149 The Oxidation of Phenol by Certain Bacteria in Pure Culture, By Gilbert J. Fowler, Edward Ardbrn, and William T. Lockett. (Communicated by Dr. A. Harden, F.R.S. Received September 30, — Read November 17, 1910.) In the course of investigations on the effect of various antiseptic substances on bacterial sewage filters, it was found that solutions of pure phenol could be oxidised on filters of this description. It was further found that the phenol apparently exerted a selective action on the bacteria present in the filter, only three or four types of organism appearing in the filtrates. Of these about half were liquefying organisms, identified as B. liquefaciens Jiuorescens. At first it was thought that the main non-liquefying, or extremely slowly liquefying, organism was B. Jiuorescens non-liquefaciens, on account of the apparently fluorescent yellow colonies it produced ; subse- quent investigation showed that this was not the case (see note at end of paper). It is therefore referred to in the paper simply as the chromogenic organism. On passing a current of filtered air through the liquid containing a mixture of these organisms, together with small quantities of phenol, the latter was slowly oxidised, which was not the case in a control experiment where organisms were absent. Preparation oj Pure Cultures. The survival of the particular organisms — B. liquefaciens Jiuorescens and the chromogenic organism — in the experiments referred to, and their constant presence in filtrates from other filters dealing with pure phenol solutions, led to experiments being made on the separate effect of each of these organisms on phenol in solution. Colonies of the organisms in question were separated; the liquefying variety was usually grown on gelatine, the chromogenic organism in broth. Cultures were obtained from different sources — from the plates made in connection with the previous experiment, and from plates made from filtrates from different filters. Later, further cultures were obtained from plates showing colonies of only one organism. The culture medium used had the following composition : — Broth. — Per litre of water. Peptone 10 grammes. Meat extract 5 or 10 >> Sodium chloride 5 „ 150 Messrs. Fowler, Ardern, and Lockett. [Sept. 30, Nutrient Gelatine. — Medium made by addition of 10 per cent, of gelatine to broth obtained as above. Diluted Broth Solution.- — Made either by the addition of about 10 c.c. of broth culture to 500 c.c. of sterile water and further incubation, or by inoculating a quantity of sterile water, containing a few cubic centimetres of " broth/' with the organism. Special Medium. — This medium is similar to one used by Percy Frankland in his earlier experiments, and had the following composition ; — In 1000 c.c. of water, with 4 grammes of pure calcium carbonate in suspension. Potassium phosphate 0*1 gramme. Magnesium sulphate (cryst.) 0'02 „ Calcium chloride (fused) 0*01 „ Glucose 0*3 Peptone 0'25 51 Action of Pure Cultures on Phenol. The preliminary experiments made in connection with the action of each of the organisms on phenolic solutions were carried out in 40-oz. bottles, fitted with rubber stoppers, tubes, etc., and arranged in series in a similar manner to the preceding experiment The procedure generally adopted was as follows : — To a sterile phenol solution (vol. 500 c.c.) containing 0*01 to 0'02 per cent, of phenol, a quantity of the culture of the particular organism under examination was added, the solution aerated and tested from time to time. The experiments throughout were conducted in such a manner as to prevent as far as possible any air or other infection. It was not supposed that the solutions were kept entirely free from infection, the extent of the contamination presumably increasing with the length of duration of experiment. I. Action of Bacillus liquefaciens nuorescens on Phenol. Several experiments were made in connection with this organism. Generally a few cubic centimetres of liquefied gelatine containing the organisms were added to a pure phenol solution, and in one case to a solution of phenol with added nitrate and nitrite. Practically no change, however, was noted in the phenol content of these solutions after two months' aeration. The numbers of organisms present at the commencement of experiments were several millions per cubic centimetre, and after two months a considerable number in active condition were still to be found in the solutions. 1910.] Oxidation of Phenol by Certain Bacteria. 151 II. Action of the Chromogenic Organism on Phenol. (a) Broth Cultures. — Experiments were first conducted with broth cultures of this organism. A few cubic centimetres of a broth culture were added to a pure phenol solution. For two or three days no change was noted in the solutions ; subsequently, however, the disappearance of phenol was rapid, the whole being completely oxidised in five or six days from the commencement of the experiments. *♦ Typical example : — Oxygen absorbed from acid permanganate solution in 3 minutes. Parts per 100,000. April 9. Experiment commenced 25 '20* „ 12 25-80 „ 14 2-00 „ 15 1-57 * Equivalent to 16 '5 parts of phenol per 100,000. The 3 minutes oxygen absorption test was chosen as the most convenient for the detection of phenol and its approximate estimation. The accurate determination of phenol in such dilute solutions would be a long and tedious process, while the amount of oxygen absorbed by the constituents of the broth or its products of decomposition bears a very small proportion to the amount taken up by the phenol. (b) Diluted Broth. — An experiment was made using about 30 c.c. of a diluted broth culture in place of ordinary broth. Again it was noticed that no change took place for a few days, followed by rapid disappearance of phenol. The time taken to complete the experiment was eight days, or rather longer than with ordinary strength of broth. Two experiments were afterwards made with diluted broth cultures (35 c.c.) with the addition of a few cubic centimetres of ordinary sterile broth. As before, no change was noted in the earlier stage of the experiment, but after four days only, complete oxidation was rapidly brought about in 24 hours. The air passing through these solutions was previously sterilised by bubbling through mercuric chloride solution. Example : — Oxygen absorbed from acid permanganate in 3 minutes. Parts per 100,000. June 27. Experiment commenced 24*8* „ 29 24-8 „ 30 24-8 July 1 22'8 „ 2 1-2 * Equivalent to 16*2 parts of phenol per 100,000. 152 Messrs. Fowler, Ardern, and Lockett. [Sept. 30, It appears, therefore, that the action is accelerated by the addition of further nutrient material. (c) Cultures on Special Media. — A few cubic centimetres of one of these cultures were added to a phenol solution. The disappearance of phenol in this case was gradual, commencing in the first day or two — the time taken, however, for oxidation was considerably longer than in the case of the broth cultures, viz. 16 days. Eesults : — Oxygen absorbed from acid permanganate in 3 minutes. Parts per 100,000. June 29. Experiment commenced 20*8* July 1 20-4 „ 2 18-0 „ 9.. 79 „ 14 1-4 * Equivalent to 13*6 parts of phenol per 100,000. Gelatine plates were made from the solutions and it was found, generally, that the numbers of the chromogenic organism present in the solutions, being several millions per cubic centimetre at commencement, were consider- ably increased during the experiment. Proportionally, the number of organisms other than the chromogenic present in the solutions were few. (d) Addition of a Culture of the Chromogenic Organism to a Solution previously unacted upon hy B. liquefaciens fluorescens. — An interesting bottle experiment was made as follows : — To a solution of phenol previously unacted upon by a culture of B. liquefaciens as described in the preceding, a broth culture of the chromogenic organism was added. Within four days of the addition of the second culture almost complete oxidation of the phenol had taken place. Eesults : — Oxygen absorbed from acid permanganate in 3 minutes. Parts per 100,000. March 2. Experiment commenced 17*40* April 12 17'00 ,, 12 after addition of second culture 19 "20 „ 13 18*60 „ 15 811 „ 16 2'46 * Equivalent to 11*4 parts of phenol per 100,000. Gelatine plates of the solution at end of experiment showed presence of large number of both liquefying and non-liquefying organisms. 1910.] Oxidation of Phenol by Certain Bacteria, 153 Flask Experiments. — Similar experiments to the preceding were made with cultures of the chromogenic organism under rigid sterile conditions. Four flasks (300 c.c. capacity), fitted with rubber stoppers, tubes, etc., and con- nected in series, were used in each experiment. The inlet of the first flask and the outlet tube of the fourth flask were connected with wide tubes drawn out at one end and plugged with cotton wool. To each of the four flasks at the outset of experiment was added 150 c.c. of phenol solution of required strength (10 parts per 100,000). The inlet tubes were raised in the stoppers so that a short space was left between the surfaces of liquids and the ends of the tubes. The apparatus connected and complete with the contained solutions was then placed altogether in a steamer and sterilised for three successive days. A culture of the organism was divided into two equal parts, one half being heated to boiling for a minute or so, and further sterilised by placing in the steamer for three days at the same time as the apparatus described above. When sterile, the second flask was carefully disconnected from its stopper and sterilised or " dead " culture added, the flask being quickly reconnected. To the third flask the remaining half of the culture which contained the living organisms was added in exactly the same manner. The tubes, which had been raised to prevent " sucking back " of the solutions during cooling periods, were then pushed through the stoppers, care being taken that no portion of the tubes which had been previously exposed to the air occupied a position inside the flasks. The contents of flasks were then as follows : — Flask I Phenol solution alone. „ II „ „ + dead culture. » HI „ „ + living „ „ IV „ „ alone. Air was slowly drawn through the solutions by means of an aspirator (rate 10 to 20 litres per day) in the direction of Flask I to IV. By this means the solution containing the living culture was kept free from air infection. Gelatine plates were made of all the solutions after the experi- ments, and the following is a general description of the bacteriological results obtained :- — Flash I. — (Phenol alone.) Very little contamination and occasionally none whatsoever. Not more than two organisms per cubic centimetre were present at any time. Flash II — (Dead culture.) Free from air infection. Plates of 1 c.c. solution usually made. In one case great numbers of the chromogenic 154 Messrs. Fowler, Ardern, and Lockett. [Sept. 30, organism were found to be present, due to " sucking back " of some of the liquid contained in the third flask ; no other organism found. Flask III. — (Living culture.) With one exception the liquid contained only organisms of chromogenic type. In the isolated case 20 moulds (per cubic centimetre of liquid) were found to be present, possibly introduced with the culture, or during plating, whilst many millions of the organism under investigation were found to be present. Flash IV. — (Phenol alone.) Generally contained the chromogenic organism. On one occasion it was entirely free. The presence of organisms in this flask is due to the carriage of organisms by the air from the flask containing the living culture. Chemical Results. — The following is a typical example of the results obtained : — Oxygen absorbed from acid permanganate in 3 minutes. Parts per 100,000. Before experiment — Solution of phenol added to flasks before sterilising 12*80* After experiment — Flask I. Phenol alone 10*80 „ II. „ + dead culture 11*80 „ III. „ + living „ 0-50 „ IV. „ alone 11*20 No intermediate tests were made. * Equivalent to 8*4 parts phenol per 100,000. While these experiments were not quite so perfectly successful as could be desired, yet sufficient evidence was obtained to show that a pure culture of the organism used was capable of oxidising phenol, whilst no change was noted in the phenol content of solutions containing " dead " cultures. Similarly, no change has been observed in the phenol content of solutions of pure phenol. The time taken for oxidation varied from two to several weeks. It should be noted that the aeration in these experiments was very much less than that obtained in the " bottle " experiments. The amount of aeration appears to make considerable difference in the rate of oxidation. Both broth and diluted broth cultures were used in these experiments. The following experiment was, however, conclusive, and confirmed the natural inference drawn from the foregoing. A solution was made con- taining approximately 0'01 per cent, of phenol, together with the following ingredients : — ■ Per litre. Ammonium sulphate 0*1 gramme. Potassium phosphate 0*1 „ Magnesium carbonate, in suspension \ — 1 „ 1910.] Oxidation of Phenol by Certain Bacteria. 155 Four hundred cubic centimetres of this solution were sterilised in a litre flask, plugged with cotton wool, in a steam steriliser for three successive days. When sterile, a few drops of a broth culture of the organism were added, particular care being taken to prevent contamination. It was intended to limit the amount of carbon in the media, it being supposed that the phenol on oxidation would supply the deficiency. The flask and contents were incubated for several days at 20° C, the liquid being aerated by an occasional shake. The solution was tested after five days with the usual precaution to prevent contamination. Eesults : — Oxygen absorbed from acid permanganate in 3 minutes. Parts per 100,000. At commencement 19*20* After 5 days 8'57 » 9 „ 1-83 * Equivalent to 12*5 parts of phenol per 100,000. On the ninth day, before submitting the solution to chemical tests, gelatine plates were made. Two plates were made taking 1 c.c. of the liquid, and one plate each for 0*01 c.c, 0*0001 c.c, and O'OOOOOl c.c. Bacteriological Eesults (3 days at 20° C). 1 c.c. original, 2 plates Crowded with organisms. All one kind. No lique- fying colonies. 0*01 c.c. „ 1 „ Crowded. All one kind. Chromogenie. 0*0001 c.c. „ 1 „ Hundreds of colonies. All one kind. Chromogenie. 0*000001 c.c. „ 1 „ 130 colonies. Chromogenie. All one kind. The solution contained 130,000,000 organisms per cubic centimetre, and there was not the slightest evidence of contamination. It may be safely concluded, therefore, that dilute solutions of phenol are oxidised by the particular organism under investigation. Experiments are being carried out to determine the exact effect upon the oxidation of phenol by this organism, of the addition of other nutrient substances, Experiment II (b) having shown that the addition of broth accelerated the action. Eesearches have also been in progress on the bacterial oxidation of sulphocyanates, which appears to be more complex, and certainly less rapidly effected than the oxidation of phenol, inasmuch as when a mixture of the two substances is submitted to bacterial oxidation the phenol is invariably the first to disappear. 156 Mr. F. W. Twort. Method for Isolating and [Sept. 30, [Note added October 20th, 1910. — Dr. Sidebotham, of the Public Health Laboratory, Manchester, has kindly identified the chromogenic organism worked with in the above experiments. He describes it as follows : — Bacillus, non-motile, no cilia ; size, 2 fjb x 0*5 ; often in pairs, no chains. Grows best at 22° C. ; growth ceases at 30° C. ; grain negative. A. P. B. — Greenish yellow ; smooth growth. G. P. B. — Growth all round stab ; growth spreads on surface ; central raised pale yellow projection, surrounded by pale pink smooth area ; gelatine very slowly liquefied. Potato. — Very bright lemon-yellow ; irregular knobby growth. Milk. — Colour, brown to pale brown ; no acid change. The characteristics seem to be similar to those of B. Helvolus (Zimmermann). The above characteristics have also been observed by one of us (G. J. F.) in the case of an organism regularly occurring in an experimental filter dealing with liquids containing phenolic compounds.] A Method for Isolating and Growing the Lepra Bacillus of Man. (Preliminary Note.) By F. W. Twort. (Communicated by Leonard Hill, F.K.S. Eeceived September 30, — Eead November 17, 1910.) (From the Laboratories of the Brown Institution, University of London.) For a number of years different investigators have attempted to cultivate the lepra bacillus of man and the allied organisms found in the rat and other animals. It is not intended in this preliminary note to discuss the numerous papers which have been published from time to time from the various English, Continental, and American laboratories. These papers deal with non acid-fast bacilli, or with acid-fast bacilli growing quickly on ordinary media, which, in the opinion of the writer, are contaminating organisms, and not the true lepra bacillus. So far, no one has produced a culture of acid-fast bacilli isolated from a leper, and showing the characters of the lepra bacillus as found in the tissues of man. It was with the object of obtaining a pure living culture of the lepra bacillus that these investigations were undertaken.