BACTERIOLOGICAL USES FOR NIGROSIN 293

Conclusion The administration of aspirin in doses of 30-45 grains per day liad no

appreciable effect on the development of agglutinins following typhoid inoculation.

Thanks arc due to the students who so kindly made the invcsstigation possible and to Dr K. E. Cooper for his constant interest and advice.

REFERENCES DEMCK, C. L., HITCHCOCK, 1927-28. J . Clin. IrLaeat., v, 427.

PERRY, C. B. . . . . . 1939. Arch. Dis. Clddh . , xiv, 32. POYNTON, P. J., AND SCHLES- 1937. Recent advances in the study of

INGER, B. rheumatism, 2nd ed.. London,

C. H., AND SWIFT, H. F.

p. 91. SCRLESINQER, 13., AND SIGNY, 1933. Quart. J . Med., N.S. i i , 255.

SWIFT, H. F. . . . . . 1922. J . Exp. Med., xxxvi, 735. A. G.

SOME USES OF NIGROSIN IN HACTERIOLOGY

ALEXANDER FLEMING

From the Inoculation Department, St Mary’s Hospital, Paddiriytorc

In the period before 1914 it was possible to obtain commercia.lly India ink which, when spread in a thin film on a microscope slide, gave a reasonably homogeneous background. After 1919 this was not in my experience possible and accordingly I looked around for other substances which gave homo- geneous dark backgrounds, and tried, among other things, nigrosin. This is an almost black dye which, when spread on a film and dried. makes a perfectly homogeneous film and does not stain bacteria.

For these reasons nigrosin is very suitable, as a substitute for India ink, for providing a dark background for unstained bacteria. It has the ad\-antage that it furnishes a smooth background and can readily be removed by water, so that a bacterial film, after examination in nigrosin, can be washcci free of this substance and sta.ined by any of the usual methods.

For over twenty years I have been teaching the usc of negative staining by nigrosin in tho preliminary examination of a culture and for othcr purposes, and the object of this article is to encourage a wider uso of t,lic method which, after long experience, I have found most valuable.

Prepurc~tion of the rbiyrosin solution Tho following solution is prepared.

Nigrosin, water-soluble (Uurr) . . l o g . Water . . 90 C.C.

Formalin - . . 10 2,

The formalin is added merely as a preservative to prevent the growth of moulds and bacteria.

While a 10 per cent. solution of nigrosin is most suitable for general JOURN. OF PATH.-VOL. Llll U 2

294 A . FLEMING

purposes there are occasions when a saturated solution is preferable, and a t other times the 10 per cent. solution can with advantage be diluted with distilled water.

Demonstration of the size, shape and arrarqement of bacteria This can be done in two ways. (a) A suspension of bacteria in water is spread on a slide as if for staining

and allowed to dry. A small drop of nigrosin is then placed on one sidc of tho bacterial film and is spread over the bacteria with a loop or another slide. This film can be dried by gentle heat over the bunsen flame and is then ready for examination.

(b ) A small drop of the nigrosin solution is placed on a slidc and into this are mixed bacteria from the culture (just as if it were a drop of water). When the mixture is complete the drop can be treated in eithcr of two ways. (i) With another slide a film is made in the same way as a blood film. This gives the most perfect results if the technician is skilled but otherwise may be disappointing, the film being too thick or too thin. (ii) The nigrosin- bacteria mixture is spread out on the slide by a circular motion of the loop, which gradually advances along the slide from its original position until i t no longer contains sufficient fluid to leave a film. This gives an uneven film-the first portion is too thick, the final portion too thin, but somewhere in the middle there is a perfect thickness. By this method several different colonies can easily be examined on the same slide.

If it is too thick the bacteria are partially overlaid by the nigrosin so that they appear too small. Suppose, for instance, we take a film of staphylococcus in nigrosin. The staphylococcus is about 0.8 p in diameter. I f the nigrosin dries in a film of the same thickness the staphylococcus can only appear as a very minute clcar spot with a hazy outline round. If the film of nigrosin is too thin thc background may not be perceptible, but in this direction there is an enormous margin. Even if the amount is so small that it dries into a hardly perceptible film the bacteria can still be seen, as around each bacterium the depth of fluid approximates by capillary attraction to the depth of the bacterium. When this dries the bacterium is surrounded by an easily perceptible dark halo inside which is a clear area denoting the size and shape of tlie organism.

It is customary in the examination of cultures, especially by students, to use for a preliminary examination a film stained with a simple stain such as methylcnc blue. The method described above has distinct advantages over simple staining. The whole operation takes less time, and is wholly per- formed on the worker's bench, thus avoiding the necessity of walking to a sink to stain the specimen. The outlines of the bacteria are more clearly defined than by any staining method. Degenerate bacteria which have lost their staining properties still show up as faint shadows in the nigrosin film. Sometimes it is difficult, with stained specimens of tiny colonies, to focus accurately on the portion of the slide on which the film is spread. If the film is spread in nigrosin solution the black colour makes it easy to find.

The thickness of the nigrosin film is important.

Use of negative staining with nigrosin in combination with positive staining methods

After a bacterial film has been stained with Gram or other stain it is sometimes an advantage to spread nigrosin over half of the stained film. This leaves half of the film for examination as a stained film while in tlie other half there is the combination of positive and negative staining. Nigrosin is, however, a decolourising agent and rapidly removes most stains from

BACTERIOLOGICAL USES FOR NIGROXIN 295

bacteria, so that if the stain is bo be preserved the nigrosin film must be rapidly dried. Even then partial decolourisation frequently occurs. This partial decolourisation in some cases gives very beautiful results in demonstrating granules in, for example, a Gram-stained film of the diphtheria bacillus.

This method involves so little extra labour that it is worth doing when stained films of material like fzcal suspensions or direct smears of diphtheria swabs are being examined. It has the advantagc of showing up orga,nisms like spirochstcs which do not show up prominently in the stained film.

2’h.e demonstration of spores The usual method for the demonstration of spores is to stain with hot

carbol-fuchsin, decolourise with weak a,cid, and counterstain the bodies of the bacilli with a stain such as methylene blue. If we consider the structure of an anthrax bacillus we see that if the spore is stained with fuchsin and the bacillary body with methylene blue the edges of the spore must be rendered to some extent indefinite by being overlaid by the blue-coloured body of the bacillus. Theoretically, therefore, the spore would be more clearly shown if the body of the bacillus were left uncoloured and its outlines delineated by negative staining with nigrosin. This theoretical consideration is borne out in practice.

The details of the methods of staining recommended are as follows. For single slides. A bacterial film is made in the usual way on a slide

and dried. Boiling carbol-fuchsin * (or carbol-fuchsin diluted with an equal amount of water) is poured on tho dido and with occasional warming is left for 5 minutes. The stain is washed off with water and the film rapidly washed with alcohol, which removes the deposit of stain adhering to the slide.

Decolourisation can be effected by covering the slide with 10 per cent. nigrosin solution for 5 minutes or longer, after which i t is washed off with tap water and the film is dried. A drop of nigrosin (10 per cent.) is then placed on one end of the slide, and with another slide is spread as a film over the stained specimen and allowed to dry. This gives bright red spores and unstained bacterial bodies, the outlines of which are clearly seen against the nigrosin background.

If it is desired to decolourise more rapidly, the usual reagents such as weak acid or sodium sulphite may be used. The decolourising agent is washed off with water, the slide is dried, and then nigrosin solution is sprea.d over the film and allowed to dry. The primary decolourisation need not be quite complete, as in the few seconds which the nigrosin takes to dry it completes the removal of the stain from the bacillary bodies.

When the stronger decolourising agents are used some of the stain may be removed from the spores ; these do not then show the intense bright red colour which is seen when nigrosin is the only decolourising agent used.

From a culture on a suitable solid medium the bacteria are transferred to water in a test-tube to form a thick suspension. To this is added an equal volume of carbol-fuchsin. Thc whole is boiled for about 5 minutes or immersed in a saucepan of boiling water for anything ovcr 5 minutcs. Tho tube is now filled up witJh water and centrifiiged, when the bacteria rapidly sedimont, as thoy arc agglomcmted in masses. The excess of stain is removed by washing once or twice with water, which process, owing to the agglomeration of the bacteria, can usually

* Basic fuchsin . . 1 g. Phenol (pure) . . 5 g .

Water (distilled) . . 100 C.C.

For large numbers of slides for class purposes.

Alcohol . . 10 C.C.

296 A . FLEMING

bo done without centrifuging. Almost, all the fluid is now removed, and with a pipette or glass rod the bacterial masses are, as far as possible, broken up into a suspension. Approximately 2 voliimes of 10 per cent. nigrosin solution arc now added and mixed with the snspension. After about ten minutes (the time varies considerably with the type of organism) decolourisation is complete, and small drops are placed on slides and spread into films of such a thickness that the nigrosin furnishes a suitably thick background. The films aro allowed to dry and are ready for examination.

Bacteria stained in this way show clear cut, intensely red spores in imcoloured bacterial bodies sharply defined in tho nigrosin. The picture is a more definite one than when a double stain is used, and the odgo of the spore is very sharp, making it obvious that it is a definite body and not merely a11 indefinite red area in a blue bacillus. This makes i t oaaior to convinco stndcnts that sporcs are real entities, and the method also makes it possible to prepare without undue trouble a sufficient number of stained specimens to present one to each student, so tha.t there is no excuse for their not having secn spores.

T h e demonstrution of enpmdes Negativt: staining for the demonstration of capsules is a common procedure.

Tho bacteria are usually mixed with a drop of India ink and spread in a film on a slide which is t.hen stained. Nigrosin cannot be used in this way as, being wattlr-solublc, it washes off the slide in the process of staining. It can be uscd, howctvcr, in another way, by spreading it in a film over the stained dide of capsulated organisms, when i t settles down and dries in a film outside the capsule, t'lins leaving an unstained area between the nigrosin and the stnincd Imcterial body which represents the capsule.

The clccolourising effect of nigrosin, so useful in the demonstration of spores, is here a disadvantage as, unless great carc is exercised, the stain may be removed wholly or partially from the bacteria. The stains which I have found to be most ri %ant to thc decolourisinp action arr Gram's stain and methylene blue.

A film of the bnctrrin i s rriado on a slide i n the usual way, staincd by Gram or mothylcnct blnc ant1 clricd. Thon a small drop of nigrosin is placed on the sliclc and with the edge of a sccond slidr is drawn into a thin film over the upper half of the stained specimen. This should be dried rapidly by heat to minimist? the risk of decolourisation by the nigrosin.

If the edge of the nigrosin-covered portion of the film is examined with an oil immersion objective one half of the film shows stained bactoria and negatively stained capsules, while the other half is merely a stained specimen of the bacterial bodies. This demonstration of the capsules in half of the field is very convincing to the sceptical student.

This method, while very beautiful, is a dangerous one for general use until we have a stain which is not removed by nigrosin, as partial decolourisation may, unless careful observation is made, closely simulate capsulation. It is, however, free from the objection which may sometimes be levelled against the method of staining the bacteria in India ink films, namely that the bacteria have shrunk away from the ink in the proccss of drying, as the nigrosin is only applied to the film after the bacteria are dry.

Summary Nigrosin solution is recommcndcd for the negative staining of bacteria.

It may be used by itself as the simplest and most effective method of demonstrating the size, shape and arrangement of bacteria, or i t may be combined witrh staining methods to clemonst,wte spores or capsules.