STUDIES ON BACTERIAL. NUTRITION.

II. GROWTH ACCESSORY SUBSTANCES IN IPHE CULTIVATION OF
           HEMOPEXLIC BACILLI.

  BY THEODOR THJ&M'A, M.D., AND 0. T. AVERY, M.D.
(From Ihe Hospitel of Tke Rockefeller I?tstitute for &fed&xl Research.)

(Received for publication, April 25, 1921.)

In a preceding paper it was shown by Thjiitta (1) that B. influenace will grow
on hemoglobin-free medium consisting of plain broth enriched by sterile suspensions
or extracts of mucoid bacteria. It was suggested that under these conditions
growth may be attributable in part to the presence in the bacterial extracts of
substances belonging to the class of so called vitamines.  It was found that the
growth-promoting action of these substances of bacterial origin was not destroyed
by boiling for 10 minutes or by passage through Berkefeld filters.  In the present
paper are presented the facts thus far acquired in the study of the growth require-
ments of the so called hemophilic bacilli: the growth-stimulating action of extracts
of yeast and of vegetable cells, and the importance of blood as a source of growth
accessory substances in the cultivation of bacteria.
The earliest reference in literature to the importance of accessory growth
factors in the cultivation of microorganisms is made by Wildiers (2) (1901).  This
investigator observed that watery extracts of yeast cells greatly enhance the
growth of yeast in a synthetic medium.

In com5rming the observation of Pasteur on the growth of yeast in a medium
composed of mineral salts and sugar, Wildiers found that the size of the inoculum
was of thegreatest importance; a small seeding would not s&ice to initiate growth,
while a larger inoculum grew abundantly in this medium.  This fact suggested
that the growth diEerence lay in some intracellular factor furnished by the heavier
seeding which on the death and disintegration of certain of the yeast cells was
liberated, and supplied to the surviving cells a necessary growth-inducing sub-
stance.  This growth-stimulating principle in yeast to which Wildiers gave the
name "bias" was contained within the cell and could be extracted by boiling
yeast cells in water.  The extract obtained in this manner and concentrated on
a water bath was a yellowish, clear, syrup-like fluid. This intracellular sub-
stance, the so called "bias," was soluble in water and 80 per cent alcohol, and
insoluble in concentrated alcohol and ether.  It was resistant to acids but sensi-
tive to alkalies.  It could be filtered and dialyzed through paratIined paper.
                        97


98           STUDIES ON BACTERIA.L NUTRITION. II

The findings of W;ildiers concerning "bios" were confirmed by Amand (3)
who showed that this substance disappeared from the culture medium during
growth. This observation Amand interpreted as evidence that "bios" was
used up by the yeast cells during growth, and was not a product of their own
metabolism.

The work of Wildiers and Amand seems to have been forgotten until quite
recently when several investigators have adapted the principle of these earlier
workers to methods for the measurement of the vitamine content of various
substances. Bachmann (4) measures the vitamines in the test material by
adding the sterilized substance to yeast cultures and measuring the amount of
gas evolved as an index of growth acceleration; Eddy and Stevenson (S), in
determining the presence of vitamine, compare the numerical increase of cells
in cultures of yeast in a medium with and without the test substance.  On the
other hand, from their experiments on the growth of yeast in synthetic media
without growth accessory substances, McDonald and McCollum (6) conclude
that yeast must be capable either of growing without vitamines or of synthesizing
these substances during growth.

While Wildiers and Amand distinctly pointed out the importance of extracts
of yeast in the nutrition and growth of yeast cells, they did not extend their
observations to the effect of these extracts upon the growth of other micruorgan-
isms, such as bacteria.  It was not until after the discovery of the value of growth
accessory substances in the nutrition of animals that the question of their signifr-
cance in the growth of bacteria was considered. Bottomley (7) showed the
existence in peat of certain substances that have a stimulating effect upon the
growth of plants and soil bacteria.  These substances he called "auximones"
and considered them of the same importance in the growth of plants as vitamines
in the nutrition of animals. These "am&ones" can be extracted from peat
in the same manner as the vitamines from plants, and this fact, together with
their apparently uniform action seems to indicate the close relationship between
these substances.

Following this discovery it was only a question of time when the principle of
growth accessory substances should be brought into full use in the cultivation
of bacteria.

In 1916, LIoyd (8) showed that the meningococcus required for its growth a
rich supply of vitamines in the medium.  Lloyd attributes the value of blood and
serous fluids in the cultivation of meningococcus to their vitamine content and
believes that Cordon's pea flour and Vedder's starch medium may also contain
vitamine which may partly account for the value of these media.  In explanation
of the fact that meningococcus after artificial cultivation tends to grow with
increasing readiness on ordinary media, this author suggests that in adaptation
to a more or less saprophytic existence these organisms become independent of
vitamine supply.

Cole and Lloyd (9) recommend as the optimum medium for gonococci one
rich in amino-acids and vitamines, or "growth hormones."  These authors


TH. THJGTTA AND 0. T. AVERY          99

believe in the existence of two difIerent "growth hormones," a substance present
in blood cells and easily absorbed from the media by filtering, which seems to
induce the initial growth, and a second substance found in tissues of plants and
animals which has the power of inducing luxuriant secondary growth. Davis
(10) pointed out that B. i~~uen~ requires in addition to hemoglo~m a vitamine
substance. He compared these two factors to the fat-soluble vitamine A and
the water-soluble vitamine B. Davis showed that the vitamine supply could
be obtained from plant and animal tissue.  By placing sterile sections of vege-
tables on blood agar seeded with influenza bacilli, Davis demonstrated that
better growth occurred around the pieces of vegetables than on the other portion
of the plates, just as Grassberger many years ago called attention to the more
luxuriant growth of Pfeiffer's bacillus around colonies of staphylococci.

Davis (10) also applied this principle in the cultivation of other bacteria.
As sources of vitamines Davis added to media polished and unpolished rice flour,
white and whole wheat flour.  If these grains were allowed to sprout the growth
was more profuse. The increasing growth on' the media made from sprouting
grain is explaiied as due to enzymatic activity in the sprouting process, resulting
in changes in proteins, and inversion of starch into sugars.

In the preceding paper (1) on the growth of Bacillus injknm
in broth containing sterile bacterial extracts, it was pointed out that
this phenomenon may be explained in part at least by the presence
of growth accessory factors, or vitamines, in the bacterial extracts.
In this paper an attempt is made to analyze further the growth re-
quirements of the so called hemophilic bacilli and to indicate the
significance of growth accessory substances in bacterial nutrition.

IXPERIMENTAL.

Growth Accessory Substances in Yeast and Vegetable CeZEs.

Although extracts from bacteria of the mucoid variety may be
readily prepared, their use is not practicable.  In the present study
extracts were prepared from yeast cells which are known to be rich
in water-soluble vitamine, and which, as previously shown by
Wildiers and Amand, have a marked stimulating effect upon the
growth of yeast cultures. Extracts of fresh ripe tomatoes, green
peas, and green beans were also used, since these vegetables are
also valuable sources of growth accessory substances.


100         STUDIES ON BACTERIAL NUTRITION. 11

Preparation of Extract of Yeast.-100 gm. of brewers' yeast' were
emulsified in 400 cc. of distilled water.  Since the vitamines will
stand boiling better in acid than in alkaline solution, the reaction of
the suspension of yeast cells was adjusted to pH 4.6, boiled over a
free flame for 10 minutes, and then allowed to sediment at room
temperature. The clear supematant extract was pipetted off and
tested for sterility.  The clear, sterile extract, unneutralized, was
stored in the ice box and added to the medium immediately before
use. The extract prepared in this manner was a clear, yellowish
fluid. Chemical analysis showed the following nitrogen content%
total nitrogen, 0.116 per cent; ammonia nitrogen, 0.011 per cent;
amino nitrogen, 0.039 per cent; peptide-bound nitrogen, 0.024 per
cent; undetermined nitrogen, 0.042 per cent. Before addition of
the yeast extract to media, the reaction may be readjusted to opti-
mum for growth of Bacillus in&enzce, pH 7.3 to 7.5.
Preparation of Extract of Tomatoes.-Ripe tomatoes were treated in
the following way: The skin surface was seared with a red hot knife
and through this area a sterile fork was plunged. The tomato was
then dipped in alcohol and flamed, then plunged into boiling water
for a minute, the skin peeled off, and the stem removed with sterile
forceps. The tomatoes were placed in a sterile enamel dish,. and
crushed with a sterile pestle.  The reaction of the tomato juice in
its natural condition was pH 4.2, and therefore required no read-
justment before boiling, as in the case of the yeast emulsion.  The
crushed tomatoes were boiled for 10 minutes and the expressed juice
either filtered through a Berkefeld filter (N) or cleared by centrif-
ugation, stored at its original acidity, and the reaction readjusted
before use. This extract was a perfectly clear, slightly yellowish
fluid. Nitrogen partition on this particular extract was as follows:
tota nitrogen, 0.14 per cent; ammonia nitrogen, 0.014 per cent;
amino nitrogen, 0.079 per cent; peptide-bound nitrogen, 0.00 per
cent; undetermined nitrogen, 0.047 per cent.

`The yeast used in these experiments was supplied through the courtesy of
Mr. F. Spitzner of the Central Brewing Company of New York.
* For the nitrogen determination on both the yeast and tomato extractswe are
indebted to Miss Alma Htier of `the Hospital of The Rockefeller Institute.


TH. THJ6TTA AND 0. T. AVERY          101

Preparation of Extracts of Green Peas and Beans.-Fresh green peas
were prepared by flaming the surface and opening the pods with
sterile forceps and crushing the separate seeds out into a sterile dish.
An equal amount of sterile distilled water by weight was added and the
reaction of the emulsion adjusted to pH 4.6 After boiling for 10
minutes the extract was strained through glass wool and then filtered
through a Berkefeld filter (N). The resulting extract was perfectIy
clear and yellowish in color.  Similar extracts were prepared from
string beans.

Stimulahng Action of V&amine-Like Substances on the Growth of
BaciUus in$uenze.-Yeast and vegetable extracts prepared in the

                       TABLE I.
Stimu&ting Action oj Growth Accessory Subdunces in Ye& and VegetubZe Extracts
          on the Growth of B. in~?uenue.

Dilution of extract
in plain broth.'

1:lO
1:loo
l:l,ooo
1:1o,ooo
Plain broth.

       Growth-stimulating action of.

Yeast extract.      Tomato ~xtrxt.     Extract of peas.

  ++         -t-t         ++
++       ++        ++
  +                         -
                            -
               -            -

* The extract-containing broth was inoculated with 0.05 cc. of the supernatant
fluid of a blood broth culture of B. infEumza.
++ indicates good growth; + moderate growth; f slight growth; - no
growth.

manner described were tested for their stimulating action on bacterial
growth by adding them in varying concentration to plain broth of
pH 7.8. This medium was inoculated with 0.05 to 0.1 CC. of the
supematant fluid of a blood broth culture of Bacillus irzjwz~.  A
bacterial whirl was often visible in the cultures seeded in this
manner after 6 hours incubation, and was always marked after growth
over night.

Titrations of the vegetable extracts were made in infusion broth to
determine the lower limit of the growth-stimulating action (Table I).
Table I shows that even in high dilutions extracts of yeast and


102        STUDIES ON BACTERLkL NUTRITION. II

of fresh green vegetabIes are able to stimulate growth of Bacillus
injuenzcl: in plain broth if seeded with a small inoculum from media
containing blood. When it is considered that the original extracts
contain little nitrogenous matter (yeast extract 0.14 per cent) and
that only one-thousandth of this amount is present in the dilution
required for growth, it becomes obvious that the extracts do not serve
merely as additional nutriment, but that their action is accessory in
nature, similar perhaps to that of vitamines in animal nutrition.
This resemblance is the more striking in that these extracts resist
boiling for at least 10 minutes and are destroyed at autoclave tern-.
peratures (120oC. for 30 minutes) as shown in Table II.

                      TABLE II.
Relative Grmuth Capacity of Yeast and Tomatu Extracts after Boiling and
               Autoclawing.

Temperature.

Tomato extract.    Yeast extract.      NO extract.

Inplain broth. I:lO.*  In plain broth.l:lO.*    Plain broth.

                                                    -
    10 min. at looec.        ++        +i-
    30 "  `1 1joe L`  I      -  I      -  I      -

fInoculated with 0.1 cc. of the supernatant fluid from an 18 hour blood
broth culture of B. in&enzc.

In the literature the statement is frequently encountered that the
growth value of culture media is greatly impaired by filtration.  This
loss is attributed to absorption of the so called hormones during the
process of filtering. In order to test the effect on the growth-stimulat-
ing value of yeast extract after absorption with bone charcoal, the
following experiment was carried out.

In each of two small Basks under sterile precautions 1 gm. of bone charcoal
and 5 cc. of yeast extract (pH 5.4) were mixed. One portion of the mixture was
heated on a steam bath for 15 minutes in order to facilitate absorption; the other
was placed at room temperature and frequently shaken. At the end of the
absorption period the charcoal was removed from suspension by centrifugation
and the absorbed extract tested for its growth-inducing action.  Extract without
charcoal was heated on a steam bath for 15 minutes to determine whether or not
the additional heating affected its potency.  The results are recorded in Table III.


TH. THJijTTA AND 0. T. AVERY          103

From Table III it is apparent that the growth-promoting power of
yeast extract is susceptible to absorption by bone charcoal. It is,
of course, obvious that the completeness of absorption is related to
the concentration of the particular extract and the length of time
allowed for absorption. It is evident that under the experimental
conditions described the &amine-like principle in yeast extract is
absorbed by bone charcoal and that absorption occurs more promptly
under the influence of heat.

From the data presented in the preceding experiments it is evident
that Bacillus injumza will grow when transferred by small inoculum
from blood media to plain broth containing extracts of yeast or vege-

                      TABLE III.
Eject af Absorf~tiun by Bone Churcoa~! on the Gknuth-Stimdating Action of Ye&
                     Extract.

Dilution of        Yeast extract absorbed.              Unabsorbed.
e=%% lain
   J  15 min~~o~,ateam    2 h-3. at room
                      temper*twc.      Unheated.   l5 tiiaz?tc-

             f
1:1o . -         +         ++        ++
  1:20                    f        ++        ++
  1:50                          ++        +

* lnoculated with 0.05 cc. of the supematant fluid of a blood broth culture
of B. influenza.

table cells but fails to grow under similar conditions in the same
broth without the addition of these extracts. However, for reasons
to be discussed later, continued cultivation fails in broth containing
only yeast or vegetable extracts.

While the chemical nature of these growth accessory substances
is not known, they are analogous in behavior to the so called vitamines.
Extracts containing these substances have been prepared from fresh
vegetables and from bacterial and yeast cells. It has been found
that they resist boiling for 10 minutes, that they are destroyed by
autoclaving, that they contain but little available nitrogen, that they
pass a Berkefeld filter with little or no impairment, but are absorbed
from water solution by bone charcoal.


104        STUDIES OF BACTERIAL NUTRITION. II

Go&h Accessory Substances in Blood.

Blood has always been considered requisite for growth of Pfeiffer's
bacillus, and the inability of certain organisms to multiply in the
absence of blood or blood derivatives has constituted an absolute
criterion for their differentiation as hemophilic bacteria.  It is im-
portant at this point to emphasize again the fact previously mentioned
that although Badus injlzceum grows luxuriantly when transplanted
from blood medium to plain broth containing yeast extracts, culti-
vation cannot be continued for more than one or two transfers in
yeast broth alone. Growth deficiency under these circumstances
suggests that possibly some other substance may be carried over
from the original blood culture in an amount sufficient to supplement
the vitamine factor in yeast broth and that growth fails in succeeding
cultures in this medium because this second substance is either-ex-
hausted by growth or lost by dilution on subsequent transfer.  For
purposes of discussion this second substance may be referred to as
the "X" factor, and the &amine-like substance as the "V" factor.
The theoretical consideration of the presence of these two essential
growth factors in blood requires for its substantiation the demon-
stration of the dual nature of the growth-stimulating property of
blood. In the following experiments the interdependence of these
two substances and their distribution in the various fractions of blood,
body fluids, and crystalline hemoglobin will be discussed.

It has already been pointed out in the first part of this paper that
one of the substances, the V factor, can be supplied from a source
other than blood, as for instance, yeast. In explanation, therefore,
of failure of continued growth of Bacillus &fluenz~~ in yeast broth
alone, it is suggested that the X substance lacking in this medium is
supplied in the 6rst instance by the inoculum from the original blood
broth, and that the amount of X furnished in this way is sufficient
when supplemented by an excess of the V factor from yeast to sustain
growth in the first transfer, but that in subsequent cultures the X
factor is quickly lost or perhaps used up by growth of the bacilli.
That this second substance, the so called X factor, is actually carried
over from blood broth with the first inoculum is shown in the following
experiment.


TE. THJijTTA AND 0. T. AVERY          10.5

The red blood cells were sedimented from a blood broth culture of B. influe~~
by slow centrifugation; the supernatant culture fluid was again centrifuged and
the sedimented bacteria were washed three time$ in large volumes of sterile
salt solution to remove any trace of X substance adherent to them.  The washed
bacilli were resuspended in salt solution to the volume of the original culture and
the relative growth capacity of the washed and unwashed bacilli from blood
broth was tested in plain bouillon containing yeast extract in 10 per cent con-
centration.

The facts recorded in Table IV and substantiated in repeated
experiments justify the assumption previously made that in addition
to the V substance in blood which iinds its analogue in yeast extract,
there is also present another substance (X) equally essential to growth

TABLE Iv.

Relative Growth Capacity of Washed and Unwashed In.uenza Bacilli from Blood
           Culture in Yeast Extract Broth.'

10 per cent yeast extract broth (V) inoculated with.

IIlOCUlUIl.

  CL.

Washed bacilli from supernatant  Unwashedbacillifrom supernatant
fluidof blc.4 broth culture      fluid of blood broth culture
      (no Xl.             (Xpreseot).

0.1
0.05
1 loop.

-              ++
-              ++

Controls: 0.1 cc. of supernatant blood broth in plain broth without yeast
extract showed no growth. 1 loop of washed bacteria on blood agar yielded
good growth.

of Bacdlus i+uenzre. This accessory X substagce, to the lack of
which in media hemophilic bacteria are peculiarly sensitive, is capable
in extraordinariIy small quantities, such for instance as may be carried
over in a singIe inoculum from blood broth, of supplying the necessary
growth conditions. Although this second substance, the so called
X factor, can function in minute amounts, it is unable by itself to
induce growth, for, as shown by the controls in Table Iv, the same
inoculum which yields growth in broth containing yeast extract fails
to grow in plain broth alone, although in both instances the amount
of X carried over in the inoculum is the same.


106         STUDIES ON BACTERIAL NUTRITION. II

These two substances may be further differentiated by their relative
susceptibility to heat.  It is known that the clear fluid extracted from
the coagulum of whole blood by short exposure to the temperature of
boiling water supports growth of Budlus~ in$uelaza?. This blood
extract, therefore, presumably contains both the X and the V sub-
stances, and its addition to plain media in small amounts suffices for
growth of hemophilic bacilli.  Since, by boiling for several minutes,
substances can be extracted from blood which still possess the growth-
stimulating properties exhibited by unheated blood, it is evident that
the factors concerned in promoting growth are not destroyed by

TABLE V.

Eject of Heal on the Growik Accessory Subsktnces of Blood.

                                               .~
                   Blow! extract.*             Blood extract.'
Dilution of blood extract       10min. at 100oC.        Aufoclaved at IZOT. for 30 tin.
  in plain broth.      Without yeast  With yeast extract,  Without yeast  With yeeat extract.
                extract.      0.5 cc. (V).      extract.     0.5 cc. (V).

   1:10          -i-i-       4-f               -t-l-
   I:100         ++       ++               -i-i-
  l:l,oGQ                 +         -        +
  1:1o,oKl                              -
  I:l~,~                   -          -

* Prepared by placing a tube containing rabbit blood in boiling water for 10
minutes and using the clear extracted fluid after removal of coagulated materisl
!3y centrifugation.

short exposure to this temperature.  In studying the effect of heat on
the growth-stimulating action of tomato and yeast extracts (Table II)
it was found that boiling for at least 10 minutes did not appreci-
ably impair their action, while exposure in the autoclave to 120oC.
for 30 minutes greatly diminished or completely destroyed their
potency. If blood, therefore, contains a substance analogous in its
behavior to the vitamine-like principle extracted frog yeast, then
blood which has been autoclaved should fail to support growth of
Bu&~lbus &$w~zE, since the V factor would be destroyed under these
conditions. On the other hand, if the X factor of blood is stable
to heat, then the autoclaved blood should be reactivated by the


TH. THJijTTA AND 0. T. AVERY          107

addition of the fresh V substance in yeast extract.  To test the va-
lidity of this assumption the experiment presented in Table V was
carried out.

From Table V it is evident that the accessory substances in blood
essential to growth of hemophilic bacilli are not destroyed by ex-
posure to 100oC. for 10 minutes, since extracts prepared from boiled
b!ood are active in promoting growth. It is further apparent that
these same blood extracts after autoclaving for 30 minutes at 120oC.
are no longer capable by themselves of supporting life of the bacilli.
The experiment further demonstrates the interesting fact that the
substance or substances in blood which are destroyed by excessive
heat can be replaced by the addition of the growth-stimulating prin-
ciple extractable from yeast. An analysis of these observations on
the heat sensitiveness of the growth-promoting substances in blood
indicates clearly that differences exist in thermostability on the basis
of which it is possible to separate these two factors.  The X factor,
so called, is heat-stable and unaffected in action by exposure to
steam under pressure at 120oC. for 4 hour.  On the other hand, the
so called V factor of blood, like the similarly reacting substance in
extracts of yeast, is relatively more labile and is destroyed at the
autoclave temperature.

Before passing to the question of the distribution of these growth
accessory substances in the various fractions of blood, it is well to
emphasize the peculiar sensitiveness of the hemophilic bacilli to a
lack of either of these two factors in media, and the relative differences
in the effective amounts of each.  It has been pointed out (Table IV)
that in a small inoculum from blood media, a sufficient amount of the
X substance may be carried over to cause growth in blood-free broth,
providing yeast extract, which supplies the V factor, is also added.
In other words, mere traces of the X substance in the presence of this
Amine-like principle suf3ce for growth, but neither separately can
function even when present in excess. This dual action, and the
relative amounts of each factor necessary, particularly the minimal
effective dose of X, are important in the technique of determining the
presence or absence of either factor, since it is possible to carry over
with the inoculum enough of the particular factor lacking in a test
medium to permit growth.  The nature of the seeding, therefore, must


108         STUDIES ON BACTERLkL NUTRITION. II

be carefully chosen so that the culture from which it is taken shall con-
tain only a minimum of the particular factor sought for in any given
medium or extract.  For example, when testing for the presence of
the V factor in a given extract or medium, the seeding shouId be made
from the supernatant fluid of a blood broth culture, since in this
instance an effective amount of the X substance is carried over with
little or no V factor.  On the other hand, in demonstrating the pres-
ence of the X substance in blood or a derivative of blood, the medium
in question should be inoculated from a yeast extract broth culture
derived as above, since under these conditions a minimal and in-
effective amount of X is transferred in the inoculum.  In testing for
the X substance an excess of the V factor should always be added in
the form of yeast extract, or its equivalent.
Distribution of the Growth Accessory Gbstances ifs Blood and Blood
Derivatives.-&4 comparative study of the distribution of these growth
accessory factors in blood and blood derivatives was made by determin-
ing the presence or absence of growth of Bacillus &j.%enzre in plain
broth enriched with graduated amounts of ascitic fluid, serum, blood
extract, and solutions of Iaked red blood cells and of crystalline
hemoglobin. The sterile ascitic fluid, untinged with hemoglobin,
was obtained from a patient suffering from cirrhosis of the liver and
had been stored without antiseptic in the ice box for several months
prior to use.  The serum, blood extract, and solution of laked blqod
cells were prepared from freshly drawn defibrinated rabbit blood.
The serum was separated from sterile defibrinated blood by repeated
centrifugation, care bein g taken to obtain a specimen with no visible
traces of blood pigment.  The blood extract consisted of that fraction
of whole blood expressed from the coagulum after boiling for 10
minutes, and separated from the coagulated proteins by prolonged
centrifugation.

The solution of laked red blood cells was made as follows:  The red
cells from 20 cc. of sterile defibrinated blood were removed by centrif-
ugation, washed three times in sterile salt solution (50 cc. each time),
and taken up in sterile distilled water to the original volume of blood.
After laking, the cell residue was sedimented by centrifuging and the
clear supernatant fluid pipetted off and used as hemoglobin solution.
By gasometric analysis 1 cc. of this solution contained 0.1 gm. of


TH. THJijTTA AND 0. T. AVERY          109

hemoglobin. The crystalline hemoglobina was prepared by the
method of Weker and Williamson (11) from ox blood.  The crystal-
line hemoglobin, as is usual with dry preparations, had lost its oxygen-
carrying capacity. A water solution of the crystals (10 per cent by
weight) was rendered sterile by passage through a Berkefeld filter (N).

TABLE VI.

Distribution oj the Growth Accessmy Substances V and X in Blood and Blood
                 Derivatives.

Dilution in plain broth.*

1:2          - ++
1:s         - ++
1:lO
1:100          - - -
l:l,Qoo
1:1o,ocKl              _ -' - -
1:100,000             - -
1:2oO,ooo

* All tubes inoculated with 0.05 cc. of an 18 hour yeast extract broth culture
of B. inflwwe.

t Contained 10 gm. of hemoglobin per 100 cc.
$ Contained 10 per cent crystalline hemoglobin.
8 Represents a final concentration of 1: l,OOO,OOO hemoglobin.
1) Represents a final concentration of 1:2,000,000 crystalline hemoglobin by
weight.

The ascitic fluid, serum, and blood derivatives prepared as described
above were added to plain infusion broth in the dilutions indicated
and inoculated with 0.05 cc. of an 18 hour yeast extract broth culture
of Bacillus &&uenza.  Growth was controlled by subculture on blood
agar and by second transfer to yeast extract broth alone.

a We are iodebted to Dr. J. P. Peters, Jr., of the Hospital of The Rockefeller
Institute for the preparation of crystalline hemoglobin used in these experiments.


110         STUDIES ON BACTERIAL NUTBITIOE;. II

An analysis of Table VI reveals certain facts concerning the distri-
bution of the growth accessory factors in blood and bIood derivatives.
In the first place, it is evident that Bacillus &JZU~~ZZCE will not
grow in serum in concentration as high as 10 per cent in broth or
in ascitic fluid diluted with equal volumes of broth.  In the presence
of yeast extract, however, growth occurred under otherwise identical
conditions in ascitic fluid broth 1: 5 and in serum broth 1: 1,000.  Sec-
ondly, blood extract and hemoglobin solution freshly prepared from
laked red cells in broth were able to stimulate growth in dilutions as
high as 1: 100 and 1: 1,000 respectively.  The addition of yeast extract
to broth containing these blood derivatives permitted growth in even
higher dilutions; namely, 1: 1,000 of blood extract, and 1: 100,000 of
the solution of laked cells, Finally, the addition of a. solution of
crystalline hemoglobin to broth in concentrations equivalent to the
solution of laked red cells failed to yield growth, while in dilutions
equivalent to 1: 200,000 of laked cells the same solution of crystalline
substance afforded excellent growth when yeast extract was added.
It shouId be noted that for purposes of comparison the concentrations
are expressed in Table VI in terms of dilution of the original body
fluids or their equivaIents.  However, since in the solutions of laked
cells and of crystalline hemoglobin the content of this substance by
weight is 10 per cent, the actual dilution of hemoglobin sufficing for
growth is, therefore, ten times greater than that recorded in the
protocol. In terms of actual amount of crystalline hemoglobin by
weight, these figures represent concentrations of 1: 100 and 1: 2,000,OOO
respectively.

In attempting to determine the distribution in blood of the so
called growth accessory factors V and X, it must be borne in mind that
the methods employed are not strictly comparable and that the results
are merely relative and of necessity must vary with each individual
specimen of serum or blood. Nevertheless, the growth differences
are sufficiently great to make it apparent that the greatest concentra-
tion of both these substances is associated with the cell fraction of the
blood: and that serum and ascitic fluid, on the other hand, contain
little or no measurable quantity of the vitamine-like principle and
only relatively small amounts of the X substance, as is evident from
the fact that by themselves they are separately incapable of support-


TH. THJijTTA AND 0. T. AVERY          111

ing growth. The quantity of X substance in pure serum is rela-
tively slight as compared with its abundance in solutions of Iaked cells
and crystals of hemoglobin. This fact makes it seem not unlikely
that the red blood cell is the source of this substance and that the
presence of X in serum or ascitic fluid is purely accidenta1, due to
conditions which permit its escape from the blood cells.  The activ-
ity of this substance in crystalline hemoglobin in dilutions as great as
1: 2,OOO,OOO by weight is strong evidence that it is some constituent
of the red blood cell which may function as a catalytic agent. It
must be borne in mind that the detection of the X substance by
itself in solutions of crystalline hemoglobin, in ascitic fluid, and in
serum is made possible only when these are used in combination with
vitamine-containing extracts from yeast or from other extraneous
sources, since the X substance alone is inactive and unable to
support growth without the complementing V factor.

In attempting to interpret the facts brought out in Table VI, it is
of further interest to observe the distribution and relative concentra-
tion of V factor, or vitamine-like principle, in the various fractions of
blood and blood derivatives.  Just as the X substance appears to be
intimately associated with the cell fraction of blood, so the V factor
is apparently found in greatest concentration in the blood corpuscles.
Because of the greater susceptibility of the V factor to heat and chemi-
cal manipulation, and owing to the fact that it is present in blood in
lower concentration than the X substance, this vitamine-like principle
is active only when the intact red cell and solutions or extracts of
these cells have not been subjected to untoward conditions. For
example, the chemical procedures incident to'the preparation of crys-
talline hemoglobin cause complete loss of this factor, while the more
stable X substance remains unimpaired. Like the V factor, ex-
tractable from yeast and vegetable cells, the corresponding substance
in blood extracts withstands boiling for at least 10 minutes, but is
destroyed by exposure to 120oC. for 30 minutes, neither of which
procedures, however, interferes with the peculiar properties of the X
substance in blood. In the specimen of blood extract used in this
experiment, the V factor exerted its growth-stimulating action in
dilution of I : 100, and in the unheated solution of laked cells, in which
the hemoglobin was physiologically active as shown by its oxygen-


112        STUDIES ON BACTERIAL NUTRITION. II

carrying capacity, the V factor was present in dilutions as high as
1: 10,000. On the other hand, as already noted, solutions of crys-
talline hemoglobin were devoid of vitamine-like property and re-
quired the addition of yeast extract to supplement the X substance
present.

DISCUSSION.

-4s already pointed out, the importance of growth accessory sub-
stances in the cultivation of bacteria has been appreciated by num-
erous observers, and in the case of the hemophilic bacilli has been
emphasized particularly by Davis. The foregoing experimental data
are presented, therefore, not merely to direct attention to the relation
of these substances to the growth of Bacillus in&enzLe, but rather
with the hope that as these studies progress, they may furnish the
basis for a more accurate understanding of bacterial nutrition, and
that the principles involved may find wider application in the culti-
vation of organisms other than those of the hemophilic group.

In preceding papers (1, 12) it has been shown that the substances
requisite for growth of Bacillus influenza can be supplied from a source
other than blood. The mucoid material elaborated during growth of
certain bacteria, together with the dead bodies of these organisms in
hemoglobin-free broth, has been found to furnish the accessory
substances necessary for the cultivation of the hemophilic bacilli.

The present paper concerns itself with an attempt to analyze the
accessory factors in blood which have to do with the pecuIiar nutri-
tional requirements of the hemophilic bacteria.  It is shown that the
growth accessory substances in blood involve two distinct and separ-
able factors, neither of which alone suffices to stimulate growth.
One of these factors is analogous in its behavior to substances belong-
ing to the class of so called vitamines, and because of this similarity
is referred to in the text as the V factor. The other factor is Iess
easily defined and is spoken of as the X substance.  On the basis of
relative differences in susceptibility to heat, these two factors may
be separated one from the other. The vitamine-like principle in
blood is destroyed by exposure to a temperature of 120oC. in the
autoclave, while the X substance resists heating under these condi-
tions. These substances, however, remain unimpaired in blood sub-


TH. THJijTTA AND 0. T. AVERY          113

jetted to the temperature of boiling water for 10 minutes.  Extracts
expressed from the coagulum of boiled blood contain both these
factors and are capable, therefore, of supporting growth of Bacillus
irtfluenza. On the other hand, autoclaved blood contains only the
more stable X substance and is incapable by itself of stimulating
growth unless reactivated by the addition of the V factor from another
source. That the vitamine-like substance in blood can be supplied
from other sources is shown by the fact that extracts of yeast, toma-
toes, green peas, and beans possess the property of reactivating an
otherwise inert medium containing only the X substance.

Furthermore, it is shown that the addition to plain broth of active
extracts of yeast or vegetable cells in dilutions as high as 1: 1,000
sm%ces to initiate growth of Bacillus &$uenzce when seeded from
blood media. However, the fact that cultivation cannot be con-
tinued for more than one or two transfers in yeast extract broth alone
suggests that in the first instance some of the X substance is carried
over in the inoculum from the original blood culture in an amount
sufficient to supplement the yeast broth and that growth fails in
succeeding cultures because this X factor is either exhausted by growth
or lost by dilution on subsequent transfer.

Moreover, study of the relative distribution of these two factors
in the constituents of blood demonstrates the fact that they are pres-
ent in greater concentration in the cellular elements than in the
serum or plasma. The facts so far acquired indicate that the red
blood cell is the carrier of the vitamine-lie principle and that this
substance, like its analogue in yeast cells, is intracellular in nature
and can be extracted by the methods described. While the chem-
ical nature of this substance is not known, it is presumably analogous
to the so called vitamines.  It has been found that similar growth-
accelerating substances capable of replacing the V factor in blood can
be extracted from bacterial and yeast cells and from fresh vegetables.
These extracts contain but little nitrogen, they are destroyed by
autoclaving, they are water-soluble, they pass a Berkefeld filter, but
are absorbed readily from heated solutions by bone charcoal.

The experimental data recorded above indicate that in blood at
least the X substance is intimately associated with or a derivative
of hemoglobin.  The fact that the X factor in crystalline hemoglobin


114          STUDIES ON BACTERIAL NUTRITION. II

can function in dilutions as high as 1: 2,000,000, when supplemented
by an excess of the V factor from yeast extract, suggests that it may
act as a catalytic agent.  Further observations on the nature of the
X substance in blood and its presence in material other than animal
tissue will be presented in a subsequent paper.

COxcLUSIONS.

The hemophilic bacteria of which Bacillus i@uenm serves as a
type require for their growth two distinct and separable substances,
both of which are present in blood and neither of which alone suffices.
These substances are (a) a vitamine-like substance which can be ex-
tracted from red blood corpuscles, from yeast, and from vegetable
cells, which is relatively heat-labile and absorbed from solution by
certain agents; (b) a so called X substance which is present in red
blood cells, is heat-stable, and acts in minute amounts.

BIBLIOGRAPHY.

1. ThjGtta, Th., J. Exp. n-led., 1921, xxxiii, 763.
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6. McDonald, M. B., and McCollum, E. V., J. Biol. Chem., 1921, xlv, 307.
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9. Cole, S. W., and Lloyd, D. J., J. P&h. and Bacl., 1917, xxi, 267.
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