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Some observations on Bacillus abortus and Bacillus Melitensis
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. X X I . No. 3. N o v e m b e r , 1927
221
SOME OBSERVATIONS ON B A C I L L U S A B O R T U S AND B A C I L L U S MELITENSIS. BY
E. H. HADDON Bacteriological Laboratory, Entebbe, Uganda. (Presented by Dr. H. LYNDHURSTDUKE.) For some years past attempts have been made at the Entebbe Laboratory to investigate the relationship existing between Bacillus melitensis and B. abortus. Unfortunately, press of routine and other work have defeated this intention, and it has been found impossible to carry out the original programme. It is now clear that research into this problem must be abandoned, for a considerable time at any rate, and these notes are therefore presented in the hope that they may be of some service to others who may be engaged on similar lines of investigation. Some years ago, an innovation was made in the method of keeping the stock cultures at the Entebbe Laboratory. It was decided, for reasons of economy both in time and material, to utilise a simple peptone agar medium. This medium consists of nothing more than a 2 per cent. " Difco " peptone and 1"5 per cent. agar. This particular brand of peptone being a standardised product, with a constant pH of 7'4, the preparation of the medium could be left to the native laboratory attendants. A trial of the medium established the fact that most of the laboratory stock cultures, including B.melitensis and B. abortus, could be satisfactorily maintained on it. After maintenance for about eighteen months on this medium, subcultures being made once a month, these two organisms began to show a striking difference in the appearance of their growth, so that they fell into two groups. The B. abortus cultures, with one exception, gave a tenacious sticky growth on the agar, whereas the B. rnelitensis, again with one exception, showed no change. Of two strains of B. paramelitensis, one gave a sticky and the other a smooth growth. All the cultures were maintained in the dark in a moist atmosphere at a mean temperature of 25 ° C. So striking was the contrast in the appearances of the growths when subcultured on fresh medium that, with the exceptions mentioned, it was quite an easy matter to distinguish an abortus from a melitensis culture. Macroscopically the fully grown cultures showed no indication of this change, which was manifest only when a portion of the growth was being removed for subculturing. The appearances displayed on the newly inoculated medium were decidedly distinctive ; that is, while the one showed no change but gave a smooth evenly spread inoculum, the other, owing to the sticky nature of the growth, presented a lumpy appearance.
9,~
OBSERVATIONS ON BACILLUS ABORTUS AND BACILLUS MELITENSIS.
In view of the interest attaching to the question of identity of these two organisms, the observed differences in the character of the growth seemed of sufficient importance to justify further investigation, which it was decided to carry out on purely chemical lines. TECHNIQUE AND EXPERIMENTAL OBSERVATIONS.
The organisms available for experiment comprised eleven strains of B.
melitensis, eight of B. abortus, and two strains of B. paramelitensis. All were old stock cultures and all the information concerning them is given in Table I. TABLE I. B. abortus. I. Old laboratory culture. Originally from South Africa. 2. Old laboratory culture. Origin not known. 3. N.C.T.C. No. 626 BANG. English strain No. 8. Isolated from stomach of foetal taft, 1919. Royal Veterinary College, London. 4. N.C.T.C. No.'1476 WEYBRIDGE I. 5. BANG. Strain B isolated 4.3.24. 6. BANe. Strain T isolated 3.9.24. 7. BANe. Strain W isolated 5.4.23. 8. N.C.T.C. No. 1785 MUHLAN, Strain I. Isolated from intestine of foetal calf, 1922. B. melitemis. 1. H u m a n strain. 2. N.C.T.C. No. 8 A~KWalGHT. Isolated from case of undulant fever in Malta, 1926. 3. N.C.T.C. No. 198. 4. N.C.T.C. No. 199. 5. N.C.T.C. ZAMMIT I. Isolated from milk of goat suffering from undulant fever, Malta,1920. 6. N.C.T.C. FENSlER MEYER. Strain 7, Group I I I Ref. J.I.D. 1920, 27, 185. 7. N.C.T.C. FENSmR MEYEa. Strain 20, Group I Ref. J.I.D. 1920, 27, 185. 8. N.C.T.C. FENSma MEYER. Strain 27, Group II Ref. J.I.D. 1920, 27, 185. 9. Strain 1197. History not known. 10. Strain 2588. History not known. 11. Strain 3218. History not known. B. paramelitemis. 1. N.C.T.C. BASSZrT-SMITH No. 82 originally from Tunis. 2. N.C.T.C. No. 200.
The majority of these cultures were sent to the Laboratory by Capt. S. R. DOUGLAS, of the Medical Research Institute at Hampstead; others were received from the Laboratory at Nairobi. OSBORNE and others have analysed a number of protein substances, and the sulphur-nitrogen ratio in these proteins, calculated from the percentages given in the analyses, are shown in Table II. It was thought that a determination of this ratio from the proteins of different species of bacteria might prove of value in the present investigation. To obtain this ratio, a method had to be devised which would be simple of application and effective with small amounts of bacterial substances. After many experiments, a method was evolved whereby the necessary estimations could be carried out on the amount of material furnished by twenty agar slope cultures. As a preliminary measure, the sulphurnitrogen ratio was obtained for a small number of miscellaneous organisms and the results obtained are given in Table III.
E. H . HADDON.
223
TABLE I I .
Protein.
Percentage Ratio of S. to N .
Vegetable.
Protein.
Percentage Ratio of S. to N .
Animal.
Amandin, almond Corylin, hazel nut Excelsin, Brazil n u t Edestin, hemp seed Globulin, cotton seed Vignin, cow-pea . . . . Glycinin, soy-bean L e g u m i n , lentil . . . . Phaseolin, kidney bean Conglutin, blue lupin Vicilin, lentil . . . . L e g u m e l i n , lentil Gliadin, wheat . . . . Glutenin, wheat . . . . Globulin, wheat . . . . Hordein, barley . . . .
...... ...... ...... ...... ...... . . . . ...... . . . . ...... ...... . . . . ...... . . . . . . . . . . . . . . . .
2'3 4"4 6'0 4"7 3"4 2"4 4"1 2"2 2"1 1"8 1"0 6"0 5"8 6'2 3'8 4"9
. .
. . . . .
Gelatin, commercial Gelatin, ligaments Elastin . . . . . . Mucin . . . . . . Mucoid, tendon . . Globulin, serum . . Albumin, serum . . Fibrin . . . . . . H~emoglobin, ox . . Casein . . . . . . Nuclio-histone . .
...... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maximum ...... Minimum ...... Difference ...... Average .........
. . . . . . . . .
3"9 3"2 2'3 6"8 19"0 7"0 11'9 6"5 2"5 5"1 2'8
19"0 1"0 18"0 4'9
TABLE I I I . WEIGHT EXPRESSED IN MILLIGRAMMES.
Organism.
Weight of Dried Bacteria.
Total Nitrogen.
Total Sulphur.
Ratio Percentage N. of S. to
2"9 1"4 0"7 1'9 1'8 2'3
0'137 0'089 0'042 0"048 0'053 0'080
4'7 6"3 6'0 2'5 2"9 3"5
t
I Staphyloccus aureus Bacillus prodigiosus B. coli communis B. abortus B. proteus I. B. proteus I I .
21"4 18"4 8"0 16"8 11'2 20'2
[ I t
The nutrient agar medium used throughout the experiments was maintained as constant in quality as was possible. It was prepared by boiling for one hour a mixture of 500 g. of minced ox heart and 3 g. o f " Difco " peptone powder in 2 litres of distilled water. The broth was allowed to stand until cold and then filtered through glass wool, 30 g. of agar powder were added, and the volume made up to 2 litres with distilled water. The whole was now steamed for one hour and then permitted to cool slowly in the steamer. The jelly was then removed in bulk to a plate and the coarse d6bris cut away from the bottom. The remaining clear portion of the jelly was melted, adjusted to pH 7"4, tubed, and sterilised in the autoclave at 15 lbs, Of each strain of organism twenty agar slope cultures grown for fourteen days at room temperature (25 ° C.) were prepared. The growth was carefully removed, by means of a platinum loop, in such a manner as not to disturb the medium itself, and suspended in 4 c.cm. of sterile distilled water. Manipulation with a glass rod sufficed to produce a fine, uniform suspension. Weight of Bacterial Substance.--This was determined by measuring accurately, in a special pipette, 1 c.cm. of the bacterial suspensioninto a tared watchglass. The same pipette was used throughout the experiment. The watch-
0~9'4
OBSE RVATIONSON BACILLUS ABORTUS AND BACILLUS MELITENSIS.
glass and contents were dried in a partial vacuum over concentrated sulphuric acid for a period of forty-eight hours and then quickly weighed. Repetitions gave identical results, which was not the case when the suspensions had been dried in the water oven. The weight of the bacterial substance was determined in a few cases only, as it was unnecessary to continue it throughout the series. Total Sulphur Content.--For this estimation, 1 c.cm. of the suspension was transferred to a platinum capsule, followed by 2 c.cm. of a saturated solution of potassium nitrate in decinormal caustic soda. The contents of the capsule were dried on the water-bath, and then carefully incinerated over a flame until all the organic matter had been completely oxidised. The residue, when cool, was dissolved in a quantity of distilled water not exceeding 7 c.cm., and transferred without loss to a cordite tube marked at 10 c.cm. Three drops of concentrated hydrochloric acid were added to clear the solution, which invariably contained a small amount of suspended carbonates. Finally, 3 c.cm. of a saturated solution of barium chloride were added, a little at a time, the tube being well agitated after each addition. Within ten minutes a maximum turbidity had developed, and the tube was matched against a series of standards. The standard suspensions of barium sulphate were prepared by taking ten carefully selected cordite tubes marked at 10 c.cm. and into each consecutive tube adding 0"1 up to 1 c.cm. of a N/100 sohition of sulphuric acid. Thiswas followed by 2 c.cm. of the potassium nitrate solution, two drops of concentrated hydrochloric acid and then distilled water to 7 c.cm. Finally, 3 c.cm. of barium chloride solution were added in the manner described above, and the tubes sealed in the blowpipe flame. The potassium nitrate and hydrochloric acid were used in the preparation of the standards, as it was found that the same quality of suspension could not be obtained in their absence. The quantity of sulphate to be estimated is so small that, for the successful application of this test, it is essential that the contents of both test and standard tube should be prepared in a manner as far as possible identical. If this is not done, the physical qualities of the precipitate for the same amount of sulphate differ sufficiently to render it impossible to match the tubes. The excess of barium chloride solution used increases the sensitiveness of the test by diminishing the solubility of the barium sulphate. The series of standards prepared in this way represent quantities of sulphur from 0"000016 g. to 0"00016 g. Total Nitroqen Content.--One c.cm. of the suspension was pipetted into a long boiling tube. Three c.cm. of concentrated sulphuric acid and a small crystal of copper sulphate were added and combustion carried out over a micro burner. At the end of one hour 0"5 g. of potassium persulphate was added and the combustion continued until a clear, faintly yellow solution was obtained. This was diluted with a small quantity of water and transferred to a Folin's absorption apparatus, with all the necessary precautions. Twenty c.cm. of
E. H. HADDON.
225
40 per cent. caustic soda were added, the ammonia removed by aspiration, and collected in 50 c.cm. of N/100 sulphuric acid. One hour was allowed for the aspiration. Excess of acidity in the collecting vessel was determined by titration with N/20 caustic soda, and the equivalent nitrogen calculated in the usual manner. The figures obtained are shown in Table IV. They are interesting when compared with those in Table III, but it is evident that the sulphur-nitrogen ratio does not serve to distinguish the one group of organisms from the other. TABLE IV. WEIGHTS EXPRESSED IN MILLIGRAMIVIES.
Organism.
B. abortus ,, ~J ~ . ~, ~,
B. melitensis ,,
Weight of Dried Bacteria. 1 2 3 4 5 6 7 8
1 2 3 4
~
5
~,
6
.
7
.
8
,, ,,
9 10
,7
11
B . paramelitensis 1 .
2
20'00 11"00
Total Nitrogen.
TotalSulphur.
Percentage ratio o f S to N .
3"36 2"10 2"66 1"89 2"45 2"73 3"99 3"01
0"080 0" 048 0'096 0"048 0"064 0'096 0"128 0"080
2"4 2'3 3"6 2"5 2"6 3"5 3'2 2"6
3'02 4"76 3"15 5"74 3"15 3'34 3"64 2'81 5"11 5"36 3"40
0'064 0"080 0"128 0"112 0'080 0'080 0'064 0"160 0" 144 0"096
2"1 3"0 2"5 2'2 3"5 2"4 2"2
3"71 3"71
0"144 0"160
0" 1 4 4
2"3
3"1 2'7 2"8
3"9 4"3
It was now decided to note the effect of transferring all the cultures on the simple peptone medium to the richer nutrient agar. This was done, and the cultures kept under careful observation. At the end of five months, during which period the organisms had been subcultured every week, only one culture ---one of the eight B. abortus examined--remained sticky. After a further period of three months this too had lost all signs of stickiness. The organisms were now transferred back again to the simple peptone medium, and, as previously, subcultured at monthly intervals. Up to date, that is after an interval of seven months, four of the abortus cultures are developing the sticky character. Evidently, in the conditions of the experiment, the tendency to acquire this character is less marked than the tendency to lose it. The conditions responsible for this change in character of growth await investigation. There is some evidence that a fairly rapid extraction of moisture
9~6
OBSERVATIONS ON BACILLUS ABORTUS AND BACILLUS MELITENSIs.
from the medium during growth hastens the development of the sticky quality. Two exceptions, however, were noticed. The observations were made on two series of cultures, one series being grown in a partially exhausted chamber containing concentrated sulphuric acid, and the other under the usual conditions, i.e., room temperature in a moist chamber exposed to the air. The influence of B. subtilis is interesting. It was found that, when this organism was inoculated at the base of an agar slope containing a five days' culture of a B. abortus, it definitely hastened the production of stickiness, and further that it retarded the change in the other direction when both organisms were grown together on nutrient agar. The inoculation of B. subtilis was necessarily always made some days after that of B. abortus. In these conditions the latter organism influenced the growth of subtilis in such a way as to prevent a rapidly spreading growth. In some cases the growth of B. subtilis was, relatively speaking, remarkably slow and consisted almost entirely of spores. The carbon dioxide tension appears to have no rble in the observed phenomenon, as experiments carried out by culturing the abortus and melitensis organisms in atmospheres containing 5, 10 and 50 per cent. by volume of this gas showed no apparent difference. Cultivation of all the organisms, in both peptone water and nutrient broth containing 0"5 per cent. glucose, showed no change at the end of a month. The sugar content was estimated by Folin's method. Further experiments were confined to B. abortus No. 8, as this organism showed the greatest difference between its two varieties of growth. Also it was necessary to make thls curtailment owing to pressure of routine work. Two points of interest were observed with regard to the smooth and the sticky growths. Judged macroscopically, the former appeared to lose its moisture more quickly than the latter, and when both varieties were subcultured on the same medium the sticky growth remained viable for a longer period, as was shown by inoculating tubes of nutrient broth at intervals. " Difference in intensity of the pentose reaction were obtained with ph]oroglucinol for the same quantity of bacterial substance, the sticky growth giving the deeper colour. A similar difference was also observed between the sporing and the vegetative forms of B. subtilis. In this case the spores gave the more marked reaction. For the small number of strains examined, the above observations seem to reveal the existence of a physical difference between the majority of strains of B. melitensis and those of B. abortus, and to suggest that this difference is one of degree. The remarkable production of a sticky tenacious growth which is observed with the majority of the B. at~ortus strains, and which is developed slowly when the organisms are cultured in conditions which may be regarded as unfavourable, may possibly be a protective effort analagous to the formation of a capsule or spore in other species, but, at the present juncture, it is impossible to decide upon the significance of this phenomenon.
221
SOME OBSERVATIONS ON B A C I L L U S A B O R T U S AND B A C I L L U S MELITENSIS. BY
E. H. HADDON Bacteriological Laboratory, Entebbe, Uganda. (Presented by Dr. H. LYNDHURSTDUKE.) For some years past attempts have been made at the Entebbe Laboratory to investigate the relationship existing between Bacillus melitensis and B. abortus. Unfortunately, press of routine and other work have defeated this intention, and it has been found impossible to carry out the original programme. It is now clear that research into this problem must be abandoned, for a considerable time at any rate, and these notes are therefore presented in the hope that they may be of some service to others who may be engaged on similar lines of investigation. Some years ago, an innovation was made in the method of keeping the stock cultures at the Entebbe Laboratory. It was decided, for reasons of economy both in time and material, to utilise a simple peptone agar medium. This medium consists of nothing more than a 2 per cent. " Difco " peptone and 1"5 per cent. agar. This particular brand of peptone being a standardised product, with a constant pH of 7'4, the preparation of the medium could be left to the native laboratory attendants. A trial of the medium established the fact that most of the laboratory stock cultures, including B.melitensis and B. abortus, could be satisfactorily maintained on it. After maintenance for about eighteen months on this medium, subcultures being made once a month, these two organisms began to show a striking difference in the appearance of their growth, so that they fell into two groups. The B. abortus cultures, with one exception, gave a tenacious sticky growth on the agar, whereas the B. rnelitensis, again with one exception, showed no change. Of two strains of B. paramelitensis, one gave a sticky and the other a smooth growth. All the cultures were maintained in the dark in a moist atmosphere at a mean temperature of 25 ° C. So striking was the contrast in the appearances of the growths when subcultured on fresh medium that, with the exceptions mentioned, it was quite an easy matter to distinguish an abortus from a melitensis culture. Macroscopically the fully grown cultures showed no indication of this change, which was manifest only when a portion of the growth was being removed for subculturing. The appearances displayed on the newly inoculated medium were decidedly distinctive ; that is, while the one showed no change but gave a smooth evenly spread inoculum, the other, owing to the sticky nature of the growth, presented a lumpy appearance.
9,~
OBSERVATIONS ON BACILLUS ABORTUS AND BACILLUS MELITENSIS.
In view of the interest attaching to the question of identity of these two organisms, the observed differences in the character of the growth seemed of sufficient importance to justify further investigation, which it was decided to carry out on purely chemical lines. TECHNIQUE AND EXPERIMENTAL OBSERVATIONS.
The organisms available for experiment comprised eleven strains of B.
melitensis, eight of B. abortus, and two strains of B. paramelitensis. All were old stock cultures and all the information concerning them is given in Table I. TABLE I. B. abortus. I. Old laboratory culture. Originally from South Africa. 2. Old laboratory culture. Origin not known. 3. N.C.T.C. No. 626 BANG. English strain No. 8. Isolated from stomach of foetal taft, 1919. Royal Veterinary College, London. 4. N.C.T.C. No.'1476 WEYBRIDGE I. 5. BANG. Strain B isolated 4.3.24. 6. BANe. Strain T isolated 3.9.24. 7. BANe. Strain W isolated 5.4.23. 8. N.C.T.C. No. 1785 MUHLAN, Strain I. Isolated from intestine of foetal calf, 1922. B. melitemis. 1. H u m a n strain. 2. N.C.T.C. No. 8 A~KWalGHT. Isolated from case of undulant fever in Malta, 1926. 3. N.C.T.C. No. 198. 4. N.C.T.C. No. 199. 5. N.C.T.C. ZAMMIT I. Isolated from milk of goat suffering from undulant fever, Malta,1920. 6. N.C.T.C. FENSlER MEYER. Strain 7, Group I I I Ref. J.I.D. 1920, 27, 185. 7. N.C.T.C. FENSmR MEYEa. Strain 20, Group I Ref. J.I.D. 1920, 27, 185. 8. N.C.T.C. FENSma MEYER. Strain 27, Group II Ref. J.I.D. 1920, 27, 185. 9. Strain 1197. History not known. 10. Strain 2588. History not known. 11. Strain 3218. History not known. B. paramelitemis. 1. N.C.T.C. BASSZrT-SMITH No. 82 originally from Tunis. 2. N.C.T.C. No. 200.
The majority of these cultures were sent to the Laboratory by Capt. S. R. DOUGLAS, of the Medical Research Institute at Hampstead; others were received from the Laboratory at Nairobi. OSBORNE and others have analysed a number of protein substances, and the sulphur-nitrogen ratio in these proteins, calculated from the percentages given in the analyses, are shown in Table II. It was thought that a determination of this ratio from the proteins of different species of bacteria might prove of value in the present investigation. To obtain this ratio, a method had to be devised which would be simple of application and effective with small amounts of bacterial substances. After many experiments, a method was evolved whereby the necessary estimations could be carried out on the amount of material furnished by twenty agar slope cultures. As a preliminary measure, the sulphurnitrogen ratio was obtained for a small number of miscellaneous organisms and the results obtained are given in Table III.
E. H . HADDON.
223
TABLE I I .
Protein.
Percentage Ratio of S. to N .
Vegetable.
Protein.
Percentage Ratio of S. to N .
Animal.
Amandin, almond Corylin, hazel nut Excelsin, Brazil n u t Edestin, hemp seed Globulin, cotton seed Vignin, cow-pea . . . . Glycinin, soy-bean L e g u m i n , lentil . . . . Phaseolin, kidney bean Conglutin, blue lupin Vicilin, lentil . . . . L e g u m e l i n , lentil Gliadin, wheat . . . . Glutenin, wheat . . . . Globulin, wheat . . . . Hordein, barley . . . .
...... ...... ...... ...... ...... . . . . ...... . . . . ...... ...... . . . . ...... . . . . . . . . . . . . . . . .
2'3 4"4 6'0 4"7 3"4 2"4 4"1 2"2 2"1 1"8 1"0 6"0 5"8 6'2 3'8 4"9
. .
. . . . .
Gelatin, commercial Gelatin, ligaments Elastin . . . . . . Mucin . . . . . . Mucoid, tendon . . Globulin, serum . . Albumin, serum . . Fibrin . . . . . . H~emoglobin, ox . . Casein . . . . . . Nuclio-histone . .
...... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maximum ...... Minimum ...... Difference ...... Average .........
. . . . . . . . .
3"9 3"2 2'3 6"8 19"0 7"0 11'9 6"5 2"5 5"1 2'8
19"0 1"0 18"0 4'9
TABLE I I I . WEIGHT EXPRESSED IN MILLIGRAMMES.
Organism.
Weight of Dried Bacteria.
Total Nitrogen.
Total Sulphur.
Ratio Percentage N. of S. to
2"9 1"4 0"7 1'9 1'8 2'3
0'137 0'089 0'042 0"048 0'053 0'080
4'7 6"3 6'0 2'5 2"9 3"5
t
I Staphyloccus aureus Bacillus prodigiosus B. coli communis B. abortus B. proteus I. B. proteus I I .
21"4 18"4 8"0 16"8 11'2 20'2
[ I t
The nutrient agar medium used throughout the experiments was maintained as constant in quality as was possible. It was prepared by boiling for one hour a mixture of 500 g. of minced ox heart and 3 g. o f " Difco " peptone powder in 2 litres of distilled water. The broth was allowed to stand until cold and then filtered through glass wool, 30 g. of agar powder were added, and the volume made up to 2 litres with distilled water. The whole was now steamed for one hour and then permitted to cool slowly in the steamer. The jelly was then removed in bulk to a plate and the coarse d6bris cut away from the bottom. The remaining clear portion of the jelly was melted, adjusted to pH 7"4, tubed, and sterilised in the autoclave at 15 lbs, Of each strain of organism twenty agar slope cultures grown for fourteen days at room temperature (25 ° C.) were prepared. The growth was carefully removed, by means of a platinum loop, in such a manner as not to disturb the medium itself, and suspended in 4 c.cm. of sterile distilled water. Manipulation with a glass rod sufficed to produce a fine, uniform suspension. Weight of Bacterial Substance.--This was determined by measuring accurately, in a special pipette, 1 c.cm. of the bacterial suspensioninto a tared watchglass. The same pipette was used throughout the experiment. The watch-
0~9'4
OBSE RVATIONSON BACILLUS ABORTUS AND BACILLUS MELITENSIS.
glass and contents were dried in a partial vacuum over concentrated sulphuric acid for a period of forty-eight hours and then quickly weighed. Repetitions gave identical results, which was not the case when the suspensions had been dried in the water oven. The weight of the bacterial substance was determined in a few cases only, as it was unnecessary to continue it throughout the series. Total Sulphur Content.--For this estimation, 1 c.cm. of the suspension was transferred to a platinum capsule, followed by 2 c.cm. of a saturated solution of potassium nitrate in decinormal caustic soda. The contents of the capsule were dried on the water-bath, and then carefully incinerated over a flame until all the organic matter had been completely oxidised. The residue, when cool, was dissolved in a quantity of distilled water not exceeding 7 c.cm., and transferred without loss to a cordite tube marked at 10 c.cm. Three drops of concentrated hydrochloric acid were added to clear the solution, which invariably contained a small amount of suspended carbonates. Finally, 3 c.cm. of a saturated solution of barium chloride were added, a little at a time, the tube being well agitated after each addition. Within ten minutes a maximum turbidity had developed, and the tube was matched against a series of standards. The standard suspensions of barium sulphate were prepared by taking ten carefully selected cordite tubes marked at 10 c.cm. and into each consecutive tube adding 0"1 up to 1 c.cm. of a N/100 sohition of sulphuric acid. Thiswas followed by 2 c.cm. of the potassium nitrate solution, two drops of concentrated hydrochloric acid and then distilled water to 7 c.cm. Finally, 3 c.cm. of barium chloride solution were added in the manner described above, and the tubes sealed in the blowpipe flame. The potassium nitrate and hydrochloric acid were used in the preparation of the standards, as it was found that the same quality of suspension could not be obtained in their absence. The quantity of sulphate to be estimated is so small that, for the successful application of this test, it is essential that the contents of both test and standard tube should be prepared in a manner as far as possible identical. If this is not done, the physical qualities of the precipitate for the same amount of sulphate differ sufficiently to render it impossible to match the tubes. The excess of barium chloride solution used increases the sensitiveness of the test by diminishing the solubility of the barium sulphate. The series of standards prepared in this way represent quantities of sulphur from 0"000016 g. to 0"00016 g. Total Nitroqen Content.--One c.cm. of the suspension was pipetted into a long boiling tube. Three c.cm. of concentrated sulphuric acid and a small crystal of copper sulphate were added and combustion carried out over a micro burner. At the end of one hour 0"5 g. of potassium persulphate was added and the combustion continued until a clear, faintly yellow solution was obtained. This was diluted with a small quantity of water and transferred to a Folin's absorption apparatus, with all the necessary precautions. Twenty c.cm. of
E. H. HADDON.
225
40 per cent. caustic soda were added, the ammonia removed by aspiration, and collected in 50 c.cm. of N/100 sulphuric acid. One hour was allowed for the aspiration. Excess of acidity in the collecting vessel was determined by titration with N/20 caustic soda, and the equivalent nitrogen calculated in the usual manner. The figures obtained are shown in Table IV. They are interesting when compared with those in Table III, but it is evident that the sulphur-nitrogen ratio does not serve to distinguish the one group of organisms from the other. TABLE IV. WEIGHTS EXPRESSED IN MILLIGRAMIVIES.
Organism.
B. abortus ,, ~J ~ . ~, ~,
B. melitensis ,,
Weight of Dried Bacteria. 1 2 3 4 5 6 7 8
1 2 3 4
~
5
~,
6
.
7
.
8
,, ,,
9 10
,7
11
B . paramelitensis 1 .
2
20'00 11"00
Total Nitrogen.
TotalSulphur.
Percentage ratio o f S to N .
3"36 2"10 2"66 1"89 2"45 2"73 3"99 3"01
0"080 0" 048 0'096 0"048 0"064 0'096 0"128 0"080
2"4 2'3 3"6 2"5 2"6 3"5 3'2 2"6
3'02 4"76 3"15 5"74 3"15 3'34 3"64 2'81 5"11 5"36 3"40
0'064 0"080 0"128 0"112 0'080 0'080 0'064 0"160 0" 144 0"096
2"1 3"0 2"5 2'2 3"5 2"4 2"2
3"71 3"71
0"144 0"160
0" 1 4 4
2"3
3"1 2'7 2"8
3"9 4"3
It was now decided to note the effect of transferring all the cultures on the simple peptone medium to the richer nutrient agar. This was done, and the cultures kept under careful observation. At the end of five months, during which period the organisms had been subcultured every week, only one culture ---one of the eight B. abortus examined--remained sticky. After a further period of three months this too had lost all signs of stickiness. The organisms were now transferred back again to the simple peptone medium, and, as previously, subcultured at monthly intervals. Up to date, that is after an interval of seven months, four of the abortus cultures are developing the sticky character. Evidently, in the conditions of the experiment, the tendency to acquire this character is less marked than the tendency to lose it. The conditions responsible for this change in character of growth await investigation. There is some evidence that a fairly rapid extraction of moisture
9~6
OBSERVATIONS ON BACILLUS ABORTUS AND BACILLUS MELITENSIs.
from the medium during growth hastens the development of the sticky quality. Two exceptions, however, were noticed. The observations were made on two series of cultures, one series being grown in a partially exhausted chamber containing concentrated sulphuric acid, and the other under the usual conditions, i.e., room temperature in a moist chamber exposed to the air. The influence of B. subtilis is interesting. It was found that, when this organism was inoculated at the base of an agar slope containing a five days' culture of a B. abortus, it definitely hastened the production of stickiness, and further that it retarded the change in the other direction when both organisms were grown together on nutrient agar. The inoculation of B. subtilis was necessarily always made some days after that of B. abortus. In these conditions the latter organism influenced the growth of subtilis in such a way as to prevent a rapidly spreading growth. In some cases the growth of B. subtilis was, relatively speaking, remarkably slow and consisted almost entirely of spores. The carbon dioxide tension appears to have no rble in the observed phenomenon, as experiments carried out by culturing the abortus and melitensis organisms in atmospheres containing 5, 10 and 50 per cent. by volume of this gas showed no apparent difference. Cultivation of all the organisms, in both peptone water and nutrient broth containing 0"5 per cent. glucose, showed no change at the end of a month. The sugar content was estimated by Folin's method. Further experiments were confined to B. abortus No. 8, as this organism showed the greatest difference between its two varieties of growth. Also it was necessary to make thls curtailment owing to pressure of routine work. Two points of interest were observed with regard to the smooth and the sticky growths. Judged macroscopically, the former appeared to lose its moisture more quickly than the latter, and when both varieties were subcultured on the same medium the sticky growth remained viable for a longer period, as was shown by inoculating tubes of nutrient broth at intervals. " Difference in intensity of the pentose reaction were obtained with ph]oroglucinol for the same quantity of bacterial substance, the sticky growth giving the deeper colour. A similar difference was also observed between the sporing and the vegetative forms of B. subtilis. In this case the spores gave the more marked reaction. For the small number of strains examined, the above observations seem to reveal the existence of a physical difference between the majority of strains of B. melitensis and those of B. abortus, and to suggest that this difference is one of degree. The remarkable production of a sticky tenacious growth which is observed with the majority of the B. at~ortus strains, and which is developed slowly when the organisms are cultured in conditions which may be regarded as unfavourable, may possibly be a protective effort analagous to the formation of a capsule or spore in other species, but, at the present juncture, it is impossible to decide upon the significance of this phenomenon.