THE. ACTION OF
CELLULOSE-DECOMPOSING
ORGANISMS ON THE SPECIFIC
POLYSACCHARIDES OF
PNEUMOCOCCE

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE

Robert J Patrick
1938

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THE ACTION OF
CWDECOMPOSING ORGANISMS ON
THE
SPECIFIC POLISACCHABIDES 0F PNEUMOCOCCI

than for thc dogma of v.3.
lichigan State Cellos:
Robert John {shtick
958

I" i”
I

THESIS

CONTENTS

Aoknowledgement.........................................
Introduction............................................
ledlum..................................................
HoBeth'o Cellulose Agar............................

Cellulose agar in which the cellulose 18
peptixed by selenium oxyohlorlde.................
Pneumoooooue capsular polysaccharide medium........
Bron Creeol Purple agaroou..................”nu

Duboo’ nodificetlon of unaliensky'o medium ........

“BthOd. ‘nd Proceduroe....o.o.....................o.....
Experimental....o.......o...............................
COIIUlOCC fermentation.............................

Specific polyesooharide fermentetion...............

T‘hl..oboeooeoe0000000000..eeeeeooeoeeeeoeeeeeeeeeeeeoeo

Diaous‘ianQOOeoeeeoeo00000000600000.00000000eee000000000

Bibliography.....................................o......

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Acknowledgment

The triter eiehee to express hie gratitude for the
helpfulness of Dre. I. E. Bunney, G. F. Forster end J. T.
Tripp of the Hichigan Department of Heelth and Dre. ward
Giltner, I. L. Hellman and C. S. Bryan of the Department

of Bacteriology, Michigan State College.

Introduction

The idea.is not a recent one that enzymes might be dis-
covered capable of acting upon the substance of disease-producing
bacteria in such a say as to render them harmless. The search.for
such an enzyme having therapeutic possibilities is as old as the
betteruknosn science of chemotherapy.

lost of the early work suggestive of this possibility,
referred to by every end Dubos, (l) sas carried out by research
corkers abroad, and includes such findings as the utilization of
the galactan capsular substance of rriedlander's bacillus by
B. vulgatus; the loss of activity of tuberculin as sell es en.-
inution of the acid resistance of tubercle bacilli in the presence
of filtered extracts of Aspergillus fumigatus. Other sorkers (2)
suggested that the lipoid substances of the tubercle and lepra
bacilli might be attacked by esterases of tissue. Robinovitch
and collaborators (3) observed that tuberculosis of the pancreas
in tuberculous animals sas relatively uncommon due perhaps to
active lipases.

In 1930 Avery and Dubos (4) investigated the yeasts and colds,
and the enzymes of‘snflmsls and plants for an extract that sould
attack the higher carbohydrates and possibly that of pneueocooci,
eithout success. However. they did find such an active principle
in an extract from pest and isolated fr. it a gram-negative pleo-
morphic. spore-forming bacillus referred to es S-III. the cell-free
filtrate of shich is effective in decomposing the specific poly.

sscohsride of type III pneumooocci, this organism having no such

4.

capacity for either type I or type II 8.3.8. nor for gum arabio
nor the carbohydrates of friedlander’s bacillus. The therapeutic
value of this enzyme has been investigated by Goodner, Dubos and
Avery, (5) who observed an early cessation of symptoms in experi-
mentally infected mice and rabbits through the administration of
the extract. trance. Terrell. Dubos and Avery (6) found consi-
derably less morbidity and mortality in Java monkeys infected by
intratracheel or intreebronchial inJections of virulent pneumo-
cocci vhen treated eith the bacterial-free filtrate of 8.111 or-
mine.

In 1932 Sicklee and Shae (7) isolated from soil, an organism
mich they call B. palustris shich has the ability to ferment type
III 8.8.3.. similar in morphology and physiology to the organism
D. SPIII described by Dubos and every. They also found a strain
of a. paluatris which ferments, specifically, the capsular sub-
stance of type VIII. They found I’Sacchlmrcbacterium ovale" to fer-
ment type II specific polysaccharide. ehile an organism, 'Saccharo-
bacteria scminetm’ sas capable of utilising type I 8.8.8. 'l'la-
vobscterium ferruginetm", on the other band, see found capable of
attacking only the ‘6’ fraction of type I pneumococci.

These findings seem sufficiently encouraging to induce fur-
ther search for ensymee vhioh sill ferment the capsular polysac-
charides of the higher types of pnemocooci and perhaps ensymea
vhich sill ferment more effectively this polysaccharide of the
types for shich an enzyme has already been found. it could seem
advisable to investigate the fermentability of the specific capsu-
lar polysaccharide by isolated organisms which have the ability to

ferment the polysaccharide cellulose, since the latter is cupossd
of a chain of d-gluccse smite bound together through the beta-
linksge and recent evidence us) has shovn that certain of the
units of the capsular polysaccharide are combined through this
same linkage. .

This possibility formed the basis for the var]: described here.

6.
Medium

Various mediums were prepared and studies made to determine
their relative usefulness. among those found to be most useful are
included umeliansky‘s mineral medium, (8) Bradley‘s casein digest,
(9) McBeth's cellulose agar (lo) and a mineral agar employing
cellulose psptised by selenium exychloride. Some difficulty was
encountered in the preparation of cellulose agar according to
BcBeth. Both copper carbonate and capper sulfate were used in
compounding Schweitzer's reagent, but in neither case did it dis-
solve paper. Finally, a procedure vas found that worked satis-
factorily. 96 grams of copper sulfatereere dissolved in 400 m1 of
water, an approximately saturated solution. To this solution 75
grams of sodium bicarbonate in 550 ml of eater \saturated solution)
were added and mixed. shis mixture wee filtered through a.Duchner
funnel and 75 grams of the moist precipitate vas dissolved in 400
ml of ammonium hydroxide solution made up by adding 125 ml of water
to 500 ml of the concentrated alkali (ep. gr. 0.90}. 15 grams of
this tissue paper sore dissolved in this ammoniacal solution of
copper hydroxide contained in a glass stoppered bottle, requiring
one-half hour and frequent shaking to dissolve the paper completely.
When completely dissolved, a.hcmogeneous, syrupy mixture is ob-
tained. ‘200 ml of this material sore put in a 2 litre lrlenmeyer
flask:and diluted slowly vdth tap water with constant vigorous
shaking until the flask was full. It see then transferred to a
20 litre bottle and diluted to 10 litres. To remove the deep blue
color (copper), dilute hydrochloric acid see added requiring 1 litre

of the 10; acid. upon standing the cellulose precipitate settled

7.

and.as much of the supernatant as possible was siphoned off.

The residue mas filtered through a.luchner funnel, using several
thicknesses of gauze in place of filter paper, and washed with 5:
H31 to remove the copper (blue) completely. The 5; acid was
followed by several rinsings of distilled water. the pure white
gelatinous peptised cellulose-was then placed in a 2 litre flask
containing 1 litre of water. this constituted an immediate supply
of peptiaed cellulose for use in the preparation of ucBeth's cel-
lulose'agar and was kept sterilized and stored in the ice box.

For the preparation of cellulose agar by using the carbohy-
drate as peptised by selenium sxychloride, no quantitative data
was found in the literature. In consequence the following proce-
dure sas carried out. 10 ml of selenium oxychloride was heated
to boiling \hcod) and pure clean filter paper slowly added from a
3 gram quantity. lhis amount of oxychloride dissolved the paper
readily, but the mixture ems thick. In the belief that the cel-
lulose would coagulate under these conditions, a.emall sample was
cooled and distilled water added; coagulation occurred. The mixp
ture see then thinned with 2.5 ml of the oxychloride to diminish
subsequent excess coagulation, and another sample tested. This
time there was less coagulation. two other quantities, 2.5 and
3.0 ml respectively, were added as above without further decrease
in coagulation. ihis factor was further reduced by adding a small
amount of water to a.ccmparatively large amount of the paper mixture.
The quantitative values for obtaining best results in dissolving
the paper were found to be 1.7 grams of paper in 12.5 ml of boiling

seleniuIonychloride. this solution see cooled and three volumes

of distilled water added slowly with constant vigorous shaking;
a dense colloidal suspension was obtained, red in color, which
settled out readily on standing. this precipitate was washed,
centrifugaliasd and decanted to remove chlorine. Considerable
chlorine was removed following the fifth washing; after the
seventh washing the colloid.vas not thrown dosn by the centri-
fuge. Apparently the chlorine ion hastened the settling out of
the colloid. the remaining chlorine was ignored and the suspen-
sion thus obtained, 200 ml, was used in the preparation of the
medium: .

Agar............................ 10.0 as

Ammonium sulfate................ 2.0 ‘

Dibasic potassium phosphate"... 2.0 "

Magnesium sulfate............... 1.0 t

Sodium chloride................. 1.0 ‘

Calcium carbonate............... trace

Iater........................... 900 ml
the medium, complete except for the peptised.cellulose ass auto-
clased at 15 pounds.pressure for 25 minutes and filtered. the
medims eas completed by adding to the still melted and hot min-
eral agar, lOOtml of the cellulose suspensoid previously heated
to the same temperature as the mineral agar. the pH was adjusted
to 1.2.

the polysaccharidb medium used.was that suggested by Dubos (l)
in ehich the carbohydrate 1. in 0.0051 concentration. m. poly-i
eaccharide media will hereafter be referred to as Rodin.
W. It would be of direct advantage to find a.

substance that would enhance the fermentability of cellulose

since this is frequently a slow process. And, since no solid

.QI‘OO

O

medium satisfactory for all cases for the detection of cellu-
lose decomposition.has been developed, some experimentation was
necessary. A bras-crescl-purple agar was prepared employing
McBeth's cellulose agar leasing out the buffer phosphate and
adjusting to neutral point. To a.litre of this 0.8 ml of a
0.41 solution of B.C.P. mas added. Another lot of McBeth's was
enriched by adding 0.5: of casein digest. This enrichment of
McBeth's medium.mas suggested by the fact that some cellulose-
ccnsuming organisms grem more readily and were more active in
casein digest medium. A third medium for investigation was the
modification of Omeliansky's suggested by Dubos (13) who found
that-0.05% concentration of sodium nitrate catalysed the paper-
splitting ferment. The results obtained from the use of these

mediums will be discussed in another paragraph.

9.

10.
Procedure

Thus far, the workers hunting for such an extract (4,?)
have turned to soils and decaying vegetable matter. ‘This source
is inexhaustible. However, it would seem that the intestinal
contents of the wood-boring, wood-consuming Arthropods might be
' a likely habitat to investigate (11,12) or perhaps the feces of
herbivore. Therefore this source of inoculum was selected ex»
cept for the use of a soil culture as a control to test the
mediums. The specimens were found in old stumps or other decay-
ing wood. In the laboratory the following routine in culturing
was carried out. One fluid and two solid types of medium
Omeliansky's, McBeth's cellulose agar, and cellulose agar pep-
tised by selenium oxychloride, 6 tubes of each being used for
each specimen found. if large enough, the specimen was incised
longitudinally to expose the intestine, and.the cultures were
made directly, but if it was small it was suspended in 5 m1 of
saline, mashed and the inoculum taken from this. 1he latter is
usually the more satisfactory procedure in either case since a con-
siderable amount of material is required for all the inoculations.
It also avoids the effect of drying of the material during the
course of inoculations. from this suspension six tubes of
Omeliansky's medium were inoculated with two loops of material.
. With the solid mediums melted and cooled to 47 to 50 degrees Gen»
tigrade, six sets of two dilutions each were poured. Dilution I
was made by inoculating three loops of material and dilution II by

inoculating two loops of material. The above 18 cultures were dis-

11.

tributed so as to obtain the following set in triplicate:
Two Omeliansky's
Two oxychloride peptized cellulose agar plates
(dilutions I and II)
Two McBeth's cellulose agar plates
(dilutions I and II)
One set was incubated at room temperature, one at 30° C. and the
third at 37° 0; These were observed for growth and cellulose de-
composition every 31to 7 days for a month. when growth appeared
in Omeliansky‘s (in 5 to 5 days usually), Gram stain preparations
were made. When the paper showed signs of decomposition, a loop-
ful of the culture was transferred to 25 m1 of saline. One loop
of this suspension was transferred to a melted and cooled tube
of McBeth’s medium to make dilution I and one loopful of dilution
I transferred to a second tube of cellulose agar for dilution II.
The dilutions were then poured and incubated at the temperature
corresponding to that at which the inoculum grew. The colonies
which appeared on the solid mediums were studied and their mor-

phology recorded. They were then subcultured to Omeliansky‘s

medium.

12.

In the order of their classification the Arthropoda inves-

tigated were the following:

Class Crustacea
Isopoda (sowbugs)
Class Myriapoda
Diplopoda (millipedes)
Chilopoda (centipedes)
Class Insecta
Orthoptsra
uryllidae
niptera
Tipulidae

(a dipterous specimen not classified
as to family) '

Siphonaptera

Carabidas
Colsoptera

Scarabeidae

Elateridae

Tenebrionidae

Cerambycidae

Cuquidae ( or Laemophloeidae)
Hymenoptera

Formicidae

The inoculation procedure used in the case of these specimens

was also used in the case of the guinea.pig, rabbit and horse

13.

fecal.material, and for samples of the contents of the calf ru~
men.

The cultures showing cellulose decomposition were saved and
transferred monthly, sterile distilled water being added from
time to time to care for evaporation. Later, they were found to
keep viable for long periods of time in the refrigerator. Plates

were incubated in a moist chamber.

14.
Experimental

Cellulose Fermentation

As indicated before, there is no solid medium satisfactory
for detection of cellulose decomposition in all cases of cellulose
fermentation. The fluid mediums, through their lack of protein
material, inhibit the growth of many organisms other than cel-
lulose decomposers, but a solid medium suitable for final iso-
lation is greatly needed. It was the writer's exper-
ience that in only one case did the organisms which apparently
decomposed the paper either in Omeliansky's or Bradley's medium,
also decompose the cellulose in cellulose agar as evidenced by
clear zones about the colonies. For this reason the following
mediums were investigated:

1. Oneliansky‘s

2. Omeliansky's
plus Brom Cresol Purple

3. Omeliansky's
plus sodium nitrate
plus Brom Cresol Purple

4. Omeliansky's
plus sodium nitrate

5. McBeth's Cellulose Agar

6. McBeth's Cellulose Agar
plus sodium nitrate

7. McBeth's Cellulose Agar
plus sodium nitrate
plus Bram Cresol Purple
Phosphates were eliminated in all except the control mediums
l, 5,-and a portion of 4 and 6. All the mediums were inoculaw

ted simultaneously with the same cellulose decomposing organisms

15.

and incubated at the temperature for optimum growth and activity
of the respective organism and observed at regular intervals for
25 days. The hydrolytic action of the organisms used was not in-
creased over that of the controls, nor did the medium with the
indicator added, make for easier detection of cellulose decompo-
sition.

No further evaluation of medium was attempted except to
observe that some cultures grew best on Omeliansky’s while others
were more active on Bradley's casein digest. During the course of
investigation twelve cultures were obtained which decomposed cel~
lulose.

Polysaccharide Fermentation

leanwhile, pneumococcus polysaccharides were isolated and
purified for use in the Shmedium. 1'0 methods were used, that
suggested by Heidelberger (14) and the calcium phosphate pre-
cipitation method recommended by Felton, Kauffmann, and Stahl (15).
Yields of 0.10 to C.l5 grams per liter of filtrate were obtained.
For testing for the presence of polysaccharide, horse antiserum
could be obtained for types I and II, but it was necessary to
immunize rabbits for the other types employed in this work, namely
III,‘V, and VIII. Six rabbits, two on each type, were immunized
in the course of eight weeks with a yield of from 110 to 140 ml
antiserum from each, and agglutinin titres ranging from 1-128 to
1-512.

In testing for polysaccharide decomposition, é ml of a
young culture showing active cellulose decomposition was added to

5 ml of S-medium and the tube thus inoculated was incubated at the

16.

optimum growth temperature of the organism. Controls of sterile
medium were incubated with the cultures. Sterile distilled water
was added to keep up volume. The presence of polysaccharide was
tested for, following one, two, and three to four weeks of in-
cubation. In the case of types I and II, serial dilutions of the
culture (1 - 2, l - 4, l - 8 etc.) were carried out four to seven
tubes depending upon the results of tests made on the Semedims at
time of inoculations. Tes§ ml quantities of these dilutions é ml
of potent horse antiserum diluted 1-4 was added and the tubes in-
cubated 2 hours at 37°. In the case of types III, V, and VIII

the serial dilutions of the culture are made with saline. Ts

i ll amounts of these dilutions, 0.4 ml of distilled.water was
added.and mixed. 0.1 ml of the antiserum is gently layered be-
neath the dilution and incubated at room temperature. Records

are taken after L or I hour incubation or at both times, observing
for a turbid layer at the interface of serum and culture dilution.
The tubes are then shaken and placed in the refrigerator for 2 to
3 days and the ”shake out' precipitin titre recorded. This method

was economical of the scarce immune serum.

17.

TABLES

The tables are records of the data obtained during the
procedure for the isolation of the organisms. The insect,
or ruminant materials included are only those from:which a

desired culture was obtained.

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23.

 

 

 

 

 

 

 

 

 

 

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26.

Discussion

Among the eight cultures isolated there were none that re-
sponded favorably on the brom cresol purple or sodium nitrate
mediums. The former did not aid in the detection of cellulose
decomposition nor did sodium nitrate catalyze cellulose fer-
mentation.

It will be seen from the foregoing tables that rather strict
medium selectivity exists among the cultures, some losing either
their viability or the ability to produce cellulose decomposing
enzymes in Omeliansky's medium and requiring casein digest medium
to retain this capacity. For others the reverse is true, better
growth and more active cellulose fermentation occurring on
Omeliansky's medium. Only one culture, that isolated from horse
manure, reacted as expected on cellulose agar, i.e. by forming
clear zones indicating cellulose digestion.

In the first test for 8.8.8. decomposition. the culture from
the Cucujidae, apparently decomposed 8.8.8. of type II pneumococcus
in three weeks' incubation. Cultures from the chujidae, Tipulidae,
Isopoda and the soil sample apparently digested 8.3.8. of type III.
Cultures from the Tipulidae, Diplopoda and from soil seemed to
attack the polysaccharides of a type V pneumococcus. Further tests,
however. failed to duplicate these results except in the case of
the culture from the Tipulidae, which retained the capacity to
digest the polysaccharide of the type III pneumococcus for four
months after which time the property was lost. During its active

period germpfree filtrates of the latter were tested for their

27.

effect on the type III carbohydrate. These results were negative.
The culture from this specimen was best maintained on casein di-
gest medium; it is entirely possible that if it were returned to
a more strictly carbohydrate (cellulose) medium, the specific

polysaccharide fermenting power might be restored.

 

 

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16.

BIBLIOGRAPHY

Every, 0. T. & Dubos, Rene, J. Exp. Med.. 54:73. 1931

Jobling, J. W.. Harvey Lectures. 12:181. 1916-17

’Robinovitch et a1. Endocrinology, 9:490, 1925

Ibid. 10:602. 1926
Avery. 0. T. a Dubos, nene. Science 72:151. 1930

Goodner. K.. Dubos, R. & Avery, 0. T., J. Exp. Med.,
56: 521. 1932

Francis, T. J., Terrell, E. E., Dubos, 8., & Avery, 0. T.
J. Exp. Med.. 59:641. 1934

Sickle. G. M. 1 Shaw. Myrtle, J. Bacty., 28:415. 1934

Wakeman, S. 3., Principles of Soil Microbiology, 2nd Ed..
Wms. & Wilkins, Baltimore. 1932

Bradley, L. A. & Rittger, L. F.. J. Bacty.. 13:321. 1927

MoBeth. J. G. & Scales, r. M., Bul. 266, B. P. J., U. 8. Dept.
Agr., 1913

Beckwith, I. D. & Rose, a. J., Proc. soc. EXp. 8101. 1 Med..
27:4. 1929

Norman. L. 0., J. Biochem.. 30:1136, 1936
Dubos, n. J., J. Bacty.. 15:223. 1928

white, Benjamin. “1he Biology of the Pneumococcus, Oxford
University Press, 1938 I

Norman, A. 0., The biochemistry of cellulose, nemicel-
luloses, Polyuronides. Lignin, etc., Oxford University
Press, 1937

Uhallinors, s. H., Haworth, w. N. & Herst, E. L.,
J. Chem. Soc.. 134:258. 1931

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