The Oxidation of Phenol by Certain Bacteria in Pure Culture

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.