong (28%) Ammonia water drop by drop with constant shaking
until only a few granules remain. About 2ml. (theoretically 1.7 cc) is required. Dilute to
80ml with distilled water. Use once. This solution should be freshly made in acid clean
glassware.

This solution gets reduced by the aldehyde groups of reticulin in tissue to a lower oxide
(which is dark brown in color).

Potassium Permanganate Solution

Potassium Permanganate ............. 0.25g

Distilled water ......................................100ml
Potaw’ifwsium
Permanganate is used for re silv rin f r ur oses of oxidat‘on. A‘_
Potassium Permanganate is used by Gordon and Sweet (1936). 4% chromic a
0.5% Periodic acid may also be used.

Oxalic acid Solution

Oxalic acid ....................... 5g

Distilled water ...................................... 100ml
Oxalic acid is used for bleaching to remove Potassium Permanganate.
W

Silver Nitrate solution

Silver Nitrate...................2g
Distilled water..../..... ............................ lOOmI/ / J
This is used as a 'sensitizer‘ instead of which Urani/um, Ferric Chloride or Iron Alum may
T'— - - . .f.
also
f be used. These sensmzers are used as(J.—
mordants as they also have an ox1dizmg
efect.
I

Formalin solution

Neutral formaldehyde (AR) 37- 40% ..................... 5m]

Distilled water ......................................95ml
F_o___alm%rm'
red_,estheuclower oxide (brown in color) to metallic silver oxide (black).
Sodium sulfite or hydroquinone can also accomplish this.
/ ———-—
.

Gold chloride Solution

Gold chloride solution 1%................................1ml

Distilled water ..........................................99ml
G‘old__’_g____~_p__tonin
is a useful ste. In untoned sections the background is yellowish because of
colloidal metallic silver, which is annoying to the eye. Toning removes the silver and
replaces it with Gold Chloride. This is reduced to metallic gold by sodium metabisulfite
or thiosulfate giving a pale grey backg______p;_g_\_ound which is leasin to the eye. Toning is
essential to the differentiate woven bone from lamellar bone as it polarizes well. Toning
also enhances countertaining ifsuch a counterstain as nuclear fast red is used.

Sodium Thiosulphate Solution

Q)
Thiosulphate in this stain has two uses. In the first step it removes“Hess
S 9—\__dium of J
MhLflUcur if Mercurial fixatives are used. In the last step, however, it removes excess 09
un’___'_ver_,orreduced
sil gold and prevents further oxidation and does not permit any
further reaction. It also reduces gold chloride to metallic gel/dag
___’____,__,__,_
Method:

1. Bring sections to water.

JO ppe
pamppv . Treat with 0.5% aqueous Sodium Thiosulphate ...........................5 min
(Skip if mercurial fixatives are not used)
Wash in distilled water.
Treat with 0.25% Potassium Permanganate Solution for...“........ 5 min
Wash in distilled water f
4. Treat with 5% Oxalic acid .............................................................. 15 min
Wash in distilled watewc'l
. Treat wit/ratesuhSllverNitr"olution................................................... 3F0min
Wash in distilled water quickly ‘
Expose to Foot's Silver Oxide solution for.................................... 20min
7. A quick wash in distilled water.
8. Treat with 5% formalin (AR) for................................................... 2 min
Wash in distilled water
9. Treat with Gold Chloride solution .................................................. 3 min
10. Fix in 5% Sodium Thiosulfate solution solution ............................ 2min
11. Dehydrate, clear and mount.

Step 10 can be totally deleted for routine purposes as Gold Chloride is

extremely expensive.

Results:

Reticulin...dark violet to black

If nuclear stain such as Nuclear Fast red or Hematoxylin is used between Steps 11
and 12, then nuclei stain red or blue.

Precautions with Reticulin Stain

1. T_____’___tL_______§____he
high alkalini of Silver solutions tends to be traumatic to sections and,
therefore, detatches the imperfectly made or fixed sections from the slides. This
A
may be overcome by:

a. Fixing sections well to albuminized slides. )‘

b.Keeping sections prior to staining in the incubator at 600C for at least 2

hours.

c. Celloidinizing the mounted sections by using 1% celluloidin after

deparaffinizing. Blot excess.

1 % Celluloidin Solution

Take one gram of celluloidin to which add 40 ml ofAbsolute alcohol and allow to
stand for 48 hours in a stoppered bottle. This procedure softens the celluloidin.
Add 60 ml of Anhydrous Ether. This dissolving procedure may take a few days.
‘”"‘“‘
 2. Sections should be well spread, of uniform thickness and from
tissues.

3. Use freshly prepared solution.

4. All salts used in this method as also other methods should be ofAR quality.

5. Facts Silver Oxide solution should also be well filtered using Whatman’s filter
paper No. 1 to avoid precipitation ofsilver on the sections.

6. The atmosphere should be dust free as any dust particles will precipitate silver.

7. Always use distilled water throughout the staining procedures.

8. All glasswares, especially that used in the preparation of silver solutions, should
be washed in 10% Nitric acid and then washed in several changes of distilled
water.

9. A silvered flask or a silver-paper coating of containers for silver solutions may be

used to avoid oxidation. These solutions should be colourless and not black or
brown at the time of usage.

10. Use alcohol alone for dehydration to get good results.

11. Explosive hazard: In the preparation of the commonly used Silver impregnation
solutions various chemical reactions occur. With ageing or exposure of
ammonical silver solutions to air or light, shiny black crystals of explosive silver
compounds eg. 'fulminating silver', Silver Nitride (AgN3) and silver azide are
formed.

Voilent explosions may occur while removing a stopper, throwing a solution

down a sink or even when holding it up to light. In order to avoid this—

a. All ammonical silver solutions should be prepared fresh just before use.

b. Any used solutions should be inactivated by adding excess of Sodium

Chloride solution or dilute Hydrochloric acid.

Microbiology Section.-

LWM
TINl

Gram staining is a procedure devised by Christian Gram (1884) to differentiate between

Gram Positive & Gram Negative bacteria.

Principle: ls based on the pH of the cytoplasm & integrity of the cell wall of the bacteria.
Gram Positive bacteria will retain the primary stain — Crystal / Methyl/Gentian Violet,
after decolorizing with acetone/alcohol. Whereas Gram Negative bacteria will get
decolorized & takes up the counter stain — dilute Carbol fuchsin/Safranin.

Reagents Used:

1. Primary stain: Crystal violet

2. Chemical mordant: Gram’s iodine
3. Decolorizer: Acetone-Alcohol mixture
4. Counter stain: Dilute Carbol fuchsin

Procedure.-

1. Make a thin smear of the clinical specimen/culture on a clean grease-free glass

slide.
Air dry & heat fix the smear
Slide is placed on the staining rack & is flooded with Crystal Violet — 1min
Wash & stain with Gram’s Iodine - 1min
Wash with tap water, decolorize with acetone-alcohol mixture — 2 to 3 sec
Wash with tap water, stain with dilute carbol fuchsin — 30 sec
Wash with tap water & air dry
89 1:9 35"

Interpretation:

Gram Positive bacteria - Purple

Gram Negative bacteria — Pink
Pus cells in clinical specimens — Pinkish purple

Uses of Gram stain:

Differentiation of Gram positive & Gram Negative bacteria required for the
identification & classification
Screening of clinical samples
In presumptive rapid diagnosis of
o Gonococcal urethritis in men
0 Acute purulent meningitis
o Pneumococcal pneumonia
0 Anaerobic infection
Selection of antibiotic treatment (Empirical therapy)
Selection of culture media

LW
FAST S ! lNlN

Acid fast staining was discovered by Ehrlich (1882). It’s a differential staining to
differentiate between acid fast & non acid fast bacteria.

Principle:

Mycobacteria do not stain easily because of the high lipid content in their cell wall. Once
stained it cannot be decolorized with mineral acid (H2804, HN03, HCl).
"—
r—‘W
This property is known as acid fastness.

Types of Acid Fast stains:

a. Hot method: Ziehl Neelsen's staining

b. Cold method: Kinyoun’s method
Gabbet's method
Results:

Mycobacterial, hair shafts, Russell bodies, mast cell granules, fungal organisms and
splendore Hoeppeli bodies around
actinomyces ................................................................................................ red
Background .................................................................................................. pale blue

Note: Decalcification using strong acids can destroy acid fastness, formic acid to be used.

RNTCP Scoring Guidelines 2015” for bacilli load.-

>10 OIF 3+ 20 Fields

NoAFB/100 100 Fields

OIF

Merits:
1] Rapid diagnosis of pulmonary tuberculosis, Nocardia, spores of the bacteria
2] To study efficacy of treatment
3] To know the degree of infection
Demerits:

1] Species identification is not possible

2] Low sensitive to get smear positive. Number of bacilli required >10,000 AFB / ml of
sputum
3] Artifacts may give false positive results.
4] Saprophytic bacteria may give false positives

Modified Fite method for M. Leprae and nocardia:

The Fite stain is used for staining of M.Ieprae which has cell walls that are more
susceptible to damage in the deparaffinization process. The avoidance of solvents (fat
dissolving agents) such as alcohol and xylene helps to conserve this fragile fatty capsule.
The Fite procedure thus includes peanut oil in the deparaffinization solvent to protect
the bacterial cell wall. The acid used for decolorization in the Fite procedure is also
weaker and generally 0.5% or 1% aqueous sulphuric acid solution is used .
Xylene-peanut oil 1 part oil: 2 part of xylene- for deparaffinisation

Ingredients: Carbolfuschin
Basic fuchsin ........................0.5 g
Absolute alcohol ....................5.0ml
5% aqueous phenol.............. 100ml
(Mix well and filter before use with filter paper)

0.5 % sulphuric acid in 25% alcohol or in water (aqueous sulphuric acid)

25% ethanol ......................................95ml
Sulphuric acid, concentrated ............5ml

Methylene Blue (stock) solutions

Methylene blue .......................1.4 g
95% alcohol ............................ 100ml

Methylene Blue (working) solutions

Methylene blue (stock)
Tap water .............................................45ml

Method
1. Deparaffinize in two changes of xylene-peanut oil, 6 minutes each.
2. Drain slides vertically on paper towel and wash in warm running tap water for 3
minutes. (The residual oil preserves the sections and helps accentuate the acid
fastness of the bacilli)
Pour carbol fuchsin solutions for 25 minutes. (solvent may be reused).
Wash well in tap water for 3 minutes.
Drain excess water from slides on paper towel.
Differentiate with 0. 5% or 1% sulphuric acid in 25% alcohol or water, two
9‘5“er

changes of 1.5 minutes each. (Do not allow the slides to dry between carbol
fuchsin and acid alcohol. Sections should be pale pink).
Wash in tap water for 5 minutes.
>1

8. Counterstain in working Methylene blue solutions, one quick dip. (DO not
overstain the slide. Sections should be pale blue).
9. Blot sections and dry in 50-55% oven for 5 minutes.
10. Once dry, one quick dip in xylene.
11. Mount with permanent mountant.

Results.-

Acid fast bacilli including M. Lepra ............... Bright red

Nuclei and other tissue elements .................. Pale blue

Li_—Q_L___L_J_PUT
MSMEAR FLU RES ENCE Ml ROS OPY

PURPOSE:
The most important tool in the diagnosis of tuberculosis is direct microscopi
examination of appropriately stained sputum specimens for acid-fast bacilli. T
technique is simple and inexpensive, and detects those cases of tuberculosis, which a
infectious.

Sputum microscopy is also useful to assess the response to treatment, and to establish 7
cure or failure at the end of treatment.

BACKGROUND INFORMATION:

Fluorescent microscopes are provided to the state designated Intermediate Reference

Laboratories (IRLs) under Revised National Tuberculosis Control programme (RNTCP)
and at present, the use of fluorescence microscopy is linked to the culture and Drug
Sensitivity Testing (DST) activities ofthe lRLs.

Fluorescence staining utilizes basically the same approach as Z-N staining, but carbol
fuchsin is replaced by a fluorescent dye (auramine-O, rhodamine, auramine-rhodamine,
acridine orange etc), the acid for decolorisation is milder and the counter stain, though
not essential, is useful to quench background fluorescence. Both sensitivity and
specificity of fluorescence microscopy are comparable to the characteristics of the Z-N
technique.

The most important advantage of the fluorescence technique is that slides can be
examined at a lower magnification, thus allowing the examination of a much larger area
per unit of time. In fluorescence microscopy, the same area that needs examination for
10 minutes with a light microscope can be examined in 2 minutes.

PRINCIPLE:

Mycobacteria retain the primary stain even after exposure to decolorizing with acid
alcohol, hence the term "acid-fast". A counter-stain is employed to highlight the stained
organisms for easier recognition. Potassium permanganate is used as counter-stain and
it helps prevent non-specific fluorescence. With auramine staining, the bacilli appear as
5 slender bright yellow luminous rods, standing out clearly against a dark background.

The identification of the mycobacteria with auramine O: is due to the affinity of the
mycolic acid in the cell walls for the fluorochromes. In fluorescent microscopy, light rays
of shorter wave length pass through smear stained by a fluorescent dye, such as
auramine O, which have the property of absorbing light rays of shorter wave length and
emitting light rays oflonger wave length. A mercury vapour lamp is used as a source of
light and by means ofsuitable filter only light rays of shorter wave lengths are allowed
to emerge and these rays are used for microscopy. The condenser of the microscope is
made of quartz which will not absorb ultra-violet rays.

SPUTUM SMEAR PREPARATION:

'
'
an
3%
[{3ng
312

The procedure for smear preparation is described below: Sputum smear should be
prepared nearer to the flame (spirit lamp/Bunsen burner). Label 3 new clean,
unscratched slide at one end with the laboratory number using diamond tipped stylus.

Use mucopurulent portion for smear preparation. Transfer an appropriate portion of

the specimen to the slide by using a broom-stick or nichrome wire loop of 5mm dm (27
SWG).

Smear the specimen over an area of approximately 2 by 3 cm. Make it thin enough to be
able to read through it. Use a fresh slide for each specimen. Allow smears to air-dry for
15 minutes. Do not use heat for drying. Fix the smear to the slide by passing it over the
flame 3 to 5 times for 3 to 4 seconds each. After making smear, burn and dispose the
broom-stick or flame wire loop thoroughly using side burner prior to re-use.

MATERIALS REQUIRED FOR STAINING:

Auramine-Phenol solution 1%, Acid alcohol 0.1%, Potassium permanganate solution

PREPARATION OF STAINS AND REAGENTS (AURAMINE TECHNIQUE)

3 % Stock solution of phenol: Phenol crystals 3.0g (if liquid: 5gm phenol solid weight =
6ml liquid volume)

Distilled water 87ml Prepared from pure crystals dissolved in distilled water and stored
in a tight fitting glass stoppered bottle.

Auramine-Phenol solution: Warm 100 ml stock of three percent phenol to 40°C. To this
add gradually 0.3 gm of Auramine with vigorous shaking for 10 minutes. Filter and store
in a dark brownbottle. The stain should not be kept for more than 3 weeks. A standard
good quality powder of "Auramine 0" should be used (see specifications).

Acid Alcohol 0.5 ml concentrated hydrochloric acid 0.5 gm Sodium chloride 75 ml

absolute alcohol 25 ml distilled water Dissolve sodium chloride in water, add the
concentrated hydrochloric acid, mix with the alcohol and store in a tight fitting glass
stoppered bottle. Always add acid slowly to alcohol, not vice versa. Store in an amber
coloured bottle. Label bottle with name of reagent and dates of preparation and expiry.

Store at room temperature for upto three months.

0.1% Potassium permanganate: Freshly prepared in distilled water and stored in a dark
brown bottle. Label bottle with name of reagent and dates of preparation and expiry.
Store at room temperature for upto three months. KMnO4 is explosive, therefore, avoid
contact with combustible materials.

Specifications for Auramine 0 and KMnO4 Auramine 0:

Auramine hydrochloride; (1,1-bis(p-dimethylaminophenyl)methylenimine

hydrochloride)
Formula: C17H21N3HCl. H20 Mol Wt. 321.85 Appearance: Yellow to brown powde
Potency (Dye content): approximately 85.0% Absorbance: 435nm Auramine O is a '
yellow fluorescent dye; very soluble in water, soluble in ethanol; used to stain acid-fag.
bacteria in sputum or in paraffin sections of infected tissue Potassium Permanganate:
Formula: KMnO4 Mol Wt. 158.04 Potency: >99% Appearance: Purple solid, dissolves i
water to give deep purple solutions. 8

STAlNlNG PROCEDURE

Place the slides on a staining rack, with the smeared side facing up, the slides not
touching each other

Flood the slides with freshly filtered auramine-phenol.

Let stand for 7-10 minutes 9 Wash well with running water, taking care to control the
flow of water so as to prevent washing away the smear

Decolorize by covering completely with acid-alcohol for 2 minutes, twice 10 Wash well
with running water, as before to wash away the acid alcohol

Counterstain with 0.1% potassium permanganate for 30 seconds

Wash as before with water and slope the slides to air dry

Precautions

- Avoid under-decolorisation with acid-alcohol. Organisms that are truly acid-fast are
difficult to over-decolorize since the decolorisation procedure with acidalcohol is
relatively milder than the 25% sulphuric acid used in Z-N staining procedure.

- Avoid making thick smears. This will interfere with proper decolorisation, and
counterstain may mask the presence of AFB. Additionally, thick smears have a tendency
to flake, resulting in loss of smear material and possible transfer of material to other
slides.

- Strong counterstain may mask the presence ofAFB.

- Smears that have been examined by PM may be restained by Z-N staining to confirm
observations. To restain the same smear for Z-N, treat with 5% oxalic acid for 2 min,
wash and proceed for Z-N. However, once smears have been stained by Z-N staining,
they cannot be used for FM.

- Fluorescent stained smear are to be read within 24 hours ofstaining because of

fading.

- Stained smears have a tendency to fade on exposure to light. The slides are to be
stored in the slide box to avoid exposure to light. Alternatively, they may be stored
wrapped in brown or black paper and kept away from light.
 at
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EXAMINATION PROCEDURE AND REPORTING OF RESULTS

Switch on the mercury vapor lamp. The bulb takes approximately 10 minutes to reach
full intensity. Using the low power objective (magnification 100-150x) first examine a
known positive slide to ensure that the microscope is correctly set up.

With auramine staining, the bacilli appear as slender bright yellow fluorescent
rods, standing out clearly against a dark background.

Rule out any artifacts. Grade positive smears into four degrees of positivity using the
20x, 25x objective along with 10x eyepiece.

Smear needs to be observed in "linear pattern". For a trained and experienced LT, each
smear would take approximately a minimum of 2 minutes for 100 fields or three
horizontal sweeps.

In the fluorescent staining, smears are examined at much lower magnifications

[typically 250x] than used for ZN-stained smears (1000x).

Each field examined under fluorescence microscopy, therefore, has a larger area than
that seen with bright field microscopy. Thus, a report based on a fluorochrome-stained
smear examined at 250x may contain much larger numbers of bacilli than a similar
report from the same specimen stained with carbolfuchsin and examined at 1000x.

For the purpose of uniformity for examination and quantitative reporting of results, a
method has been suggested (Reference 1. WHO Manual on Microscopy Part [1)
whereby the number of acid-fast bacilli observed under fluorochrome staining could be
divided by a "magnification correction factor" to yield an approximate number that
might be observed if the same smear were examined under 1000x after carbol fu’chsin
stain. To adjust for altered magnification of fluorescent microscope, when using
objectives of x20 or x25 powers, divide the number of organisms seen under FM by the
factor of 10.

Similarly, ifone using a 40 x objective the magnification correction factor is 5, and if one
using a 45 x objective it is 4.
 Table 1: Comparative grading

grading (using 100x oil staining grading (using

immersion objective and 20 or 251: objectlv'e and
10x eye piece) 10x eye piece)

-
>10 AFBlfield after >100 AFB/field after Posm"ve. 3+

-
1-10 AFBifieid afler 1 1-100 AFBlfieid after Posm"ve. 2+

——
10-99 AFBI100 field

1—9 AEBI100 field

1-10 AFBI field after

1-3 AFB/100 fields

No AFB per 100 fields
Posrti"ve, 1+

doubtful posm"ve [repeat

No AFB per 100 fields

Table 2: Magnification correction factor

FM objective Magnification
magnification correction

' To obtain the comparative grading, divide the observed court of AFB under the FM objective with this factor before
grading.

L__—LERT'S
STAINING

It is a special stain used to demonstrate the granules - volutin or metachromatic in

Corynebacterium diphtheria for the identification of bacterial species.

Reagents used:

Albert's A: Toludine blue, Malachite green, Glacial acetic acid in 95% Alcohol &
deionised water

Albert's B: Iodine dissolved in Potassium Iodide & deionised water.

Procedure:

1] Prepare the smear from the sample & heat fix

2] Stain with Albert‘s A for 3-5 min & wash with Albert's B
 ‘/

.v‘.

"I 3] Stain with Albert’s B for 1-2 min

4] Wash with water & blot dry
5] Observe under oil immersion

interpretation.-

Bacilli — Body of the bacilli appear green

Granules - appear bluish black

Other methods for staining Metachromatic granules.-

1] Neisser’s method: Bacilli — pink, Granules — deep blue

2] Pugh's method: Bacilli — light blue, Granules — reddish purple

_G—__[\U—____MS_]NE
ATIVE STAINING ITH INDIA INK 0R NI R IN

India ink or Nigrosin can be used to demonstrate the capsule of microorganisms.

1] Nigrosin stain: Nigrosin — 100gm/ L; Formalin — Sml/ L in water

2] India Ink Stain: Black Pelican drawing ink N o 17; Deionised water; Thimerosol

Uses:

To demonstrate and identify capsulated organisms - Cryptococcus / Klebsiella sps /

Streptococcus pneumonia

Principle:

The stain is a suspension of carbon, formed in India ink or Nigrosin. The carbon
particles are negatively charged as in the cell membrane. As they repel the dye, cells
remain clear & the background looks black of dark green.

Procedure:

1] Place a drop oflndia ink / Nigrosin on a clean slide

2] Using aseptic precautions, add a loopful oforganism to the drop ofstain & mix
3] Using another slide (spreader) at 45° angle spread it along the first slide making a
thin smear. 4] Add a cover slip and observe under light objectives] (Low & High power)

Staining procedure for demonstration of bacteria or yeast cell.-

1] Take 1-2 drops ofstain on a clean slide & make a smear ofthe organism
2] Allow the smear to air dry; do not heat fix
3] Add Safranin / Crystal violet / Methylene blue — leave for 3 minutes
4] Observe under oil immersion

Interpretation:

Capsules - appear clear/ remain as unstained zones

Background — Black / Dark background of the respective stain (India ink/ Nigrosin)

If Safranin / Crystal violet / Methylene blue is used for staining the cell, it takes up the
respective color

L_____[____—1PORE
STAINING SCHAFFER FULTON

Spores do not take up stains readily, appears as a refractile colorless objects in gram
stain. They can be stained with Schaffer Fulton stain or by Dorner method.

Reagents Used:

Schaffer Fulton stain A: Malachite green — SOg/ L water

Schaffer Fulton stain B: Safranin — 0.5g/ L in water

Uses:

To demonstrate the morphology & position ofthe spores in Clostridia 8: Bacillus

species.

Principle.-

This differential stain distinguishes between vegetative cells and Endospores.

The spore coat takes up Malachite green (Primary stain) when heat is applied, which
cannot be decolorized later.

Malachite green is water soluble and has low affinity for cellular material, so vegetative
cells will get decolorized. Vegetative cells are counterstained with Safranin.

Procedure:

1] Make smear, air dry & heat fix.

2] Use positive & negative controls — E. coli / B. subtilis
3] Flood the smear with Malachite green
4] Cover the flooded slide with filter paper & steam for 10 min
5] Drain the slide & rinse for 30 sec with deionised water
6] Flood the smear with Safranin — leave for 1 min
 MNV lw'l
IV ,2”

'
1‘1
t’ ,'

/.‘ Drain the slide & rinse for 30 sec with deionised water
’ 8] Blot dry & observe under oil immersion

Interpretation.-

Spores — Green colored

Vegetative cells — Red / Pink colored

_——____(_]LACTOPHENOL
COTTON BLUE LPCB

Reagents used: [LPCB with polyvinyl alcohol]

Polyvinyl alcohol (PVA) powder — 15gm

Distilled water - 100ml
Phenol, Lactic acid & Cotton blue

Staining solution:

PVA stock solution — 56ml

Melted Phenol — 22ml
Lactic acid — 22ml
Cotton Blue - 0.05gm

The preparation has three components: phenol, which will kill any live organisms; lactic
acid which preserves fungal structures, and cotton blue which stains the chitin in the
fungal cell walls.

To prepare permanent stained preparations, PVA can be added to Lacto phenol cotton
blue (LPCB) stain

Uses:

Lacto phenol cotton blue is a stain which is used to stain the fungi & study its
morphology in routine mycology laboratory for identification of fungal species.

Procedure:

Tease out fungal culture on a glass slide in a drop of LCB stain using teasing needles,
put up cover slips & examine under dry objectives of the microscope.
 Stains for fungi:

Most fungi that infect the subcutaneous and horny layers of skin and hair shafts, belong-l:-
to microsporum and trichophyton groups and appear as yeasts, or mycelia forms. Thes'
can be readily demonstrated with the common special stains, Gomori’s methenamine
silver (GMS), Gridley's fungus (GF), and periodic acid-Schiff (PAS), also referred to as
“broad spectrum" fungal stains.
The fungal stains such as alcian blue and Mayer's or Southgate's mucicarmine, that
readily demonstrate the mucoid capsule of Cryptococcus neoformans can be termed as
“narrow-spectrum" stains for fungi. This staining reaction differentiates Cryptococcus
neoformans from other fungi of similar morphology such coccidiodes, candida and
histoplasma.
When these fungi grow in tissue, they display asexual forms, and appear as spherical
yeast or spore forms. Some grow as tubular hyphae that may be septate and branched. A
mass of interwoven hyphae is called a fungal mycelium. Rarely, spore producing fruiting
bodies called sporangia or conidia are produced.

WA\ ( Grocott methenamine silver (GMS) stain for fungi:

W I 8 -=='-._.—’

//\’1’9 ‘ Principle: Fuwn

“ll/J22) 1' for example have polysaccharides in their cell walls which on oxidation
/ 'vgreleasedialdeh de groups. These aldehyde groups besides being stained by the
saw" Schiffs reagennmywesVecanalsobedemonstratedbthelrablhtoreducemethenammIlr'""
W. nitrate in an alkaline solution. The G'________—_grocott's
alkaline methenamine silver nitrat
, i fiso‘\___b______g_g_%~Ldp_________nlution
reresents a vehicle which uon reduction, reciitates nascent silver ios,
MW” thus blackening the site.
isa Wuw “J “m
[W7 Advantages: GMS is preferred for screening, because it gives better contrast, and stains
118 NW ‘ even degenerated and flT—T‘onwaeungi tat are sometimes refractory to H & E. GMS also
leowstains algae, intracytoplasmic granular inclusions of Cytomegalovirus, Actinomyces
‘ Israeli, Noc‘ardia, Mycobacterium, and nonfilamentous bacteria with polysaccharide
Moi/Low ,capsules such as Klebsiella pneumoniae and Streptococcus pneumoniae. Prolonged
W staining in the silver nitrate solution may be required to adequately demonstrate
degenerated fungal elements such as the yeast-like cells of Histoplasma capsulatum.

Disadvantages: The stain masks the natural color of pigmented fungi, making it
impossible to determine whether a fungus is colorless, hyaline or pigmented. Such a
determination is crucial in the histologic diagnosis of mycosis . GMS does not adequately
demonstrate the inflammatory response to fungal invasion.

Ingredients:
Solutions:
a. 5% sodium tetraborate in distilled water
b. Methenamine solution
5% silver nitrate in distilled water ......... 5ml
3% Methenamine in distilled water .......... 100ml
Add silver nitrate to Methenamine silver, gently shaking until formed precipitate
\, ’ dissolves.
VH1:6W,
H
W,

W”
MI
incubating solutions:
Solution a (Borax) ........................... Sml
Distilled water .................................. 25ml
Solution b Methenamine silver... .25ml

Ideally, the Methenamine silver/water solution and the borax should be preheated to
560C and mixed prior to use, as the silver solution starts to degenerate once borax is
added.

Method 41%
1. Bring sections to distilled water. filo
2. Oxidise with 5% aqueous chromic acid for 1 hr W "'
3. Wash in water for a few seconds. //7‘ W
4. Treat sections with 1% sodium metabisu'l'phite 1 min
5. Wash in running tap water 5 mins
6. Rinse thoroughly in distilled water.
7. Place in pre-heated working silver solution in a water bath at 56 °C for 30 to 40 mins
until section turns yellowish-brown. (The incubation time is variable and depends
upon the type and duration of fixation —may also be 1-3 hours at 37 to 45°C ).
8. Rinse well in distilled water. 3
9. Tone sections with 0.1% gold chloride for 4 mins.C$ ’9 “A
10. Rinse in distilled water.
11. Treat sections with 3% sodium thiosulphate for 5 mins (to remove unreacted silver).
12. Wash with running tap water 5 mins.
13. Counterstain in working 1% light green in 0.1% acetic acid for 15-30 sec or
H & E.
14. Rinse excess light green off slide with alcohol 7
flog/cw.
15. Dehydrate, clear and mount.

429%
Results

Leishmania, toxoplasma .............................................................negative NW @7064ch 4

Mucin..................................................................................................... dark grey
Red blood cells................. .. . . . . ..................yellow
Background........................................................................................ pale green

Gomori ‘s technique : is similar to that of Grocott's; infact the latter is an adaptation of

the Gomori's technique with a difference in incubation times.

Other silver stains: Silver stains have varied applications in histopathology, they stain
not only reticulin fibres but also neuroendocrine granules, micro-organisms as well as
have applications in renal biopsies. This depends on their unique ability to pre cipitate
metallic silver which is subsequently reduced to give a black colour. Microorganisms
like spirochetes also have the capacity to bind to silver which can be reduced by other
a ents to black silver /. "’J
g
. 0,0.2/ J
~
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\ c Mg
/C/‘7/.
“Every???” pl},
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Silver impregnation is often spoken of as if it were a single, unvarying process, i.e.
depending on a singlegchemical basis which underlies all the different impregnation
procedures, but that is' probably not the case. The various methods for different
structures may manipulate silver deposition by different mechanisms and be based on
different chemical underpinnigs. The only general underlying principle is that finely
divided silver is deposited and appears as dark brown or black deposits.

When discussing silver impregnation it is usual to refer to substances as being

argentaffin or argyrophil. Argentaffin means that the tissue structure causes silver to be
deposited without a reducing agent whereas argyrophil means a chemical reducing
agent should be used for the silver deposition. Thus melanin impregnated from an
ammonical silver solution is said to be argentaffin and, using the same silver solution in
conjunction with dilute formalin to impregnate reticulin, would be said to be argyrophil.
To these two another term ("induced argentaffin") may be added i.e. chemical
production of a argentaffin material which is then used to reduce silver compounds
without using a supplementary reducing agent. The gomori and jones procedure are
examples of this, both of which use an oxidising agent(chromic and periodic acid
respectively) to produce aldehydes which then reduce ammoniacal or methamine silver
solutions without any additional chemical reducing agents.

Therefore, the "argentaffin reaction" is the ability of a silver complex solution to

blacken a tissue element without the need ofa reducing bath. The term is adjectival and
is applied to many methods. The term "argentaffin reaction" should therefore not be
used as a proper name. It is most commonly used to describe the brown black
granularity observed in the carcinoid tumours. The tumours arise from the Kulchitsky
cells or neuroendocrine cells. The cells manufacture the 5-HT (5 hydroxytryptamine)
from tryptophan. When S—HT is produced in excess, it may lead to peculiar changes in
the right heart and lungs. It is also associated with the excretion in the urine oflarge
amounts of the degradation products of 5-HT, ie., S-hydroxyindole acetic acid (5—HIAA).
Probably by virtue of the content of 5-HT, or a precursor substance which may be
bound to protein, the enterochro'maffin cells give a number of histochemical reactions.
They reduce ammonical silver solutions by virtue of the phenolic structure of 5—HT or
its precursor ie. they are argentaffin. Carcinoids arising in the common sites ie.
appendix and small intestine [mid gut carcinoids) almost always react and are
argentaffin. Those arising in the unusual sites eg. Rectum, stomach, bronchus, more
frequently fail to give argentaffin reaction but are argyrophilic. All argentaffin
carcinoids are also argyrophilic.
ll1‘; 1

Examples of silver stains used :

1. Gremilius stainL-(Argyrophil stain): shows positive granules in carcinoid tumours-

Foregut and hindgut carcinoids(Bronchial, stomach, 1st part of duodenum,
oesophagus, pancreas, transverse colon last 3“, descending colon, rectum)
2. Argentaffin stain (See Masson's Fontana silver stain) — midgut carcinoids 1'.e.
second part of duodenum onwards to 1st 2/3rd of transverse colon
3. Gomori’s Methenamine silver stain .............fungus, pneumocystis lerovski
4. Gridley’s silver stain ..................................... stains fungus and chalymatobacteria
granulomatis '
5. PAS-methenamine silver stain .....................basement membrane in kidney
6. Levaditi's stain..............................................spirochaetes
7. Warthin starry sky Helicobacter pylori

Fixation : Bouin’s or formalin TK‘L

0v”
Q,

Sections.-Parafin embedded a/W, % {NM/LL.

Reagents.- M, E 600°
a)_ Silver solution?!
(If
o Acetate buffer, pH5.6 10ml
0 Double glass distilled water 87 ml
0 1% aqueous silver nitrate (Fresh) 3ml
b) Reducing solution
/&nlwfl
My 0 Hydroquinone 18m WWW/w
I‘WV ,
ab 0 Sodium sulphite crystals ng M Tyr 1U”
J's
UKK' o Distilled water (freshly prepared) 100ml 99‘“
.9)" ~
Acetate buffer - Solution a) Acetic acid 1.2m] 94R yo 94‘ I C We MN,
‘I O‘L fl“
Distilled water 100ml / CW
Solution b) Sodium acetate 2.7gm
Distilled water 100ml

Mix 9ml of (a) + 91 ml of (b) ; adjust pH to 5.6

-c:::.:
Procedure:

1) Bring sections to distilled water.

2) Transfer sections to pre heated silver solution at 60°C
flh—U‘
3hors_
3) Drain silver solution from slides thoroughly.
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