STUDIES ON OXIDATION AND REDUCTION BY
          PNEUMOCOCCUS.

III. REDUCTION OF METHYLENE BLUE BY STERILE EXTRACTS
             OF PNEUMOCOCCUS.

BY OSWALD T. AVERY, M.D., AND JAMES M. NEILL, PH.D.
(From the Hospital of The Rockefeller Institutefor lCledical Research.)

(Received for publication, October 11, 1923.)

The two preceding papers of this series have dealt with
the phenomenon of peroxide formation by pneumococcus. The
first of these studies (1) concerned itself with the conditions under
which this compound is formed when air is admitted to cultures of
pneumococci which have been grown anaerobically. Under these
conditions peroxide is formed at reactions and at temperatures in-
hibiting active cell multiplication. These facts suggest that the
mechanism of peroxide production is dependent upon an interaction
of oxygen and cell constituents, and that under experimental condi-
tions, at least, this reaction may proceed independently of cell growth.
This view is further supported by the data presented in the second
paper (2) on the formation of peroxide by sterile extracts of pneumo-
cocci. The fact that soluble extracts of pneumococcus cells form
peroxide when exposed to molecular oxygen is convincing evidence
that this function is not dependent upon the presence of living,
formed cells.

Just as the admission of air to sterile pneumococcus extracts was
found to give rise to the prompt formation of peroxide, so in the
present experiments it will be shown that the addition of methylene
blue to these same extracts results in rapid reduction of the dye
to its colorless base. The admission of air or the addition of dye
to pneumococcus extracts, in the absence of living cells, induces
reactions which although yielding dissimilar products, may be alike
in nature, depending upon whether molecular oxygen or methylene
blue serves as hydrogen acceptor.

543


544     OXIDATION AND REDUCTION BY PNEUMOCOCCUS. III

  In the following experiments, certain conditions are de&red which
influence the reduction of methylene blue by pneumococcus extracts;
and certain similarities are pointed out which serve to relate dye
reduction and peroxide formation as functions of the same or closely
,  allied oxidation-reduction systems in the cell.

EXPERIMENTAL.

Methods.

Preparation of&tracts.-The sterile extracts of pneumococci used in the present
experiments were prepared according to the methods previously described (2).
They consisted either of broth extracts of unwashed pneumococci or of washed
cells extracted in phosphate solution.

Technique of Methy.len.e Blue Reduction Tests.-A stock solution of Merck's
medicinal methylene blue, in concentration of 1: 1,000 in water, was sterilized in
the autoclave and stored in the ice box.  At the time of testing, dilutions of the
stock solution were freshly prepared in sterile 0.1 M phosphate solution, pH 7.5.
Exhaustion of air from the test fluids was not carried out prior to the addition of the
extract, but the amount of molecular oxygen present was kept uniform throughout
by saturating all solutions with air at a constant temperature of ZS'C., and further
admission of air was prevented by sealing the tubes with Vaseline.  The sterile
extracts of pneumococci used in the tests were treated as indicated in the protocols.
The solutions containing the complete reduction system were placed in narrow
tubes of uniform bore, sealed with Vaseline, and incubated at 37oC.

Reduction of Methylene Blue by Sterile Broth Extracts of Unwashed
               Pzeumococti.'

In repeated experiments it has been found that small amounts of
the sterile cell extract possess the property of rapidly reducing large
amounts of methylene blue.  For example, 0.3 cc. of the sterile
extract added to 1 cc. of 1: 20,000 methylene blue solution effected
complete reduction in 40 minutes at 37oC.; the same amount of
sterile extract added to 1 cc. of 1:2,000 methylene blue (a concen-
tration of approximately 1 mM) brings about complete reduction
in 24 hours.

Peroxide may be considered as a product resulting from the trans-
fer of hydrogen to molecular oxygen in the same way that the leuco

l Unless otherwise specified, the term pneumococcus extract refers to broth
extracts of unwashed cells.


OSWALD T. AVERY AND JAMES hf. NEILL       545

base of methylene blue may be regarded as a product resulting from
the transfer of hydrogen to the dye.  Thus, from the nature of the
processes involved in peroxide formation and methylene blue reduc-
tion, it is possible that the same system or systems in the extract
may be responsible for both activities; if molecular oxygen is ad-
mitted to an active extract peroxide is formed, if methylene blue is
added to the same extract, methylene white, or the Ieuco base of the
dye, is formed.  In both cases, some constituent of the extract is
oxidized, with the simultaneous formation of peroxide or of methy-
lene white, depending merely upon whether molecular oxygen or
methylene blue serves as hydrogen acceptor.

With these relationships in mind, the effects of various agents on
the activity of these two functions of pneumococcus extracts have
been investigated in the following experiments.

Effect of Heat upon Methylene Blue Reduction by Extra&s of Przeu-
                       mOcOCCUS.

In order to determine the effect of heat upon the reducing action
of pneumococcus extracts, the following experiments were carried
out. For purposes of comparison the heat sensitiveness of the
peroxide-forming and methylene blue-reducing activities of the
same extract was studied under similar conditions.

1. Injluence upon the Potency of the Extracts of 5 Minutes Exposure
to Temperatures Varying from 5%100oC.-In the following experi-
ment a broth extract of unwashed pneumococci was heated for
5 minutes at the stated temperatures. The ability of the heated
extract to reduce methylene blue and form peroxide was then deter-
mined.

1.5 cc. portions of cell extract were placed in a series of small, agglutination
tubes, sealed with Vaseline, and incubated 30 minutes at 37'C.  The tubes were
then immersed for exactly 5 minutes in a constantly agitated water bath at the
temperatures shown in the tables.  After heating, each tube was cooled immedi-
ately in ice water and held under seal until all the heating tests were completed.

1 cc. portions of extract which had been heated at the different temperatures
were then aerated by shaking, and further exposed to air in a thin layer at room
temperature in the dark. Peroxide tests were made on the heated extract after
1 hour's exposure to air.


546    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. III

0.5 cc. of cell extract which had been heated at these temperatures was added to
1 cc. of a 1:2,000 methylene blue solution in ~430 Pod.  The solutions were mixed
thoroughly in small, narrow tubes and then carefully sealed with Vaseline.  The
reduction tests were incubated in a water bath at 37oC. The results are given in
Table I.

It is evident (Table I) that heating pneumococcus extract for 5
minutes at 55oC. does not seriously impair either its ability to form
peroxide or to reduce methylene blue. On the other hand, heating
the same extract for 5 minutes at 65oC. results in practically com-
plete loss of both of these properties, although in the case of the

                      TABLE I.
Coflzparfso?r of Eject of Heating for 5 Minutes at Dijerent Temperatures U~OVZ the
Perox-ide-Forming and Alethylene Blue-Reducing ActiCty of Sterile Ex-
             tracts of Pneumococcus.

Extract.

Peroxide formation after
  1 hr. aeration.

Methylene blue reduction
after 2 hn. incubation.

Unheated control. . . . . . . . . . . . . . .     ++++*        ++++t
5 min. at  WC.. . . . . . . . . . . . . . . . .      +++          +++
5 " (( (jj" (1                       -                -
           . . . . . . . . . . . . . . . . . .
5 " "  75" Cl                       -
           . . . . . . . . . . . . . . . . . .
5  " `I  8j"" ..,...............         -                -
5  `I   `I  100" " . . . . . . . . . . . . .         -
Broth (unheated) control. . . . . . . .         -                -
-

* + + + + indicates strong peroxide reaction; + + +, marked peroxide
reaction.

7 + ++ + indicates complete reduction; + + f, almost complete reduction.

methylene blue reaction, delayed and slight reduction was evident
after 72 hours.

Extracts heated for 5 minutes a.t or above 75oC. failed to show
any evidence of reducing action even after 3 days. These results
indicate that the methylene blue-reducing and peroxide-forming
powers of pneumococcus extracts are both susceptible to the de-
structive action of heat and that each of these activities shows the
same rapid increase in rate of destruction when an extract is exposed
to temperatures between 5.5" and 65oC.

2. Eject of Heating at 55oC. upon the Methyleue Blue-Reducing
J'ower of Puetmococcm Extracts.--In the following experiment the


        OSWALD T. AVERY AND JAMES Y. NEILL       547

rate of destruction of the reducing power is followed by comparisons
of the final amount of methylene blue reduced by an extract which
had been previously heated for periods of from 5 to 60 minutes at
55oC.

0.5 cc. of the sterile cell extract was placed in a number of narrow tubes, sealed
with vaseline, and incubated at 37oC. for 40 minutes.  These tubes were then
heated anaerobically for different periods at 55oC. in a constantly agitated water
bath.  Each tube was cooled at once after the heating period and kept under seal
until all tubes had been heated.

The concentrations of methylene blue used were 1:3,000,1:7,500, and 1: 15,000.
These solutions were brought to constant oxygen tension by saturation with air at

TABLE II.

Injuence of Heating Sterile Pneumococcus Extract upon the Total Amount of
      Methylene Blue Reduced at Final Equilibrium.

Cell extract heated af 55oC.
     (0.1 cc).

     Reduction of metbykne blue (1.4 cc.).

1:3,000   I

--

Unheated. .............    +++*
Heated  5 min .........     ++
"   10 "               -
          ........
"   30 `(               -
          ........
"   60 "               -
          ........
Broth control               -
         ..........
PO, solution, control.          -
              ...

1:7,500

++++
++++
++
  -


  -

_ -









-

1: 15,000

++++
++++
++++
++
+

* ++++ indicates complete reduction; ++, partial reduction; +, slight
reduction; and -, indistinguishable from control.

25oC.  0.1 cc. of heated extract was then added to 1.4 cc. of each concentration of
methylene blue solution.  As controls, 0.1 cc. of broth, or 0.1 cc. of phosphate
solution, was added to each dilution of the dye.  All of the reduction tests were
sealed with Vaseline, and incubated at 37'C.

After 72 hours incubation, the reduction of the dye was determined by compari-
son with the respective controls.

The data in Table II show that the longer a pneumococcus ex-
tract is exposed to 55'C. the less is the amount of dye reduced by
equivalent amounts of extract.  The gradual decrease in the dye-
reducing power of the heated extract may be considered as due to a
gradual destruction of the total oxidizing-reducing activity of the


548     OXIDATION AND REDUCTION BY PNEUMOCOCCUS. III

extract.  In an analogous experiment in the preceding paper (2),
heating an extract at 55OC. was found to effect a gradual decrease
in its power to form peroxide.  It'is evident, therefore, that heating
pneumococcus extract for 1 hour at 55oC. results in's gradual loss,
but not total destruction, of both its peroxide-forming and methylene
blue-reducing properties.

Activatirm of Sterile Extracts of Washed Pneumococci.

In the preceding paper (2) it was pointed out that thorough washing
of pneumococci before extraction deprived the extracts of their capa-
city to form peroxide on exposure to oxygen.  It was further shown
that these inactive extracts acquire this property on the addition
of the cell washings, muscle infusion, or yeast extract.  In the follow-
ing experiments methylene blue was added to washed cell extracts to
determine whether they still retained their reducing action; or if this
activity were lost, whether it could be restored again by adding
substances known to reactivate the peroxide-forming function of
these same extracts.

0.6 cc. of unfiltered, sterile extract of washed pneumococci was added to an
equal volume of either muscle infusion or yeast extract which had been previously
adjusted to pH 7.5.  The test solutions were all brought to uniform oxygen ten-
sion by saturation with air at 25oC.  To this mixture 0.1 cc. of 1:2,000 solution
of methylene blue was added.  The small tubes containing the test system were
sealed with Vaseline and incubated at 37oC. As controls double quantities of
washed cell extract alone, and 0.6 cc. quantities of yeast extract or muscle infusion
without pneumococcus extract, were added to 0.1 cc. amounts of methylene blue.
The total volume was kept constant by the addition of sterile phosphate whenever
necessary.  The results of this experiment are shown in Table III.

Table III presents evidence that sterile extracts of washed pneu-

mococci are unable by themselves to bring about the reduction of
methylene blue.  It is further demonstrated in this experiment that
washed cell extracts in which reducing activity has ceased, may be
stimulated to carry on reduction processes by the addition of muscle
infusion and yeast extract.2 The presence of these activating sub-
* Muscle infusion: The infusion was prepared by the routine procedure followed

in the laboratory in the preparation of meat infusion broth.  500 gm. of lean beef
were allowed to infuse in 1 liter of distilled water overnight at ice box temperature.


OSWALD T. AVERY AND JAMES M. NEILL       549

stances, which do not themselves reduce the dye, restores to washed
cell extracts the power to decolorize methylene blue.  This comple-
menting action is wholly analogous to the activation by these same
substances of the peroxide-forming power of extracts of washed
pneumococci. Harden and Zilva (3) have found that saline sus-
pensions of washed living colon bacilli are by themselves unable to
reduce methylene blue, but exhibit this property upon the addition
of various substances, among others cell washings, broth, glucose,
and peptone.

In$uence of Exposure lo Air.-When a broth extract of unwashed
pneumococci is exposed to air, it suffers a rapid loss of its ability to
decolorize methylene blue; if the exposure to oxygen is continued

TABLE III.

Acfiz~atiotion of Reducing Actiwity in Sterile Extracts of Washed Pneumococci.

Sterile extract   Activating substance.
of mashed
pneumococci.   MUSCk     Yeast
         infusion.    extract.

  cc.        ct.       6.5.

  1.2
  0.6       0.6
          0.6
  0.6             0.6
                0.6

Stde
phosphate
solution I I
    Me%?         Result.


PH 7.5.     1: 2,000.    Reduction.     Time.

cc.




0.6


0.6

cc.
0.1
0.1
0.1
0.1
0.1

Negative.   5 days.
Complete.   23 hrs.
Trace.     5 days.
Complete.    6 hrs.
Trace.     5 days.

for 12 to 24 hours the methylene blue-reducing action of the extract
is completely destroyed.  Reference has already been made to the
fact that extracts prepared from washed cells are unable to induce
reduction' of methylene blue in the absence of certain activating
substances. Extracts of this nature may be exposed to molecular
oxygen for considerable periods with relatively little effect on their

After titration through coarse paper, the infusion was sterilized for 20 minutes at
1OO'C. on 3 successive days.  The reaction was adjusted to pH 7.5 before use.  .
Yeast extract: 100 gm. of brewer's yeast in 400 cc. of distilled water were

adjusted to pH 4.5 and boiled for 10 minutes.  The cellular material was allowed
to sediment at room temperature and the clear supematant was pipetted off and
tested for sterility. The extract was stored in the ice box and the reaction adjusted
to pH 7.5 before use.


550     OXIDATION AND REDUCTION BY PNEUMOCOCCUS. III
                 F

reducing action when subsequently activated by the addition of these
complementing substances.  If, on the other hand, these same ex-
tracts are rirst activated and then exposed to air, they show a pro-
gressive decrease in dye-reducing activity, although the loss is not
so pronounced as that which occurs on exposure of the unwashed cell
extracts.

. The inability of previously oxidized extracts to cause reduction
of the dye may be attributable to the loss in activity resulting
from the irreversible oxidation of certain constituents of the extract.
The loss of this power may also be due in part to the deleterious action
on the reducing system, of the peroxide which accumulates in the
extract during exposure to air.

Consumption of Molecular Oxygen by Sterile Extracts of Pneumo-
                       coccus.

From the data in this paper it is evident that sterile extracts of
pneumococci cause complete reduction of methylene blue in solu-
tions previously saturated with air.  This fact indicates that these
extracts are able to consume dissolved oxygen in the presence of
methylene blue. However, the production of peroxide by these
extracts upon exposure to air is evidence of `their ability to consume
or accept molecular oxygen in the absence as well as in the presence
of methylene blue.  Quantitative analyses of oxygen consumption
are now being made in a study of this function of the extracts.

DISCUSSION.

In the present work, as in the experiments recorded in the preceding
paper, the sterile extracts of pneumococcus employed have been of
two types: one type represents broth extracts of unwashed pneumo-
cocci; the other, extracts of washed pneumococci which have been
extracted in phosphate solution.  These two types of extract exhibit
marked differences in behavior.  The extract prepared from unwashed
cells extracted in broth contains a complete oxidizing and reducing
system, capable in itself of forming peroxide, or of reducing methyl-
ene blue upon the admission of molecular oxygen or the addition of
the dye. The second type of extract, prepared from the washed


OSWALD T. AVERY AND JAMES M. NEILL       5.51

bacteria cannot by itself initiate either of these reactions without
the addition of certain substances found in meat infusion and yeast
extract.  The presence of these complementary substances "com-
pletes" the potentially active system or systems in the washed cell
extract and renders it reactive both with molecular oxygen and with
methylene blue.

Both types of pneumococcus extract are thermolabile and lose
their activity on exposure to 65oC. However, the complementing
substances in meat infusion and yeast extract which serve to com-
plete the unheated extracts of washed cells are thermostable.  These
facts indicate that the active perorfide-forming and methylene blue-
reducing systems in pneumococcus extracts consist of two component
parts, one sensitive to heat, and the other relatively heat-resistant.
Further analysis indicates that the thermolabile component is derived
from the bacterial cell and may be ferment-like in nature. The
thermostable component can be artificially supplied from sources
other than those of bacterial origin.  Neither of these components
by itself is reactive with molecular oxygen or methylene blue, each
being separately inactive and both being essential for the formation
of peroxide and the active reduction of the dye.  The union of these
extract constituents provides an active and complete system in which
or by which some substance is oxidized with the formation of peroxide
or the reduction of methylene blue, depending upon whether molecular
oxygen or the dye functions as hydrogen acceptor. The complex
nature of the extracts themselves and especially the unknown charac-
ter of the substances actually oxidized in the extracts, impose certain
limitations on the interpretation and analysis of the phenomena.

In the bacterial extracts employed in these studies, the systems
responsible for peroxide production and methylene blue reduction
have so many characteristics in common that it seems probable that
they are either identical or closely related.

SUMNARY.

1. Sterile broth extracts of unwashed pneumococci, entirely free
from living or intact cells, actively reduce methylene blue.
2. Sterile extracts prepared by extracting washed pneumococci
in phosphate solution are unable by themselves to reduce methylene


552     OXIDATION AND REDUCTION BY PNEUMOCOCCUS. III

blue. Upon the addition of meat infusion or yeast extract, these
washed cell extracts actively reduce methylene blue.

3. The system or systems in pneumococcus extracts responsible
for methylene blue reduction are destroyed by exposure to tempera-
tures practically identical with those which have been previously
found to destroy the peroxide-forming activity of the same extracts.

4. It is suggested that peroxide formation and methylene blue
reduction by pneumococcus extracts are functions of the same or
closely related systems, the particular reaction induced depending
upon whether molecular oxygen or methylene blue serves as hydrogen
acceptor or oxygen donator.

BIBLIOGRAPHY.

1. Avery, 0. T., and Neil& J. M., J. Exp. Med., 1924, xxxix, 347.
2. Avery, 0. T., and NeiIl, J. M., J. Exfi. Med., 1924, xxx-ix, 357.
3. Harden, A., and Zilva, S. S., Biochem. J., 1915, ix, 379.