JPCS Vol(1) ● April-June 2011
www.arpapress.com/Volumes/JPCS/Vol1/JPCS_1_04.pdf
A NEW HAEMATOXYLIN STAIN FOR THE DEMONSTRATION OF
NUCLEAR AND EXTRA NUCLEAR SUBSTANCES
Sina Iyiola1,* & Godwin Avwioro2
*Email: [email protected], Tel: 2348066003863
1
Department of Histopathology, Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria
2
Faculty of Basic Medical Science, Delta State University, Abraka, Nigeria
ABSTRACT
Haematoxylin is the most important and most used dye in histology, histochemistry, histopathology and in cytology.
Several haematoxylin staining techniques exist, based on their mode of preparation and their action on tissues. The
improved method of preparation of alum haematoxylin stain described here was to add to the compendium of
existing methods. 1g haematoxylin, 50g ammonium alum, 1g citric acid, 0.15g sodium iodate and 50ml glycerine
were boiled in 1000ml distilled water. The resulting mixture was used to stain sections of kidney, liver, intestine,
lung, intestine and the heart, and counter stained with aqueous eosin according to the standard technique. The result
was compared with standard haematoxylin and eosin techniques using Ehrlich's, Mayer's, Cole's, Harris, Delafield's
and Carazzi's haematoxylin as the nuclear stain. No difference was noticed microscopically between the improved
haematoxylin and the existing techniques. The major advantages of the improved technique are in its usage. It has
the ability to stain progressively and regressively depending on the staining time. There is also absence of scum on
the surface of the stain when it was left unused for several weeks. Therefore, filtration for the removal of scum
which is necessary in certain haematoxylin preparations is not necessary in the improved technique.
Key words: Haematoxylin, histochemistry, staining, histology
1. INTRODUCTION
Haematoxylin has been used extensively in cytology, histology, histopathology and histochemistry [1]. Its most
important use in histopathology is as a nuclear stain for the diagnosis of malignant lesions. It has also been used for
the diagnosis of non malignant lesions. Being able to stain several intracellular and extracellular substances makes it
an indispensable dye in the histology laboratory [2]. Haematoxylin is obtained from the logwood, haematoxylon
campechianum [3]. The crude product is obtained from the milled heartwood by hot water or steam, purified by
ether extraction, dried and recrystallized from water [4,5,6]. Alternatively, the aqueous extract is precipitated from
solution with urea [7]. Haematoxylin has also been prepared synthetically [8], but the natural dye is mostly used.
Haematoxylin has to be oxidized to haematein [5,9] before it can be used as a stain. The oxidation process can be
achieved naturally and artificially. In the natural process, prepared haematoxylin solution is exposed to air and
sunlight for 6 to 8 weeks before it is used [10]. The advantage of the natural process of oxidation is that the solution
lasts longer because oxidation is slow and gradual.
Fig. 1
Haematoxylin C16H14O6
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Haematein C16H12O6
JPCS Vol(1) ● April-June 2011
Iyiola & Avwioro ● Demonstration of Nuclear and Extra Nuclear Substances
In the artificial process of oxidation, oxidising agents are added to the solution of haematoxylin to convert it to
haematein and the solution may be used immediately, although with a shorter life span because dye solution is
quickly over oxidized. Haematein cannot stain a tissue unless a mordant is incorporated into the solution of the dye
[11,12]. A mordant is a metallic salt, which acts as a bridge between the stain and tissue enabling staining to take
place [1]. The colour of the end product depends on the type of mordant used. Iron alum is black, potassium alum is
blue while salt of tin is red [2]. Examples of alum haematoxylin are Ehrlich's [13], Mayer's [14], Cole's [15], Harris
[16], Delafield's [17] and Carazzi's [18] haematoxylin. Weigert's [19], Verhoeff's [20] and Heidenhain's
haematoxylin are examples of iron haematoxylin [21]. Haematoxylin may be used progressively where
differentiation is not required or regressively where differentiation is required [1]. It has been used extensively
either as a primary stain or as a counter stain for the demonstration of several substances such as lipoproteins [22],
myelin [23,24], phospholipids and cytoplasmic granules in B cells of the anterior pituitary, pancreatic islet [25],
collagen [26,27], neural tissue [28]. fatty acids [22,29], mitochondria [30], amyloid [28,31], glycogen [30], mucin
[32], phospholipids [33], astrocytes, myoglia and fibroglia [14] microfilaria and amoeba [34], chromosomes,
chromatin, nucleoli, mitochondria, centriole, cross striations of muscle fibers [21], fibrin [35], lead, copper and iron
[36], immunohistochemstry for The demonstration of tissue antigens [37,38] and in cytology [16,35]. The objective
of this study was to add to the compendium of existing alum haematoxylins, an improved scum free alum
haematoxylin stain for the rapid diagnosis of malignant and non malignant lesions as well as for the demonstration
of intracellular and extracellular substances.
2. MATERIALS AND METHODS
Six different alum haematoxylin solutions were prepared as controls; Ehrlich's alum haematoxylin [15]
(Haematoxylin 6g, absolute alcohol 300ml, distilled water 300ml, glycerol 300ml, glacial acetic acid 30ml and
ammonium alum to saturation), Harris alum haematoxylin [35] (Haematoxylin 2.5g, absolute
alcohol
50ml,
potassium alum 50g, distilled water 500ml, mercuric oxide 1.5g and glacial acetic acid 20ml), Mayer's haemalum
[34] (Haematoxylin 1g, distilled water 1000ml, ammonium alum 50g, sodium iodate 0.2g, citric acid 1g and chloral
hydrate 50g), Cole's haematoxylin [17] (Haematoxylin 1.5g, 1% iodine in 95% alcohol 50ml, saturated aqueous
potassium alum 700ml and distilled water 250ml), Gill's haematoxylin [39] (Haematoxylin 2g, distilled water 730ml,
ethylene glycol 250ml, sodium iodate 0.2g, aluminium sulphate 17.6g and glacial acetic acid 20ml) and Carazzi's
haematoxylin [18] (Haematoxylin 0.5g potassium iodate 0.01g, potassium alum 25g, glycerol 100ml, distilled water
400ml). Tissues, 3 mm thick were obtained from the kidney, liver, intestine, lung, intestine and the heart. They were
fixed in 10% formol saline for 24 hours and processed by the paraffin wax method through ascending grades of
alcohol for 2 hours each, two changes of xylene for 2 hours each, two changes of paraffin wax at 70ºC for two hours
each. Tissues were subsequently embedded in paraffin wax at about 70ºC. 10 serial sections of 3µ thick were cut
from each of the tissues with the rotary microtome. 6 sections from each block were stained with haematoxylin and
counter stained with 1% aqueous eosin according to the methods of Ehrlich, Harris, Mayer, Cole, Gill and Carazzi.
Another haematoxylin solution was prepared as follows:
Table 1 Iyiola and Avwioro’s haematoxylin
Heamatoxylin
1g
Citric acid
1g
Ammonium alum
50g
Glycerine
50ml
Distilled water
1000ml
Sodium iodate
0.15g
The reagents were added to about 500ml of distilled water, mixed and made up to 1000ml with distilled water. The
mixture was boiled, removed from flame and sodium iodate added immediately and mixed gently. The mixture
which turned deep red was cooled rapidly in running water.
The remaining four sections were stained with the prepared haematoxylin for 2, 5, 10 and 20 minutes and counter
stained with eosin according to the standard method.
3. RESULTS
Similar staining reaction was observed when the new haematoxylin stain was used on the different sections and
compared with existing alum haematoxylin of Ehrlich, Harris, Mayer, Cole, Gill and Carazzi. Several intracellular
and extracellular substances were stained in different shades of blue. Malignant cells were well differentiated from
non malignant cells when the stain was counter stained with eosin. Sections stained well between 2 minutes and 20
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JPCS Vol(1) ● April-June 2011
Iyiola & Avwioro ● Demonstration of Nuclear and Extra Nuclear Substances
minutes and differentiation was not necessary when it was applied onto sections for less than 10 minutes. Scum
which frequently forms on the surfaces of some haematoxylin solutions when they are left unused for a few days
was not observed in this preparation. The alum haematoxylin was used immediately after preparation of the stain.
The active staining ingredient was haematein obtained from oxidation of the heamatoxylin. The mechanism of
staining was through indirect method with the ammonium alum acting as the mordant.
4. DISCUSSION
Sections stained well between 2 minutes and 20 minutes and differentiation [1] was not necessary when the new
haematoxylin stain was applied onto sections for less than 10 minutes. This means, it can be used as both a
progressive stain and as a regressive stain. Therefore, it has combined the advantages of a progressive stain [35] and
a regressive stain [15]. The oxidizing agent which it contains enables its usage immediately after preparation which
makes it similar to Harris’s [35], Mayer’s [34] and Cole’s [17] haematoxylin. It is a good companion for rapid
diagnosis of diseases being able to stain tissues very well within 2 minutes as a progressive stain. Generally
haematoxylin is the most important and most used dye in the medical laboratory being able to differentiate
malignant cells from non malignant cells makes it an excellent tool in the diagnosis of diseases affecting tissues. Its
ability to stain several intracellular and extracellular substances in shades of blue to black also makes it very useful
in histochemistry and histology [34,17]. We have found this quality in the new haematoxylin stain. The stain keeps
well like the other haematoxylin stains which contain oxidants but a major advantage of the new haematoxylin is
that it does not form a scum when it is stored making filtration unnecessary before usage. The active staining
ingredient was haematein obtained from oxidation of the heamatoxylin. The citric acid helped to sharpened the
nuclear staining, while the mordant was ammonium alum. The glycerine was the stabilizer, while immediate
oxidation of haematoxylin to haematein was achieved with the addition of sodium iodate. Water was the solvent in
which the reagents were dissolved. The mechanism of staining was through indirect method with the ammonium
alum acting as the mordant. We have not experimented on the use of this dye as a counter stain on specific methods
such as in the demonstration of lipids, carbohydrates and immunohistochemical procedures [1]. This is a necessary
step because Erhlich’s haematoxylin [13] which is often used routinely for the demonstration of general structures is
not used as a primary stain in the demonstration of neutral carbohydrates [30,40]. The new haematoxylin stain is a
promising stain that can compete favourably with other alum haematoxylin preparations.
5.
REFERENCES
[1].
[2].
[3].
[4].
[5].
[6].
[7].
[8].
[9].
[10].
[11].
[12].
[13].
[14].
[15].
[16].
[17].
[18].
Avwioro OG. Histochemistry and tissue pathology, principles and techniques. Claverianum press, Nigeria.
2010
Lillie RD, Fullmer HM. Histopathologic technic and practical histochernistry. New York: McGraw-Hill.
1976
Willis JC. A dictionary of the flowering plants and ferns. Cambridge: Cambridge University Press. 1951.
Corm HJ. The haematoxylin problem. Stain Technol. 1927; 2;1-3.
Duff DH. Logwood colouring matters. Dyer 1950; 103;271-3.
Lalor GC, Martin SL. Studies on haematoxylin and haematein. The colouring principles of logwood. IAbsorption spectra of pure compounds in various solvents and a spectrophotometric method of analysis for
haematoxylin and haematein. J. Soc. Dyers Colourists 1959; 5;513-517.
Stevens A. The haematoxylins. In: Bancroft JD, Stevens A, eds. Theory and practice of histological
techniques. Edinburgh: Churchill Livingstone, 1990.
Morsingh F, Robinson R. The synthesis of diazilin and haematoxylin. Tetrahedron. 1970; 26;281-289.
Culling CFA. Handbook of histopathological and histochemical techniques. 3rd ed. London: Butterworth,
1974.
Drury RAB, Wallington EA. Carleton's Histological Technique. 5th ed. Oxford University Press, Oxford
1980
Marshall PN, Horobin RW. The oxidation products of haematoxylin and their role in biological staining.
Histochem J. 1972; 4;493-503.
Clark G. Comparison of various oxidants for alum hematoxylin. Stain Technol. 1974; 49;225-227.
Scott SG. On successive double staining for histological purposes. J. Pathol. Bact. 1912; 16;390-398.
Mallory FB. On certain improvements in histological technique. J. Exp. Med. 1897; 2;529-533.
Ehrlich P. Hamatoxylinl osung. Z. Wiss. Micr. 1886; 3;150.
Papanicolaou GN. A new procedure for staining vaginal smears. Science. 1942; 95;438-439
Cole EC. Studies on hematoxylin stains. Stain Technol. 1943; 18;125-142.
Carazzi D: Eine neue Haematoxylinlbsung. Z Wiss Mikr 1911, 28:273-4.
22
JPCS Vol(1) ● April-June 2011
Iyiola & Avwioro ● Demonstration of Nuclear and Extra Nuclear Substances
[19]. Weigert K. Eine Kleine Verbesserung der haematoxylin-van Gieson-Methode. Z Wiss Mikr 1904; 2;1-5.
[20]. Verhoeff FH. Some new staining methods of wide applicability. Including a rapid differential stain for elastic
tissue. JAMA 1908; 50;876-877.
[21]. Heidenhain R. Eine neue Verwendung des haematoxylin. Arch. Mikr. Anat. 1885; 24;468-470.
[22]. Gurr E. Staining animal tissues: practical and theoretical. London: Leonard Hill, 1962.
[23]. Pal JW. Med. Jahrb., NF. 1886; 1;619-631
[24]. Loyez M. Coloration des fibres nerveuses par la méthode à l'hématosyline au fer après inclusion à la
celloidine. C R Soc. Biol (Paris) 1910; 69;511–513
[25]. Gomori G. Observations with differential stain of human Islet of Langerhans. Amer. J. Path. 1943; 17; 395406.
[26]. Bulmer D. Observations on histological methods involving the use of phosphotungstic and phosphomolybdic
acids, with particular reference to staining with phosphotungstic acid /haematoxylin. Q. J. Microsc. Sci. 1962;
103;311-323.
[27]. Mallory FB. Pathological technique. Philadelphia: WE; Saunders, 1938.
[28]. Mallory FB. Phospho-molybdic acid haematoxylin. Anat. Anz. 1891; 6;375-376.
[29]. Casselman WGB. Histochemical technique. London-New York: Methuen & Co. Ltd
[30]. Thompson SW. Selected histochemical and histopathological methods. Springfield: Charles C Thomas, 1966.
[31]. Hignman B. Improved methods of demonstrating amyloid in paraffin wax sections. Arch. Path. 1946; 41;559562
[32]. Mayer P. Mitt. zool. Stn. Neapel. 1896; 12;303
[33]. Baker JR. The histochemical recognition of lipine. Quart. J. Micr. Sci. 1946; 87; 441-471
[34]. Mayer P. Ueber das Forben mit haematoxylin. Mitt Zool Stat Neapel 1891;10;170-186.
[35]. Harris HR. On the rapid conversion of haematoxylin into haematein in staining reactions. J. Appl. Microsc.
1900; 3;777-780.
[36]. Culling CFA, Allison RT, Barr WT. Cellular pathology technique 4th ed. Butterworths, London, 1985.
[37]. Costa CAX, Brito KNO, Gomes MA, Caliari MV. Histopathological and immunohistochemical study of the
hepatic lesions experimentally induced by Entamoeba dispar Eur. J. Histochem. 2010; 3;
[38]. Nadworny PL, Wang JF, Tredget EE, Robert E. Anti-inflammatory activity of nanocrystalline silver-derived
solutions in porcine contact dermatitis Burrel J. Inflammation 2010; 7;13
[39]. Gill GW, Frost JK, Miller KA. A new formula for a half-oxidised hematoxylin solution that neither overstains
nor requires differentiation. Acta. Cytol. 1974; 18;300-311.
[40]. Pearse AGE. Histochemistry, Theoretical and Applied, 4th ed. Churchill-Livingstone, Edinburgh. 1980
23