Notes and queries
This section contains anonymous questions and signed peer-reviewed answers to questions on topics within the scope of the journal. Send questions or answers by mail to: Dr. JA Kiernan, Department of Anatomy and Cell Biology, The University of Western Ontario, London, Canada N6A 5C1, or by e-mail to
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Which buffer for immunohistochemistry? How should I choose the type of buffer to use for immunocytochemistry? Can an antibody react well in phosphate buffered saline (PBS) but not in TRISbuffered saline (TBS), or the other way around? As far as I know it makes no difference whether PBS or TBS is used for washing or for diluting antibody solutions. Immunohistochemistry is a very tolerant technique in this respect! There is one situation in which phosphate buffers must be avoided; this is when using alkaline phosphatase (AP) as a marker enzyme, as in many labeled secondary antibodies. The activity of this enzyme is strongly inhibited by the phosphate ions in PBS. After incubation in an AP-labeled immunoreagent, you must rinse your slides three times with a nonphosphate buffer (e.g., TRIS buffer or TBS) before starting the AP visualization reaction. Chris van der Loos Department of Cardiovascular Pathology Academic Medical Center Amsterdam / The Netherlands
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Acid-fast bacilli in tap water This is completely anecdotal, but I have heard of various mycobacteria lurking in tap water that show up on sections washed in that water, causing acid-fast false positives. Is this an urban legend? – Biological Stain Commission Biotechnic & Histochemistry 2004, 79(2): 107 /109.
DOI:10.1080/10520290410001729359
Mycobacteria lurking in taps is not an urban legend; it is real. I have had it happen. We traced some Ziehl /Nielsen /positive bacteria to the crud (a technical term) on the inside of a vacuum trap at our special stains bench. Nutrients in the water and in the air evidently allowed the growth of plates of the organisms. Fragments of these plates broke off and adhered to sections, giving false positives. The organisms were thicker and longer than tubercle bacilli and were strongly beaded, but who knows what others could look like. After that, I regularly cleaned the inside of all the taps in the lab with brushes, and it never happened again. Bryan Llewellyn Section Head (retired) Anatomic Pathology Prince George Regional Hospital Prince George, BC, Canada
[email protected] The acid-fast bacilli that come out of the water faucet are supposedly Mycobacterium gordoneae (gor-DOH-nee-ee, after microbiologist Mary Gordon). A synonym is M. aquae (ACK-wee, ACK-wye or ACK-way, according to your schooling). They aren’t pathogenic except maybe in situations of extremely compromised immune function. Mycobacteria of tap water stain with both light microscopic and fluorescent acid-fast techniques. The trick to recognizing them is that usually they are slightly out of the plane of the section, and in tissue sections they turn up randomly rather than in places in tissues where a pathologist would expect to see them. Robert S. Richmond Department of Pathology Gaston Memorial Hospital, Box 1747 2525 Court Drive Gastonia, NC 28053-1747, USA
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The M’Fadyean reaction: a stain for anthrax bacilli What is the M’Fadyean reaction for anthrax bacilli, and how should the technique be carried out? 107
M’Fadyean (1903a) used 1% methylene blue to stain smears of blood from dead farm animals. The darkly stained anthrax bacilli were within capsules that were colored pink rather than blue. Later, the same author reported that the pink staining did not occur with aqueous solutions of ‘‘fresh, new methylene blue.’’ Bright pink staining was seen, however, with a solution made by boiling the new dye powder in 0.5% aqueous sodium bicarbonate (M’Fadyean 1903b). The pink (metachromatic) color of Bacillus anthracis capsules evidently requires dyes formed by oxidative demethylation of methylene blue, which happens with aging or when an alkaline solution of the dye is heated (Lillie and Fullmer 1976). The capsule of B. anthracis is composed of a linear polymer of D-glutamic acid (Mock and Fouet 2001). The stacked side-chain carboxylate groups of this material may account for the metachromatic staining property. The technique recommended by the WHO (World Health Organization, 1998) is as follows: Two alternative formulations of the staining solution (Loeffler’s methylene blue) are given. For the simpler one, dissolve 0.3 g methylene blue chloride (C.I. 52015) in 30 ml 95% ethanol, then add this solution to 100 ml of 0.01% potassium hydroxide. Finally, add 0.65 g potassium carbonate (K2CO3).
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Fix the air dried blood film in methanol or ethanol for 30 /60 sec. Stain with Loeffler’s methylene blue for 30/60 sec. Wash with water; collect the washings into sodium hypochlorite solution (bleach). Blot and examine.
Mock M, Fouet A (2001) Anthrax. Ann. Rev. Microbiol. 55: 647 /671. World Health Organization (1998) Guidelines for the Surveillance and Control of Anthrax in Humans and Animals , 3rd ed. http://www.who.int/emc-documents/ zoonoses/docs/whoemczdi986.html World Health Organization (2003) Model C Microscopic Examination for Anthrax . http://w3.whosea.org/bct/ anthrax/modelcf.htm
Michael P. Owen FDA Pacific Regional Laboratory Northwest 22201 23rd Drive SE Bothell, WA 98021, USA
[email protected] John A. Kiernan Department of Anatomy and Cell Biology The University of Western Ontario London, Canada N6A 5C1
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Staining sections with Giemsa Can paraffin sections be stained with Giemsa? If so, how is it done? The method used for blood smears stains everything in the section blue. Giemsa’s stain is a mixture containing azure B, methylene blue and eosin Y (Wittekind 2002). It gives excellent results with ordinary paraffin sections once you have found the ideal pH for the staining solution. Fixation affects the color balance, so you must experiment the first time you use the method. After adequate formaldehyde fixation (at least 24 h) the best pH is often in the 4.0 to 4.5 range.
Solutions Chains of blue-black bacilli are surrounded by pink capsules. Putrefaction of the specimen can lead to loss of the capsules. For a colored illustration see World Health Organization (2003). Some nonvirulent strains of anthrax bacilli lack the capsule.
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References
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Lillie RD, Fullmer HM (1976) Histopathologic Technic and Practical Histochemistry, 4th ed., McGraw-Hill, New York. p. 133. M’Fadyean J (1903a) A peculiar staining reaction of the blood of animals dead of anthrax. J. Comp. Pathol. 16: 35 /41. M’Fadyean J (1903b) A further note with regard to the staining reaction of anthrax blood with methylene blue. J. Comp. Pathol. 16: 360 /361.
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Acetate buffers, 0.2 M, pH 3.5 to 7. (Only one of these will be needed when the optimal pH is known.) Buffered water, made by 10-fold dilution of the 0.2 M buffer. The pH needs adjustment using drops of 5% acetic acid after dilution. Giemsa stock solution. Usually this is bought ready-made. It should contain dyes certified by the Biological Stain Commission. (Ask the supplier.)
Method 1. 2. 3.
Biotechnic & Histochemistry 2004, 79(2): 107 /109
Bring paraffin sections to water. Put slides in acetate buffer for 5 min. Pour off the buffer. (It can be reused.)
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Add 1 ml Giemsa stock solution to 50 ml of buffered water in a beaker. Stir, then pour the diluted stain into the staining jar containing the slides. Staining time is about 2 h at 208 C or about 15 min at 50 /608 C in a microwave oven. Rinse the slides in buffered water, blot dry, and check with a microscope. If blue predominates, differentiate carefully in 0.01% acetic acid, rinse in buffered water and blot dry again. Too much blue means the pH of the buffer was too high. If red predominates, use a higher pH next time. Either thoroughly air dry the slides or dehydrate the almost dry sections in two changes, each 3 min, of n-butanol. Clear in xylene and coverslip.
In a correctly stained preparation, nuclei and cytoplasmic RNA are blue to purple. Erythrocytes, collagen and keratin are pink. The different types of leukocyte are recognizable by their nuclei and cytoplasmic details. Bacteria are dark blue to purple. The preparations are more informative than sections stained with hemalum and eosin, because more cytoplasmic detail is visible in a wider range of colors. The late RD Lillie perfected methods of this type as routine staining techniques
for use on a large scale (mechanized) in histopathology (Lillie and Fullmer 1976).
References Lillie RD, Fullmer H (1976) Histopathologic Technic and Practical Histochemistry. 4th ed., McGraw-Hill, New York. pp. 193 /197. Wittekind DH (2002) Romanowsky /Giemsa Stains. In: Conn’s Biological Stains. A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. Horobin RW, Kiernan JA, Eds., 10th ed., Bios, Oxford. Chapter 22, pp. 303 /312.
John A. Kiernan Department of Anatomy and Cell Biology The University of Western Ontario London, Canada N6A 5C1
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Answers needed I would like to know how formalin fixation works. Does formalin enter the extracellular space and replace water? Does it enter both extra- and intracellular space? Is shrinkage of formalin fixed tissue indicative of water loss and/or collapse of intracellular/extracellular space?
Notes and queries
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