(SnCk2), zinc chloride (ZnCk2) and tannic acid from the Merck. sulphate, ferric chloride, mercuric chloride, osmic acid, potash

Transcription

1 THE STAINING OF FLAGELLA OF BACTERIA, WITH SPECIAL REFERENCE TO MORDANTS W. E. MANEVAL Department of Botany, University of Missouri Received for publication November 3, 930 Many mordants have been used in the process of staining bacterial flagella. Among the chemicals most frequently employed in mordants are chromic acid, copper sulphate, ferrous sulphate, ferric chloride, mercuric chloride, osmic acid, potash alum, tartar emetic, tannic acid, basic fuchsin and gentian violet. Casares-Gil's mordant contains the chlorides of aluminum and zinc. Wright (98) substituted chlorides of cobalt, copper, magnesium, manganese, mercury, nickel and zinc for ferrous sulphate in Loeffler's mordant with very poor results. Ziehl- Neelsen's carbol-fuchsin has probably been used most frequently for staining. Usually mordants have been prepared cold and allowed to stand for some time to "ripen." Heating has often been resorted to in mordanting. Most mordants contain considerable dye and it is probable that many of the best ones contain much material in a colloidal state. CHEMICALS AND SOLUTIONS USED In 99 the writer published a method of staining bacterial flagella. Since then he has tried many mordants in an attempt to find a better one. The following chemicals were used in their preparation: aluminum chloride (AlCl. H0) and silver nitrate from the J. T. Baker Chemical Company; antimonous chloride (SbCl3), cobalt chloride (CoCk), cupric chloride (CuCl), ferric chloride (FeCl3), nickel chloride (NiCk) stannous chloride (SnCk), zinc chloride (ZnCk) and tannic acid from the Merck Chemical Company; basic fuchsin, Coleman and Bell. In addition chromic chloride, lead acetate, lead chloride, manganese 33

2 3 W. E. MANEVAL chloride, mercuric chloride and tartar emetic were used, but unsuccessfully. All solutions and mordants were prepared with distilled water in Pyrex, Erlenmeyer flasks of 50 cc. capacity. Percentage solutions were made by adding the weight of chemical indicated by the per cent in grams to 00 cc. of distilled water. For example, a 0 per cent solution contained 0 grams of chemical added to 00 cc. of water. Two colloidal sols were used in some of the mordants: () a ferric hydroxide sol prepared by adding slowly 7.5 cc. of 30 per cent ferric chloride solution to 00 cc. of vigorously boiling water, the boiling continuing till all the ferric chloride had been added; () a silver sol, prepared by adding ammonum hydroxide, drop by drop, to 5 cc. of a per cent solution of silver nitrate until the precipitate formed just disappeared; this was diluted with 00 cc. of distilled water and then added slowly, with constant shaking, to an equal volume of a 0.5 per cent tannic acid solution. A similar sol, which proved excellent in mordant no. below, was made by adding the ammoniacal silver nitrate solution to boiling taniic acid. These sols are quite stable and will keep several weeks. The tannic acid solutions were always filtered as soon as made and used fresh. The filtered solutions of the chlorides mentioned may be kept as stock solutions and used over a considerable period of time. Saturated solutions of SnCl and SbCl3, a 30 per cent solution of FeC3, and 0 per cent solutions of all other chlorides were used. Alcoholic basic fuchsin contained 0 grams of the dye in 00 cc. of 95 per cent alcohol. MORDANTS The first satisfactory mordant prepared contained 0 cc. of tannic acid (0 per cent), 6.5 cc. of ferric hydroxide sol and 0 drops of alcoholic fuchsin. The solution of tannic acid was heated to boiling over an asbestos pad. The ferric hydroxide sol was added rather slowly (thirty seconds to one minute) and then the alcoholic fuchsin, drop by drop. Boiling was continued two minutes and then the mordant was cooled and filtered. This mordant was best when one to three days old and gave very good results.

3 STAINING OF FLAGELLA OF BACTERIA 35 Later it was found that one could substitute cc. of 30 per cent ferric chloride for the ferric hydroxide sol, the procedure for preparation being the same as above. This mordant in some respects was better than the first. These results suggested trying other chemicals in the same way. Excellent mordants were prepared by substituting solutions of AlCl3, CuCl, NiCl and ZnC for the ferric hydroxide sol. CoCl, SbCl3 and SnCl were much less satisfactory, while CrCl, lead acetate, PbCl, MnCl, HgCl and tartar emetic could not be used as substitutes for the ferric hydroxide sol. The fact that Casares-Gil's mordant contains both AlCls and ZnCl suggested substituting combinations of two inorganic salts for the single one in the mordants previously prepared. Of the thirty-six possible combinations in pairs of the chlorides of Al, Co, Cu, Fe, Ni, Sn and Zn and of the ferric hydroxide sol, all but two, CoCl plus NiCl and NiCl plus SnCl, were tried. The six combinations containing CoCl and the six containing SnCl were unsatisfactory or, in most cases, entirely worthless. The best of these, if used fresh or not more than one day old, were CoCl combined with CuCl, FeCl3 or ZnCl, and SnCl combined with AlCl3. All combinations of the chlorides of Al, Cu, Fe, Ni and Zn and of the ferric hydroxide sol, fifteen in number, proved very good to excellent. All pairs containing AlCl3 or FeCl3 were excellent as was CuCl plus ZnCl. Those containing NiCl were not quite so good as some others, while CuCl plus ferric hydroxide sol was the poorest. Besides those already mentioned, three other very good mordants were prepared. These contained alcoholic fuchsin and FeCl3, AlCl8 and ferric hydroxide sol used singly as above; but instead of tannic acid alone a mixture of equal volumes of tannic acid and silver sol. The silver sol was also used in preparing several of the other mordants, but they were more satisfactory without it. The entire list of mordants is given in tabular form (table ) and also the general procedure for their preparation. When SnCl or SbCl8 was used in mordants only 3 or drops of saturated solutions were added to 0 cc. of tannic acid, while of CoCl cc. of a 0 per cent solution were added.

4 36 w. E. MANEVAL The preparation is practically the same for all of these mordants. The tannic acid, or else the tannic acid and silver sol mixed (nos. -), is heated to boiling in a Pyrex flask. The inorganic salt (nos. -6 and -), or the two inorganic salts mixed (nos. 7-) is added, a few drops at a time, as boiling continues. Then the MOEDANT NUMBER TANNIC ACID,. 0 PER CENT ALCOEOLIC FUCH}IN, 0 PER CENT drops TABLE Formulas for mordants AMCs, 0 PER CENT CuCh, 0 PER CENT FeCl, 30 PER CENT Fe(OH)s SOL NiCIl 0 PER CENT ZnCls, 0 PE:R CENT SILVER SOL alcoholic fuchsin is added and the mixture boiled two minutes longer. After cooling the mordant should be filtered (I use Whatman no. filters). Refiltering on successive days, just before using, is generally necessary. These mordants are generally most satisfactory when one to four days old, with the exception of nos. -, which are best 0 0 0

5 STAINING OF FLAGELLA OF BACTERIA when a few hours or a day old. After two to five days they usually fail to work but may be "renewed" by bringing to the boiling point and adding cc. of inorganic salt or salts and drops of alcoholic fuchsin per 0 cc. of mordant. "Renewed" mordants are sometimes better than when first made. They should be filtered after standing a day and on succeeding days just before using. All of these mordants leave some film on the slide when used. This seems necessary for one cannot stain flagella, it seems, when there is no film at all. The thickness of the film depends on the composition of the mordant, its age and the length of time it is allowed to act. Those containing the silver sol (nos. -) give the least film when fresh, but the others give less as they age until none is formed. "Renewed" mordants may give less film than others. Soon after their preparation (fresh to one day old) some of these mordants may not require more than one and one-half to two minutes for mordanting, but when older (two to three days) they generally require at least ten minutes. Those containing AlCl3, excepting no., or ZnCl do not work when fresh. Apparently all of these mordants contain considerable material in a colloidal state. After standing four or five days a heavy precipitate forms and they often fail to work; i.e., they no longer form a film on the slide. They are always heated when prepared but not for mordanting. They differ from many other mordants in containing less dye. Some dye is necessary but not much.- Any one of them may be prepared in a few minutes as the only solution that needs to be fresh is the tannic acid. Large numbers of stained preparations would have to be compared to determine their relative merits. For general use nos., 3 and would doubtless prove sufficient and very satisfactory. However, it is interesting to know that all mordants in this list gave excellent results with the organisms used in this work. CULTURES 37 For most of the stained slides of flagella, mixed cultures of bacteria were used. The cultures were prepared by putting one or

6 38 W. E. MANEVAL two beans in a preparation dish of 00 to 50 cc. capacity, covering with about 50 cc. of tap water and allowing to stand one to two days. Excellent results were also obtained with material taken directly from the teeth and from pure cultures on agar slopes of the following: Bacillus carotovorus; B. cereus; B. megatherium; B. mycoides; B. phytophthorus; B. tumefaciens; B. vulgatus; Pseudomonas campestris; Ps. fluorescens; Ps. pyocyanea; Spirillum rubrum; and an unidentified organism isolated from diseased Geranium leaves. Clean, flamed slides were used. On each, four small drops of water ( to 6 mm. across) were placed, arranged about cm. apart in the form of a square. If bacteria for staining were taken from the mixed cultures mentioned above, two drops of water were placed on a slide, a bean touched to the water until slight cloudiness appeared and then the slide was allowed to stand one to two minutes. A loopful (loop mm. in diameter) of the bacterial suspension was then transferred to the four drops of water on a slide, touching each in succession. This gives four dilutions. The slide was then dried at room temperature in the absence of air currents. When pure cultures on agar slopes were used the procedure was practically the same. A bit of bacterial growth was transferred to two drops of water on a slide, but not mixed with the water; and after two to five minutes a loopful from the edge of the water was touched to four droplets of water on another slide as described above. Sometimes the bacteria were allowed to diffuse, as is so commonly done, for ten to thirty minutes in 5 to 0 cc. of water in a test tube before transferring to the water drops on a slide; but this procedure was generally much less satisfactory than the preceding one. Apparently many flagella break off when the bacteria diffuse for a considerable time in a rather large volume of water. Of course the usuial precautions for obtaining actively motile bacteria were observed. Daily transfers were made to nutrient broth, or to nutrient broth and agar slopes alternately, and finally to agar slopes. Best results were generally obtained with cultures about twenty-four hours old; but with some kinds of bacteria (Bacillus carotovorus; B. phytophthorus; B. tumefacies; B. vulgatus; Pseudomonas

7 STAINING OF FLAGELLA OF BACTERIA campestris; Ps. fluorescens; Ps. pyocyanea) cultures two or three days old were as satisfactory as younger ones. Excellent preparations of Ps. pyocyanea and Ps. campestris were made from cultures seven days old. No other cultures so old as this were tried. In all cases if the bacteria were actively motile the flagella could be stained readily. It is very important to have the bacteria quite free from the nutrient medium, especially proteinaceous material, in all staining of flagella. If more than a trace of the medium is present the flagella stain poorly or not at all. Making dilutions in four drops of water on the slide will usually result in at least one, sometimes all, being satisfactory. STAINS AND STAINING Although several staining solutions were tested, no other, not even carbol fuchsin, proved as satisfactory as anilin-alcoholfuchsin (Maneval, 99). This stain contains: cc. Water, distilled Basic fuchsin, 0 per cent alcoholic... 0 Anilin oil ( part) and 95 per cent alcohol (3 parts) mixed... 5 Acetic acid, per cent Mix in the order given, filter once or twice and again before using. It is best after three to six days and may sometimes be kept several weeks, but it finally deteriorates. Good results have been obtained by substituting rosanilin or methyl violet for the basic fuchsin in this formula. Anilin fuchsin and anilin gentian violet were poorer. The air-dried slides were placed on a level surface, treated with to 8 drops of mordant and allowed to stand two to ten minutes. (One or two trials indicated the time to use.) They were washed with 6 to 0 changes of water, being careful to hold them level when the water was first poured on. After the second change of water the preparations were wiped carefully with a clean towel to remove the edge of the film of mordant which may harden somewhat, and upon completing the washing treated with 3 to 5 drops of stain for three to five minutes. After washing thoroughly and drying they were examined with the immersion objective without a cover glass.

8 30 W. E. MANEVAL If preparations are satisfactory there will always be some film present. The position of the four drops of water may be seen against a white background. Just inside the edge of each drop there will be a clearer zone in which the flagella will generally be stained better than elsewhere. When these mordants are used fresh or a day old the stained flagella usually appear rather thick but thinner if the mordants are older, until finally they will appear scarcely visible. This depends not only on age, but also on composition of the mordant and time used for mordanting. Sometimes the stained film will appear under the microscope as made up of very numerous small droplets. This may indicate that the mordant is not old enough or that it or the stain should be filtered. It seems very likely that the excellence of the mordants recommended here depends largely on the existence in them of much material in a colloidal state; and that some solutions fail to act as mordants because a suitable sol is not obtained in preparing them. The tannic acid and the sols of Fe(OH)8 and Ag are colloidal. It is possible that when the various chlorides used successfully are added to boiling tannic acid they pass over into a colloidal condition, the same as FeCls does when added to boiling water. After standing several days heavy precipitates generally form and the efficiency of the mordants is much decreased. It seems probable that staining results mainly from adsorption by the flagella, so that a layer of mordant and dye is formed over them which may make them appear much thicker than they really are. No doubt the size and electrical charge of the colloidal particles, as well as H-ion concentration are important factors in the results obtained, but they have not been investigated experimentally. SUMMARY Hundreds of excellent stained preparations of flagella of numerous kinds of bacteria were made. The air-dried "smears" were mordanted in most cases with a solution containing tannic acid, alcoholic basic fuchsin and a chloride of Al, Cu, Fe, Ni or Zn, a ferric hydroxide sol, or else combinations of these chlorides

9 STAINING OF FLAGELLA OF BACTERIA 3 in twos with each other or with the Fe(OH)3 sol. Three mordants contained a silver sol. Formulas are given for twenty-four mordants. The stain used was an alcoholic-anilin-fuchsin. It seems probable that the efficiency of these mordants depends largely on their colloidal condition. Their use makes the staining of bacterial flagella relatively easy. REFERENCES MANEVAL, W. E. 99 Some staining methods for bacteria and yeasts. Stai Technology,, -5. WRIGHT, WILLIAm H. 98 A discussion of some of the factors causing variable results with flagella stains. Stain Technology, 3, -7. Downloaded from on March 9, 09 by guest