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Technical Method J Clin Pathol: first published as 10.1136/jcp.38.9.1073 on 1 September 1985. Downloaded from J Clin Pathol 1985;38:1073-1084 Technical method Use of microwaves for acid and alcohol fast staining S HAFIZ, RC SPENCER, MARGARET LEE, organism depends on several factors but remains an HILARY GOOCH, BI DUERDEN Department important component in the identification of a ofMedical Microbiology, Sheffield University Medi- restricted group of organisms. cal School, Sheffield The technical problems associated with the classic Ziehl-Neelsen method include the dangers of heat- ing by flaming torch in laboratories in which volatile Certain bacteria that are characterised by a high organic solvents are used, the deposits that accumu- content of lipid in their cell walls cannot be stained late on the underside of the slide as a result of heat- by simple stains, and either heat or prolonged con- ing, and the crystalline deposits of stain that collect tact is required to drive the stain into the cells; once on the film as a result of evaporation and drying. stained they resist decolourisation by mineral acids This method also requires 20-30 minutes of or acid and alcohol. These organisms are designated laboratory time. Numerous minor modifications acid fast or acid and alcohol fast. They include have been made to the Ziehl-Neelsen method by Mycobacterium tuberculosis and related mycobac- increasing the concentration of carbolic magenta or teria, Mycobacterium leprae, and certain of the by cold staining techniques,'0-'2 but the basic prob- actinomycetes. Koch first stained the tubercle bacil- lems remain. lus by immersion in an alkaline solution of Some of the problems may be overcome by the copyright. methylene blue for 24 hours.' Erlich used warm sol- use of microwave energy, a type of high frequency utions of magenta (fuchsine) or methyl violet for (2450 MHz) radiowave. It is absorbed by molecules 15-30 minutes; the bacilli then resisted decolourisa- of water, causing them to vibrate at a very high fre- tion with strong mineral acids.2 He believed that the quency and produce heat, which radiates out; con- bacilli were resistant to ordinary stains because they sequently, there is an increase in the turgor pressure were surrounded by a capsule permeable only to within the cells and the bacterial cell becomes more alkalis. Ziehl, however, showed that they could be permeable, allowing easy passage of soluble pro- stained with an acidic dye or 2% alcoholic methyl ducts as shown by the increased permeability of bac- violet solution in carbolic acid water.3 Later, Ziehl terial enzymes.'3 Prolonged exposure results in dis- http://jcp.bmj.com/ used carbolic magenta. Neelsen increased the ruption and destruction of the cells. strength of phenol in the stain,4 and the Ziehl- We describe a new rapid approach to the Ziehl- Neelsen method became the standard one for gen- Neelsen stain using a microwave oven for the stage eral use. of heating in carbolic magenta. The procedure also The property of acid fastness seems to be due to eliminates the need to treat histopathological sec- the presence in the bacilli of unsaponifiable wax,5 tion with xylol and alcohol and sheds some light on first isolated by Aronson in 1898.6 It was referred to react with the way in which acid fast organisms on October 2, 2021 by guest. Protected as one of the higher alcohols by Bulloch and Mac- stains. leod.' Tamura isolated an alcohol from mycobac- teria that had the property of acid fastness and named it "mycol."8 It was at first regarded as an Material and methods unsaturated fat but is now generally believed to be lipoidal or waxy in nature. Not everyone agreed on MICROWAVE OVEN the simple chemical explanation of acid fastness. A domestic oven (Sharp model 9410E) with a max- Sordelli and Arena, for example, stated that it imum output of 640 watts, four power settings, and depended on a semipermeable membrane around a digital timer was used. the organism that allowed magenta to diffuse inwards but did not allow acid magenta to diffuse SPECIMENS outwards.9 The degree of acid fastness of an Clinical smears of sputum from patients with pul- monary tuberculosis and from non-tuberculous con- Accepted for publication 24 April 1985 trols were prepared in the normal way and dried in 1073 J Clin Pathol: first published as 10.1136/jcp.38.9.1073 on 1 September 1985. Downloaded from 1074 Hafiz, Spencer, Lee, Gooch, Duerden air. Histological sections of lung from patients with organisms, the limited number of clinical slides were tuberculosis and patients with infections with stained by the following methods: one set of sputum Nocardia asteroides prepared in paraffin wax were smears was stained by the standard Ziehl-Neelson provided by the department of histopathology. method; a duplicate set was flooded with carbolic magenta, placed in the microwave oven for 30 sec- ORGANISMS onds at full power, decolourised with acid alcohol, Pure cultures of M tuberculosis, non-acid fast bac- and counter stained with malachite green. One set of teria including Bacillus spp, clostridia, corynebac- histological sections from patients with tuberculosis teria, staphylococci, streptococci, Haemophilus spp, was stained by the standard Ziehl-Neelson method. enterobacteriaceae, and neisseriae, N asteroides A second set was flooded with carbolic magenta NCTC 11293, andActinomyces israeli NCTC 10236 without having been treated with xylol and graded were cultured on recommended media and grown in alcohol, placed in the microwave oven for 30 sec- optimum conditions. Smears were made in the onds at full power, decolourised with acid alcohol, normal manner and dried in air. Pure cultures of M and counterstained with malachite green. The third tuberculosis were mixed with other organisms and set was treated as the second, except that the smears made. primary stain used was saffron. Sections of lung from a patient with nocardia STAINS FOR MYCOBACTERIA infection were flooded with carbolic magenta, Carbolic magenta, magenta, and saffron were used placed in the microwave oven for 30 seconds at full for initial staining. Carbolic magenta was prepared power, decolourised with acid alcohol, and counter according to the method described by Cowan and stained with malachite green. Steel.4 A solution of saffron (1%) was made in distilled water. Magenta was made by dissolving 1 g magenta in 10 ml absolute alcohol, which was then Results made up to 100 ml with distilled water. Table 1 shows the results with pure cultures, sputum COUNTER STAIN smears, and histological sections. There were no dif-copyright. Malachite green was prepared according to the ferences between the results obtained with the stan- method described by Cowan and Steel.'4 dard Ziehl-Neelsen staining technique and the method based on microwave treatment in terms of STAINING METHODS proof of acid and alcohol fastness. The slides were Standard Ziehl-Neelsen method Smears were cleaner, however, after treatment with microwaves, flooded with carbolic magenta and heated until and there were no crystalline deposits of stain in the steaming with a torch for 10 minutes, washed with smears. Table 1 shows only the results obtained water, decolourised with acid alcohol (hydrochloric when the decolourising agent was acid alcohol. The acid 3% in 95% alcohol), and counter stained with mycobacteria were consistently acid and alcohol http://jcp.bmj.com/ malachite green. fast, unlike the other bacteria examined. Microwave method Smears were flooded with Table 2 records the effects of different decolouris- carbolic magenta, placed in a sandwich box with the ing solutions. Only Mycobacterium spp were resis- lid on, and exposed to microwave irradiation for 30 tant to decolourising by acid alcohol or 20% sul- seconds at full power (640 watts). They were then phuric acid. N asteroides was resistant to 1% sul- decolourised with acid alcohol and counterstained phuric acid and about 50% of cells in the smears with malachite green. were resistant to 5% sulphuric acid. A israeli and on October 2, 2021 by guest. Protected A series of 15 sets of slides was made from the other non-acid fast species were decolourised by 1 % cultured organisms. One set was stained by the stan- sulphuric acid. Identical staining reactions were dard Ziehl-Neelsen method, two sets were stained achieved by substituting magenta or 1 % saffron for with magenta or saffron, heated by the conventional carbolic magenta and exposing the smears to mic- torch method, decolourised by acid alcohol, and rowave energy, although the acid and alcohol fast counter stained by malachite green, four sets were species were not stained by these particular stains flooded with one of three stains (carbolic magenta, when conventional heat was used. magenta, or saffron) and exposed to microwave The staining of sections of tissue (without dewax- irradiation in the oven for 30 seconds at full power; ing) by the microwave method with carbolic one set treated with each stain was then decolour- magenta or magenta was comparable with the ised by 1%, 5%, and 20% sulphuric acid and by acid results obtained by the conventional Ziehl-Neelsen alcohol. method (after dewaxing), but 1% saffron was not On the basis of the results with pure cultures of suitable for histological sections as the dye was not J Clin Pathol: first published as 10.1136/jcp.38.9.1073 on 1 September 1985. Downloaded from Technical method 1075 Table 1 Comparison ofacid and alcohol fast staining by standard Ziehl-Neelson and microwave methods Type ofspecimen No ofspecimens No positive by: examined Ziehl-Neelsen method Microwave method Tuberculous sputum 30 30 30 Non-tuberculous sputum 30 0 0 Nocardia sputum 1 0 0 Cultures of Mycobacterium tuberculosis 30 30 30 Cultures of Mycobacterium bovis 1 1 1 Cultures of Mycobacterium kansasi 1 1 1 Cultures of Nocardia asteroides 1 0 0 Cultures of Actinomyces israeli 5 0 0 Cultures of non-acid fast bacteria 10 0 0 Histological sections of tuberculosis 4 4 4 Histological sections of nocardiasis 2 2 2 retained when the sections were treated with acid is not simply a result of heating but also reflects alcohol.
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