STUDIES ON BACTERIAL NUTRITION.

IV. EFFECT OF PLANT TISSUE UPON GROWTH OF P~~u~ococcus AND
               STREPTOCOCCUS.

    BY H. J. MORGAN, M.D., AND 0. T. AVERY, M.D.
(From the Hospital of The Rockefeller Institute for Medical Research.)

(Received for publication, April 12, 1923.)

Study of the growth requirements of Bacillus injhenza! (l-4) indi-
cated that for the cultivation of this organism at least two substances
must be present in the culture medium. Both of these substances
are present in red blood corpuscles.  One, a vitamine-like substance,
can be extracted not only from red blood corpuscles but from yeast and
vegetable cells as well, and is relatively thermolabile.  This substance
has been called the V factor.  The other substance is also present in
red blood corpuscles and in plant tissue, but it is thermostable and is
effective in amounts so small as to suggest the possibility that it
functions as a cat'alyst.  It has been called the X factor. These
observations on the growth of Bacillus influenzce in the absence of
blood have been extended (5).  The organism can be readily grown
in plain broth in which small pieces of sterile, unheated yellow or
white turnip, carrot, beet, parnsip, or sweet potato have been placed.
Knowledge of the growth-stimulating property of unheated plant
tissue for organisms as sensitive to cultural conditions as the bacteria
of the hemophilic group encouraged an extension of the study to other
groups of bacteria.  The present paper presents the results of a study
of the effect of plant tissue upon the growth of certain Gram-positive
cocci.

Material.

Media.-The plant tissues used in this study were white potato,
sweet potato, carrot, white turnip, yellow turnip, parsnip, beet, and
banana.  Care was observed in selecting fresh young specimens.
With the same technique described in a previous communication (4),
                       207


208         STUDIES ON BACTERIAL NUTRITION. IV

sterile pieces of these vegetables were placed in phosphate broth of
pH 7.8. When air is not excluded from media prepared in this way
oxidation of the plant tissue results in brown discoloration of the
vegetable and of the supematant broth.  To prevent this the media
were sealed with sterile Vaseline.  The media could then be stored in
the ice box for many weeks without discoloration.

Organisms.-Three strains of pneumococci, three of hemolytic
streptococci, and five of non-hemolytic streptococci were employed in
these experiments.

Grouth of Pneumococcus in Media Contailtirtg Sterile Plant Tissue.

The rate of growth of pneumococcus in broth is dependent upon
several factors, the most important of which are (a) the growth activity
of the bacteria used for seeding (6), (b) the size of the inocula, and (c)
factors pertaining to the media such as its pH (7), its content in
nutritive substances, etc. Depending upon the balance between
these factors, growth as determined by the number of viable organisms
present at different intervals during the life of the culture is divisible
into four periods: (a) period of lag, during which the number of viable
organisms in the culture decreases, remains constant, ,or slowly
increases; (b) logarithmic period, in which the culture maintains its
maximum rate of growth; (c) stationary period, in which the maximum
rate of growth is not maintained, the organisms present, though
viable, reproducing more slowly or not at all; and (d) period of decline,
in which the number of viable organisms decreases rapidly, the
pneumococcus culture eventually becoming sterile (6).

Eject of the Addition of Sterile Unheated Potato on the Growth of

Pneumococcus.

With the above facts in mind, an experiment was planned to show
what effect the addition of sterile unheated plant tissue to fluid media
would have on the growth of pneumococci.  The period of lag was
first studied. Plain broth containing sterile unheated potato was
used as the test medium.  As a control, broth to which 1 per cent of
dextrose had been added, was employed. Dextrose is readily fer-
mented by pneumococcus, and is known to accelerate the growth of
this organism.


H. J. MORGAN AND 0. T. AVERY         209

Experiment l.-Into a 300 cc. flask containing 150 CC. of phosphate meat infusion
broth (pH 7.6) were placed four cylindrical pieces of unheated, sterile potato,
each about 1.5 cm. long and 0.5 cm. in diameter.  The tlask was then incubated
at 37oC. to insure sterility.  A control flask, containing 1.50 cc. of broth of the same
lot, to which 1 per cent dextrose had been added, was prepared.  With the media
at 37oC. each flask was seeded with 0.1 CC. of a 6 hour broth culture of Pneumococ-
cus Type I.  The culture used for seeding was actively growing, as indicated by
the presence of long chains in the stained &lm.  At frequent intervals over a period
of 9 hours, fractions of both the dextrose broth and the potato broth cultures were
removed and estimations made of the number of viable organisms present in each.
In making these estimates the following method was employed.  Several accurate
dilutions of the culture were made.  0.5 cc. from each of two or more of these
dilutions was added to a tube of 1 per cent dextrose agar and plates were poured.
The necessary calculations for the different dilutions having been made, the average

TABLE I.

.Acceleraiion of Growth of Pneumococcus in Broth Containing Sterile Unheated

Time after seeding.

krs.

0
1
2
3
4
5
6
7
9

Potdo.

Log of No. of colonies per 0.5 cc. of culture.

Plain broth + potato.

3.7
4.0
4.2
4.9
5.1
5.8
6.3
7.1
7.9

Plain broth + 1 per cent dextrose.

3.8
4.1
4.0
3.8

4.0
4.0*
4.0*
4.0*

* No colonies in the dilutions plated.  Therefore the exact determination was
not made.  The log is less than 4.

of the number of colonies present in the plates after 48 hours at 37oC. was used for
the colony count per 0.5 cc. of culture for that hour.  The results of these estima-
tions as expressed in the logarithms of the number of colonies present at the time
of each examination, are given in Table I.  By using these logarithms as ordinates
and the hours of examination as abscissae, Text-fig. 1 was constructed.

From Table I and Text-fig. 1 it is evident that the organisms in
broth containing sterile unheated potato grew steadily and at a con-
stant rate.  There occurred no period of lag.  In contrast with this,


210        STUDIES ON BACTERIAL NUTRITION. IV

organisms seeded into broth containing an abundance of an easily
fermentable substance, dextrose, but PO plant tissue, failed to attain
this maximum rate of growth for a period of at least 9 hours.

A second experiment was now performed in order to extend these
observations and to determine the effect of the presence of unheated
plant tissue upon the rate of growth during the entire cycle of a
pneumococcus broth culture and also the effect upon changes in

reaction which

usually occur in the medium during growth. It is
                                         .

8

m-IO 2 4 6 8 IO
  Tnne after inoctiaticm

TEXT-FIG. 1. Influence of sterile unheated plant tissue on the acceleration of
growth of pneumococcus. A indicates less than 4.

known that during growth of pneumococci in broth an increase in the
pH occurs.  When the medium is enriched by the addition of readily
fermentable substance;, as dextrose or certain other sugars, the acid
productiontby the growing pneumococcus is still more marked (8).

Er@sGnent Z.-The medium was prepared as in Experiment 1.  Two 300 CC.
flasks, one containing 150 cc. of 1 per cent dextrose broth, the other 150 cc. of
plain broth to which had been added about 5 gm. of sterile unheated potato, were
seeded with small identical amounts of a growing broth culture of Pneumococcus
Type I. At intervals, as noted in Table II, samples were removed from each of
the ilasks. Estimations of the number of viable organisms were made, as in
Experiment 1. Determinations of pH, by means of the calorimetric method, were
made at the same time.  The results of these observations are shown in Table II,
and are graphically rcpresznted in Text-fig. 2.


H. J. MORGAN AND 0. T. AVERY         211

From Table II and Text-fig. 2 it is seen that, under the conditions
of the experiment, no period of lag occurred in the broth culture con-
taining unheated potato.  The control culture in dextrose broth, a
medium quite favorable for prompt and abundant growth of pneu-
mococcus when the inoculum is sufficiently large, showed a period of
lag of 18 hours, during which the viable organisms in the culture
apparently decreased in number.  However, when once the culture

TABLE II.

Comparison of Growth of Pneumocotcus in Potato Broth and Dextrose Broth.

The after seeding.

krs.

1
2
3
4
5
6
8
9
11
13
15
18
28
30
32
34
36

Broth containing unheated potato.

I.08 of No. of
colomes per 0.5 a.
  of culture.

5.1

.%$?5
IL r: 5f Actual number of colonies not determined.

4.5
5.0
5.5
6.1
6.7
7.3
8.2
8.1
8.1
8.1
8.1
8.0
5.8
5.1

pH of culture.

Bmth containing 1 per cent dextrose.

Log of No. of
colonies per 0.5 cc.
  of cllltllre.

4.2


3.3

2.3

2.3

2+*
2+*
2.3
6.5
7.4
8.4
8.7
4.1

pH of culture.

7.7

7.7


7.65

7.6

7.6
7.1
6.3
5.3

entered upon the logarithmic phase of growth, the generation time
was approximately the same in the dextrose broth culture as in the
potato broth culture; that is to say, cell division, when once initiated,
proceeded at about the same rate in the two cultures. It is note-
worthy that the stationary phase of the pneumococcus culture in
potato broth extended over a period of 10 hours, while that of the
culture in dextrose broth was of extremely short duration.


212       STUDIES ON BACTERIAL NUTRITION. IV

Other observations have shown that in the potato broth culture
there is a prolongation of the period of decline in the growth curve;
that is, the period in which cell death is proceeding at a more rapid
rate than multiplication. It is especially prolonged when compared
with a culture in dextrose broth. Moreover, when young actively
growing potato broth cultures are removed from the incubator and are
placed on ice, the bacteria may remain viable for very long periods

nr;, 0 2 4  6  6  10  12  14  16  18  20  22  24  26  28  30  32  34 36
            Time apter inoculation

TEXT-FIG. 2. Influence of sterile unheated plant tissue on the growth curve
of pneumococcus.                                         ,  /
                                                    T& -.a,
of time, even 8 months. These facts indicate that not only is `the
initiation of growth accelerated in the potato broth, but death of the
bacteria occurs more slowly in this medium.

EJect of the Addition of Other Sterile Unheated Plant Tissues on the
         Grozuth of Pneumococcus.

The following experiment illustrates the effect on growth of pneumo-
coccus when vegetable tissue, other than potato, is added to p'.ain
broth.


H. J. MORGAN AND 0. T. AVERY         213

Experiment 3.-Sterile pieces of parsnip, sweet potato, carrot, yellow turnip,
white turnip, and banana, each weighing about 0.5 gm., were placed in tubes con-
taining 10 cc. of plain broth and these were sealed with Vaseline.  After incubating
several days to insure sterility, the seals were removed and 2 cc. of the broth were
taken from each tube for calorimetric pH determinations.  Each tube was then
seeded with 0.05 cc. of an actively growing broth culture of Pneumoccocus Type
II. A tube containing 8 cc. of broth of the same lot, but no vegetable tissue, was
seeded with the same amount of the same culture.  The tubes were placed in the
incubator at 37oC.  Observations on the degree of clouding present in the media
were made after 5 hours, and again after 17 hours incubation.  The results are
given in Table III.

TABLE III.

Acceleration of Growth of Pneumococcus in Broth Containing Sterile Unheated
              Vegetable Tissue.

Broth.

cc.

8
8
8
8
8
8
8
8

7-

--









-

Plant tissue.

None.          7.8
Parsnip.          6.3
Banana.          6.4
Sweet potato.       6.7
Carrot.           6.9
Yellow turnip.      7.1
White "        7.2
Potato.          7.4

-

pH at time
of seeding.

ZZ
I

-

IWCUlU~.



cc.
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

=
I

-

             -
Growth of pneumccoccus:

5 hrs.

+:+
++
t-f
++
+T+

-


_-









-

17 hrs.

t-l-
++++
++++
+-I-++
+++-l-
++++
++++
+++-I-

*- indicates no macroscopic growth; *, faint haze confined to vicinity of
plant tissue; +, faint clouding throughout media; ++, moderate clouding
throughout media; + + f, marked clouding throughout media; + + ++,
maximum growth attained.

From Table III it is evident that other plant tissues have the same
growth-stimulating effect upon pneumococcus that potato possesses.
It is also evident that this stimulation operates in spite of the fact
that the initial reaction of the media may be unfavorable for growth
of this organism.

The Limiting pH for the Growth of Pneumococcus in Potato Broth.-
It has been shown that the optimum hydrogen ion concentration for
growth of pneumococcus is pH 7.8 and that growth in plain broth


214          STUDIES ON BACTERIAL NUTRITION. IV

can be initiated only if the hydrogen ion concentration is be-
tween pH 7 and 8.3 (7). The observations made in Experiment 3
indicate that if there is present in the medium a bit of unheated
vegetable tissue growth of pneumococcus can be initiated even
though the hydrogen ion concentration is only pH 6.3.  An attempt
was made, therefore, to determine the limiting hydrogen ion concentra-
tions for the initiation of growth of pneumococcus in broth con-
taining sterile unheated potato.

TABLE IV.

Growth of P?teumococcus at Diferent Hydrogen Ion Concentrations in Broth Contain-

         -   -
ing Sterile Unheated Plant Tissue.

Indum.


  cc.
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

pH of media.

Growth of pneumococcus
in plain broth after
    18 hrs:

5.8
6.2
6.8
7.1           +
8.3           +
8.6
8.8              -

9.0              -

*- indicates no macroscopic growth; +, growth.

Growth of pneum-
in plain broth + potato
   after 18 hn:

6.0   6.2  6.4  6.6   6.8  7.0   7.2  7.4  7.6  7.8  8.0  8.2  8.4  8.6  8.8  9.0  92
                  PH

TEXT-FIG. 3.  Influence of sterile unheated plant tissue on the range of hy-
drogen ion concentration for initiation of growth of pneumococcus.

Experiment 4.-Tubes containing 10 cc. of potato broth were prepared in the
usual way and incubated to insure sterility.  In a number of tubes the hydrogen
ion concentration was adjusted, by the addition of suitable amounts of sterile N
and 0.1 N HCl or NaOH, so that a series of tubes was obtained in which the reaction
ranged between pH 5.8 and 9.  In the same way there was prepared a series of
tubes of plain broth, without potato, in which the hydrogen ion concentration
ranged between the same points.  All these tubes were then seeded, each with


H. J. MORGAN AND 0. T. AVERY         215

0.05 CC. of a growing culture of Pneumococcus Type I, and incubated at 37oC.'
Growth of pneumococcus occurred as is recorded in Table IV, and as represented
diagrammatically in Text-fig. 3.

The results in Table IV indicate that the presence of sterile unheated
potato in broth greatly increases the range of pH in which the growth
of pneumococcus can be initiated.  This increased range extends
between pH 6.2 and 8.8.

E'ect of the Addition of Sterile Unheated Potato on the Growth of
      Hemolytic alzd Non-Hemolytic Streptococci.

In order to determine whether unheated plant tissue might exert a
growth-stimulating effect on other cocci, strains of hemolytic and of
non-hemolytic streptococci were grown in plain broth alone and in the
same broth to which unheated plant tissue had been added.  Differ-
ences in growth activity of the same organisms in the two types of
media were noted.

TABLE V.

Acceleration of Growth of Hemolytic and Non-Hemolytic Streptococci in Broth
         Containing Sterile Unheated Plaftt Tissue.

OrgCUlhlS.                Growth in 10 cc. of   Growth in 10 cc. of
                    plain broth:     potato broth:

    Results after 14 hrs. incubation.

1. Hemolytic Streptococcus 3/22. ........... _.
2. Li       "    23/20 ..............
3. "       `I    84/18. ..............

Reklts after 18 hrs. incubation.

4. Non-hemolytic Streptococcus A141. . . . . . . . . .       -
5. "         `I     A13.5. _ . . . . . . . .       -
6. "         "     B39............    + ,   +++
7. "         `L     38d.............       *        +++
8. I`         "     A149. . . . . . . . .       *        ++

*- indicates no macroscopic growth; *, faint haze; +, bacterial whirl; + t,
marked growth; + + +, maximum growth.

r A set of uninoculated tubes containing potato broth at reactions between pH
7.2 and 4.6 was also incubated to determine whether plant tissue alone, at 37"C.,
might cause a change in the pH. After 3 days at 37oC. the pH determinations on
the broth in these tubes showed that no changes had occurred.


216         STUDIES ON BACTERIAL NUTRITION. IV

Exjwiment 5.-The media were incubated for 3 days to insure sterility.  24
hour blood broth cultures of three strains of hemolytic streptococci and five strains
of non-hemolytic streptococci were used for seeding.  In each instance the tip of a
platinum wire was inserted the distance of 1 cm. into the supernatant fluid of the
blood broth culture and then carried into the test medium.  The latter was then
placed in the incubator at 37oC.  The degree of clouding of the medium was noted
at the hours given in Table V.

From the results shown in Table V it is evident that sterile unheated
plant tissue has a marked growth-stimulating effect upon hemolytic
and non-hemolytic streptococci.  Heavy clouding of the potato broth
cultures of the three strains of Streptococcus hamolyticus and, to a less
degree, of two of the five strains of non-hemolyzing streptococci
occurred before there was macroscopic evidence of growth of these
organisms in the plain broth without potato. In the remaining
cultures of non-hemolytic streptococci at the end of 18 hours growth
was much more abundant in the potato broth tubes than in the
tubes of plain broth.

SUMMARY.

In previous papers it has been shown that unheated plant tissue, in
the form of potato, contains the two factors necessary for the growth
of organisms of the hemoglobinophilic group. Further studies (5)
confirmed these findings and showed that yellow and white turnip,
carrot, beet, parsnip, and sweet potato can replace blood in the
cultivation of Bacillus influertz~.

In the present paper it has been shown that vegetable tissues also
greatly facilitate and stimulate the growth of other organisms entirely
unrelated to Bacillus i@uenzte.  Three varieties of Gram-positive
cocci have been used in the present study, pneumococcus, .%`eptococcus
Jzamolyticus, and Streptococcus viridans. With pneumococcus it has
been previously shown that prompt and luxuriant growth will occur
in broth containing unheated potato even though the seeding be so
smaI1 that no growth whatever will occur with the same seeding in
plain broth (5).  In the present study it has been shown that even in
dextrose broth this minimal inoculation is foIlowed by a proIonged
period of lag, whereas in potato broth this same inoculum serves to
initiate immediate and rapid growth.  When pneumococci are grown
in potato broth not only is the period of lag abolished, but the sta-


H. J. MORGAN AND 0. T. AVERY         217

tionary period of growth is extended and cell death is delayed.  More-
over, in plant tissue medium the zone of hydrogen ion concentration
within which growth of pneumococcus can be initiated is consider-
ably extended beyond the acid and alkaline limits of the optimal
range in ordinary bouillon.

It has been found also that the presence of unheated plant tissue in
the media likewise stimulates growth of hemolytic and non-hemolytic
streptococci.

In this investigation no attempt has been made to determine the
exact nature of the substances in plant tissue upon which these
properties depend. That they are not of the nature of readily
fermentable carbohydrates, however, is made evident by the fact that
no increased production of acid occurs in the pneumococcus culture
when potato is present.

CONCLUSIONS.

1. The initiation of active growth in broth culture of pneumococcus,
Streptococcus hamolyticus, and Streptococcus tiridans is accelerated by
the presence of unheated plant tissue.
2. Cultures of pneumococcus in broth containing unheated plant
tissue show a prolongation of the stationary phase of growth.
3. Death of the individual organisms in pneumococcus broth cul-
tures containing unheated plant tissue does not proceed so rapidly as
in cultures without plant tissue.
4. The zone of hydrogen ion concentration `within which Lgrowth of
pneumococcus in ordinary broth can be initiated is considerably
extended, both on the acid and on the alkaline side, by the addition
of unheated vegetable tissue to the media.

BIBLIOGRAPHY.

1. ThjGtta, T., J. Exp. Med., 1921, xxxiii, 763.
2. Thjiitta, T., and Avery, 0. T., Pm. SOG. Exp. Biol. and Med., 1920-21, xviii,

197.

3. ThjGtta, T., and Avery, 0. T., J. %p. Med., 1921, nxiv, 97.
4. ThjGtta, T., and Avery, 0. T., J. Ezp. Med., 1921, xxxiv, 455.
5. Avery, 0. T., and Morgan, H. J., Proc. Sot. Exp. Biol. and Med., 1921-22, xix,

113.

6. Chesney,A. M., J. Exp. Med., 1916, xxiv, 387.
7. Demby, K. G., and Avery, 0. T., J. Exp. Med., 1918, xxviii, 345.
8. Avery, 0. T., and Cullen, G. E., S. Exp. Med., 1919, xxx, 359.