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: Gan 10 1998.

 

 
The author wishes to acknowledge the

kindly suggestions of Mr. C. W. Brown whose

sympathetic supervision has been of great

assistance in this investigation.
VIABILITY OF PSEUDOMONAS RADICICOLA UNDER

AEROBIC AND PARTIAL ANAEROBIC

CONDITIONS.

By

F. 0. Ockerblad

June, 1916.
THESIS”

 
TABLE OF CONTENTS.

Introduction and purpose.

Literature review.

Methods.

Experimental work.
a. Viability in culture bottles.

b. Tolerance of acids and alkalis.
c. Thermal Beath Point.

Discussion of results.
Summary.

Literature cited.

165202

<r
INTRODUCTION AND PURPOSE.

The work with Ps. radicicola up to the present

time deals largely with the relation of the organiam to
its host plant; whether it is a parasite or a beneficial
intruder, and with its mode of entrance into the plant.

Another side of the problem which has been studied to a
considerable extent of recent years is the study of the
organism in the soil. The thermal death point has been
determined, but it has not been shown whether the various
strains have a common or a different thermal death point.
It is not the intention of the author to give an
exhaustive study of Pseudomonas redicicola under various
conditions but rather to present the results of his work
on the viability of different strains in culture bottles
under aerobic and partial anserobic conditions, on their
toleration of acids and of alkalis and on their thermal

death point.

LITBRATURE REVIEW.

A review of the literature upon Ps. radicicola
shows that comparatively little work has been done with
ite viability. Harrison and Barilow(6) show that, after
being grown on a medium consisting of ash-leachings, maltose,
potassium phosphate and agar for a short time at 20° to 25°C.

and kept at room temperature, Ps. radicicola remains alive
for nearly two years. Edwards(3) carried on a similar

experiment growing the organism on ash-maltose agar in
a darkened closet at laboratory temperature. He found

that out of the nineteen strains used in the experiment
fifteen were alive after four years. Kruijff(8) and
Chester(2) state that the organiam will not withstand
drying on cotton. Edwards and Barlow(4) tested the re-

sistance of the nodule-forming bacteria to desiccation

on seeds -- peas, beans and red clover. It was found
that the organisms died quite rapidly so that after four-

teen days a small percentage were alive. They also demon-
strated that the organism would not survive desiccation
for more than twenty-four hours on filter paper and would
succumb in less than twenty-four hours on glass. Giltner
and Langworthy(5) have similar data regarding its ability
to withstand desiccation on glass.

The optimum temperature for this organism has
been given by several authors at different times. One of
the most recent to work on this problem is Zippel(13) who
states that the optimum temperature is from 18° to 20°C.
He reports that the organism will not grow below 3°C. nor
above 45°C. According to deRossi(11), however, it will
grow slowly at 4° to 6°C., but ceases growth altogether at
0° to -1°C. and at 37°G@ Beyerink(1) obtained his first
negative results at 47°C., in contrast to Schloesing and
Laurent(12) wao found the maximum temperature to be 30°C.
and to Maze'(9) who states the upper limit of growth is
36°C,
METHODS.
Media.
Ash-sugar solution is prepared by adding 10 gms.

ordinary granulated sugar to 1000 c.c. ash leachings <--«
place 5 gms. wood ashes ina liter of tap water and filter

through paper after 5 minutes. The mixture was boiled,
filtered, bottled and sterilized intermittently in flowing

etean.

Ash-sugar agar is prepared the same as the liquii
medium except that 10 gms. agar is added and digested durimg
the boiling.

Ashby's solution is prepared according to the
following formula:

Water (distilled) 1000 c.c.

KgHPO, 0.2 gms.
Mg80.4 QO.2 gms.
NaCl 0.2 gms.
CaSO, O.1 gms.
CaCO, 5. gms.
Mannit 10. gms.

The mixture was boiled, filtered, tubved and sterilized
intermittently in flowing stean.

Ashby's agar as prepared by adding 15 gms. agar
to the solution and digesting by boiling. It was then
tubed and sterilized by the Tyndall metnod.
Inoculation and Incubation.

The ten cultures used in this work are those
employed in the distribution for legume inoculation. A
suspension in physiological salt solution (about 50 c.c.
of salt solution in a bulb pipette) was prepared from a
two weeks old agar siant. Ash-sugar agar slants and ash-
sugar solutions (about 30 c.c. in 2 oz. bottles) were in-
oculated with three to five drops of the suspension and
inoubated at room temperature (20° to 23°C.). Thirty-two
bottles of each medium was inoculated with each or the ten

cultures.

Other Treatment, Analysis and

Determinations.

The cultures were prepared for this experiment

after two weeks incubation by replacing the cotton plugs
in one-half the number of both the liquid and the solid
cultures with cork stoppers which had been soaked previously
in a 11,000 solution of mercuric chloride and dried, then
flamed at the moment of insertion.

Living organians. At intermals of ten days one
bottle from each set of conditions was analysed. In the
case of the agar cultures the entire growth was washed off

into 100 c.c. sterile physiological salt solution. With

the liquid cultures one cubic centimeter was withdrawn and
placed in 99 c.c. of sterile salt solution. Higher dilutior

were made and plated at once in ash-Sugar agar.
 

Total numbers. The total numbers were determined
by use of the Thoma haemocytometer using the liquid cultures

direct and the first suspension from the agar cultures.
Toleration of acids and alkalis. Ashby's agar
was prepared and titrated with methyl red to determine the

acidity. Sterile normal acetic acid or sodiim hydroxide
was added to the sterile agar by means of a pipette cali-

vrated to deliver one hundred drops per cubic centimeter
until the desired amount of acid or alkali was present.
The mediim was then melted and poured into sterile petti
dishes. After it had solidified the bacteria were trans-
planted on the surface by making a stroke with a platinum
needle. Those strains capable of making a perceptible
growth are recorded positive for that reaction.

Thermal Death Point. The thermal death point of
a 48 hour culture in Ashby's solution was determined by

the capillary tube method. The exposure was for ten minutes

at definite temperatures, then they were cooled immediately.
The contents of the tubes were placed on Ashby's agar slants
and recorded positive for that temperature if growth char-

acteristic of Ps. radicicola occurred.
EXPERIMENTAL WORK.

Attention is called to the plan of the investi-
gation: The liquid and the solid media (ash-sugar solu-

tion and ash-sugar agar) inoculated with a suspension of
the bacteria and incubated at 20° to 23° C. for two weeks

were prepared for the experiment by replacing the cotton
plugs in one-half of each of the liquid and the solid
Cultures with cork stoppers. The cultures remained at a
temperature of 20° to 25° C. in a dark room where electric
light was used occasionally until they were used for anal-
ysis. We have, therefore, for each strain of Ps. radicicola
studied four sets of conditions; (1) liquid cultures under
cotton plugs; (2) liquid cultures under cork stoppers; (3)
solid cultures under cotton plugs; and (4) solid cultures
under cork stoppers. At intervals of ten days one culture
from each set of conditions was removed and analysed. Our
purpose for giving two weeks incubation was to approximate
commercial methods. In commercial work the cultures are
grown under cotton plugs which are replaced with flamed
corks just before sending out. It is desirable to know how
rapidly the organisms died off in the cork stoppered bottles
in order to obtain an idea as to how long the cultures may

ve considered good.
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"409084 IOS BTASezZOV
Viability in Culture Bottles.

The growth in the liquid medium was turbid
throughout at first and later settled to the bottom.

When this stage was reached it was difficult to distribd-
ute the organisms evenly throughout the medium as they

goon settle upon standing. The growth on agar is thick,
hyaline to pearly and rather well distributed. There is

a much smaller number of organisms in the liquid than in
the solid cultures. This was noticeable throughout the
experiment. When we compare the viability of the strain
for alfalfa with that for sweet clover we find that at
twenty days there is 84 percent living (Table I) and 85
percent living (Table II) on ash-sugar agar under the cork
stoppers. After 100 days the strain for alfalfa has 26
percent living while that for sweet clover has 34 percent.
And then taking the data at 150 days we find that while
alfalfa has 6 percent living. sweet clover has 11 percent
In the same manner let us compare the strains for red
clover and for alsike clover. We find that the sealed
agar culture for red clover haa 62 percent living and

that for alsike clover has 58 percent after 20 days. Going
on to 100 days we find that the red clover strain has 9
percent living and that the alsike clover strain has 12
percent. After 150 days we find that the red clover strain
has decreased to 0.5 percent and alsike clover strain to 2
percent. Comparing the strain for white clover with shat
for field bean we find that 69 percent and 64 percent
- 28 -

respectively are living after 20 days. After 100 days we
find 28 percent and 39 percent living, and at the end of
150 days both the cultures have 10 percent living. The
total number in a culture of any strain of Ps. radicicola

on the agar under the cotton plugs is no greater than
under the cork stopper. <A point of note is that the
cotton plugged cultures did not die off as rapidly as

the sealed cultures. The liquid cultures at all times
had a amaller total number of organisms, that is, Ps.
radicicola is unable to increase in 30 c.c. ash-sugar
solution (about 10 square centimeters surface exposed to
air) to as great a number as on an ash-sugar agar slant
(about 18 square centimeters surface). The death rate <--

10 to 75 percent within 10 days -- is high in the liquid

cultures.

Some of the factors which caused a decrease in
the number of living organisms are, (1) partial anaerobic
conditions, (2) accumulation of metabolic and toxic pro-
ducts, etc., (3) plasmolysis caused by the concentration
of the ash-sugar solution through evaporation. In the case
of the unsealed liquid cultures which constantly show a
much smaller number of organisms it is probable that the
partial anaerobic conditions in the medium was a factor in
the decreased numbers of living bacteria. The accumulation
of metabolic products, toxic products, etc., are another
possible factor in reducing the living organisms although
no bacteroidal forms, which are supposed to be the result

of unfavorable conditions, were observed at any time. It
e 29 -«

is of note that while Edwards(3) obtained sub-cultures
after four year he did no quantitative analysis and it

is possible that toxins killed four of his cultures.
Plasmolysis could not be a factor in the dying off of

the cultures as the rate of evaporation was quite slow
(about one cubic centimeter in ten days). In the unsealed
agar cultures desiccation was probably a factor in redwing
the living bacteria. But with the sealed cultures of both
media the partial anaerobic conditions, which tended to
become wholly anaerobic, were the principal cause of the
dying off of the bacteria.

Upon comparing the graphs of the different strain
it will be seen that in several instances there is a sharp
rise in the percent of living bacteria. This may be accounted
for by the fact that a different bottle, which may have had
different but unknown conditions entering, was used for each
analysis. It will be observed that the total numbers are
fairly constant from the beginning to the end of the experi-

ment.

Tolerance of Acids and Alkalis.

The ashby's agar used in this work was titrated
while hot with several indicators and the results recorded

as follows:

Methyl red +1.7°
Methyl orange +#2.5°
Cochineal +1.0°
Litmus +1.8°

Phenolphtnalein -0.5°
Table XI.

Growth on Acid and Alkaline Media.

 

 

Acetic acid Sodium hydroxide

Bacteria

for 20° 10° #£§° °§® 10° -20°
Alfalfa = ~ ~ + + +
Sweet clover - ° + + +
Red clover - - - + + +
Alsike clover - - - + + +
White clover - - - + + +
Field bean - ° - + + +
Garden bean ° - ° + + +
Soy bean - - - + + +
Garden Pea - - - + + +
Field pea - - - + + +
Cow pea - - - + + +
Ve toh e - - + + +

 

*A degree is equivalent to a percent N/10.

The reaction of the tuves of medium used for the experi-
ment was adjusted to the methyl red. It will be seen by
referring to Table XI that none of the strains were able
to withstand an acidity of +5° Fuller's scale, whereas all
the strains were able to make a good growth in the medium
with a reaction of -20°. This fact is significant in the

culture of legumes since both the plants and the bacteria
-31-

Table XII.

THERMAL DEATH POINT DETERMINATIONS.

 

Temperature of Exposure

 

 

Bacteria
for 58°C. 59°C. 60°C. 61°C. 62°C. 63°C. 64°C.
Alfalfa + + - - - . -
Sweet clover + + + : . - -
Red clover = - - - - - °
Alsike clover + + + - . - -
White clover + + + -  - - -
Field bean + + - ° ° - .
Garden bean + - . ° ° - -
Soy bean + + ° - ° - -
Garden pea + + + - - - -
Field pea + - . - - - o
Cow pea + + ° - - ~ ~
Vetch + + - - ° - ~

 

require an alkaline condition for their best growth. It

is probable, however, that Ps. radicicola can withstand a
greater acidity in the soil than in an artificial medium
since the soil particles will adsorb and the organic matter
in the soil will absorb a large part of the acid. These

phenomena are not pronounced in the medium used.
Thermal Death Point.

By referring to Table XII it will be noted
that nine of the strains will reproduce after an ex-
posure of ten minutes to a temperature of 59°C. While
four of the twelve will stand a temperature of 60°C.,
none of them are able to grow after being exposed for
ten minutes to a temperature of 61° C. This shows that
the thermal death points of these strains of Ps. radi-
cicéla fall within narrow limits.

GENERAL DISCUSSIONS.

The mass of growth on the surface of the agar
cultures is not composed entirely of bacterial cells. It
is estimated that from 50 to several hundred percent of
this mass is slime. In each culture there is présent a
number of bacteria sufficient to allow, when 60 pounds of
seed is treated, several thousand bacteria per seed.

A number of experiment stations and commercial
firms distribute cultures of nodule-forming bacteria in
liquid media. The results obtained with ash-sugar solu-
tion are not favorable for liquid media. Even solid media,
ash-sugar agar, does not keep gs large number of bacteria
alive for prolonged periods of time. By observing the
curves in this work it is seen that the bacteria die off
rapidly: At the end of thirty days the unsealed liquid
cultures have an average of 38.5 percent living and the

sealed liquid cultures have only 18.90 percent, while the
- 33 «

unsealed agar cultures have an average of 87.0 percent
living and the sealed agar cultures have 55.8 percent.

It, therefore, is imparative that a time limit for the

use of the cultures be observed.

SUMMARY.

1. Ash-sugar agar supports a better growth of Ps.
radicicola than ash-sugar solution.

2. Cultures of Ps. radicicola in cork stoppered
vottles die quite rapidly: the organisms living after 160
days average 0.21 and 3 percent in the liquid and solid
cultures respectively.

3S. The viability in ash-sugar golution is much shorter
than on ash-sugar agar.

4. On ash-sugar agar under cotton plugs the different
strains of Ps. radicicola die off gradually: An average
of 91.1 percent living after 20 days, of 46.2 percent after
100 days and of 12.5 percent after 160 days.

5. The strains of Ps. radicicola are sensitive to
acids, that is, they fail to grow on Ashby's agar to which
0.5 percent l/l acetic acid was added. The tolerance of
alkalis is greater, that is, they make a good growth on
Ashby's agar to which 2.0 percent N/l sodium hydroxid was
added.

6. The thermal death point of twelve strains of Ps.
radicicola determined in Ashby's solution fall between 59°
and 61° C.
 

- 34°
Literature Cited.

(1) Beyerinck, M. W.
1890. Kanetliche Infection von Vicia Faba mit Bacillus
radicicola.
in Bot. Zeitg. Bd. 48, pp. 837 -843.

(2) Chester, Frederick D.
1907. The effect of desiccation on root tubercle bacteria.
Del. Expt. Sta., Bul. 78, p. 15.

(3) Edwards, S. F.
1911. Viability of Ps. radicicola on ash-maltose agar.
in Science n. ser. Vol. 33, pp. 543-544.

(4) Edwards, S. F. and Barlow, B.
1909. Legume bacteria.
Ontario Dept. Ag., Bul. 169, p. 32.

(5) Giltner, W. and Langworthy, H. Virginia.

1916. Some factors influencing the longevity of soil
microorganisms subjected to desiccation, with
special reference to soil solution.

in Jour. Agr. Research, Vol. V, pp. 927-943.

(6) Harrison, F. C. and Barlow, B.

1907. The nodule organism of the Legumnosae,- its
isolation, vultivation, identification and
commercial application.

dn Centrbl. f. Bakt. Abt. II, Bd. 19, pp.426-441.

(7) Hiltner, L.

1900. Ueber die Bakteroiden der Leguminosenknollchen
und ihre willkirliche Erzetgung auserhalb der
Wirtapfilanzen.

in Centrodl. f. Bakt., Abt.II, Bd.6, pp. 273-281.

(8) Kruijff, B. de. |
1907. Onderzoekingen over - en entproeven met de z.g.
Knolletsbacterién.
in Jaarboek van net Department van Landbourv in
Nederlandsh Indi8; Batavia, G. Kolff and Co.,
pp e 112-114 e

(9) Mazé, M.
1897. Fixation de l'azote libre par les bacille des
nodosites des Legumineuses.
in Annales de l'Institut de Pasteur, Tm. XI,
pp e 44-64 e

(10) Prazmowski, A.
1889. 0 istocie i znaczenuin biologiczgnem brodanek
korzeniowych grochn.
Bot. Centrbl. Bd. 39, pp. 386-362.
- 35-

(11) Rossi, Gino de.
1907. Ueber die Likroorganismen, welche die Wurzel-
kndllchen der Leguminosen erzugen.
Centrbl. f. Bakt. Abt. Il, Bd. 18, pp. 481-483.

(12) Schloesing, Th. and Laurent, En.
1892. Retherches sur la Fixation de l'azote libre par
les plantes.
in Annales de l'Institut Pasteur, Tm. VI, pp.
65-115.

(13) Zipfel, Hugo.
1912. Beitrage sur Morphologie und Biologie der
Knélichenbakterien der Leguminosen.
Centrbl. f. Bakt. Abt. II, Bd. 32, ppe 97-137.
‘uti