THE EFFECT OF GROWTH STNULATING
SUBSTANCES IN THE MENU)! ON THE

PRODUCTION OF BACTERJOPHAGE
firms 72203 THE DEGREE a? a. s.

Beatrice Leora Kelly
1933

 

 

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THE EFFECT OF GROWTH

STIMULATING SUBSTANCES IN THE Mpm
ON THE PRODUCT ION OF BACTERIOPHAGE

Submitted to the
Faculty of the Michigan State College

Thesis for Degree of M.S.
Beatrice Leora giggly

1933

THESIS

 

Acknowledgement

It is with Sincere appreciation
I acknowledge the helpful guidance of
Dr. N. W. Larkum, and the kind assist-
ance of the other members of the Eiohigwn

State Department of Health Laboratory.

10.16% ’7

Contents

Page
Introduction 1
Experimental
Part I. Work with Various Peptones 5
A. Effect of’Peptone on Rate and 7
Degree of Lysis by BacteriOphage
B. Effect of’Peptone on Serial 23
Transmission of Bacteriophage
Part II. Work with Synthetic Medium 31

A. Rate and Degree of Lysis by Bacteriophage 31
in.Synthetic Medium.

B. Serial Transmission of Bacteriophage in 43
Synthetic Medium.
Discussion 53

Conclusion 55

Bibliography 57

Introduction

The experiments hereinafter described were
undertaken with the view of learning certain facts
concerning the availability of various types of
mediums for the produCtion of bacteriophage.

The phenomenon of bacteriOphagy was first
described by d'Herelle (8) in 1917. His investigae
tions on the subject continued over a period of
years and brought him to the conclusion that the
bacterioPhage was a living principle, particulate
in nature. This autonomous virus theory is sup-
ported by much evidence but today has few adher-
ents. A considerable number of hypotheses on the
nature of bacteriOphage have been advanced from
various sources.

Kabeshima (11) suggests that a catalytic agent,
arising in the tissues of an infected animal, acti-
vates a proferment contained within the bacterial
bodies; the activated ferment lyses the bacterium
from which it arises, is then liberated and passed
on to other bacteria to liberate the proferment which

'they contain.

Bordet and Ciuca (3) believe that a nutritional
disorder of bacteria brings about autolysis and the
liberation of an agent capable of transmitting the af-
fectiOn to other bacteria.

Bail (2) says that the bacteriophagous agent,
(splitter) arises from the bacteria themselves.

Hadley (7) suggests that phage is a filterable
state in the cyclogeny of organisms.

These theories vary widely one from the other
and so far none seems to be backed by conclusive proof.
The net result of these conflicting findings, inter-
pretations, and views is that knowledge of the nature
of bacteriophagy is in a somewhat confused state. It
is not possible at the present time to make a final
decision concerning the nature and source of the bact-
eriOphagous agent.

In spite of these uncertainties, a great deal of
pragress has been made in the application of bacterio-
phage to various bacteriological and clinical problems,
among which its use in the field of clinical medicine
has received marked attention and has resulted in a
literature of literally thousands of articles from

which one can only conclude that the possibilities of

its use in the field of clinical medicine are limit-
less. With the development of bacteriOphage for clin-
ical use there has arisen a.necessity for a clear under-
standing of the mechanism of its action both in the
body and in the test tube in order that the principle
may be suitably administered and that it may be pre—
pared in its most potent and readily available form.
Neither problem has been solved, but it has been recog-
nized for a long time that the medium exercises a pro-
found influence upon the deve10pment of a bacterio-
phage. An analysis has shown that of the various con-
stituents of the medium, peptones are probably the

most significant. This is of interest not only be-
cause of the influence of peptone upon the production
of the principle, but also because when bacteriophage
is applied through inoculations as a therapeutic agent,
the presence of peptone is undesirable in general.
Hence, various investigators have attempted to pro-
duce bacteriophage in the absence of peptone, es-
pecially when.the product was to be administered intra-
veneously. It has been observed by Larkum and others

(personal communication), that a bacteriophage of weak

potency only resulted from the use of mediums poor

in peptone, and most workers in this field have

failed to achieve the results reported by MacNeal(loc.oit.)
and Applebaum through the use of a snythetic medium
containing no peptone.

Although the general observation had been made
that peptones influence the development of bacterio-
phage probably because of their influence on the rate
of growth of bacteria which has a direct relation to
the development of bacteriOphage, no attempts have
been made to quantitatively determine Just what con»
centration of peptone represents the optimum in a
given medium. It was in order to shed some light on
this question that the first group of experiments to
be reported in this paper was undertaken.

A.chance observation on the development of a
growth stimulating substance for bacteria by Hughes
(9) suggested the possibility that the objectionable
peptone might be avoided through the incorporation
in a synthetic medium of this growth stimulating sub-
stance which would have the further advantage of re-

ducing the total nitrogen content of the medium with-
out inhibiting the effective growth of bacteria. These
experiments constitute the second portion of this study.

PART I. Work with Various Peptones

Section A. The Effect of'Peptone on the Rate
and Degree of Lysil.

General Procedure:

The general procedure for the following experi-
ments consisted in the preparation of mediums contain-
ing variable concentrations of the selected peptones
and making a study of the rate of lysis of Staphylo-
coccus aureus after the seeding of the staphylococcus
cultures with a constant amount of staphylococcus
bacteriophage. In all cases the medium was aerated
to expedite growth and bacterial dissolution. The
rate of lysis was determined by a comparison of the
turbidity of these broth cultures at stated intervals
with a set of turbidity standards containing known
numbers of bacteria. In this manner it was possible
to get a fairly accurate measure of both the rate of
growth and the rate of lysis of the organisms. The
medium.used was a beef extract broth with the followb
ing formula in which, obviously, the concentration of

peptone varied:

Dist. water 1000 g.
Beef extract (Bacto) 3 g.
Peptone

NaCl 5 g.

-6.-

The standard turbidity suspensions were prepared
for each experiment by adding graduated amounts of
culture su3pension to broth mediums containing peptone
in the amount used in the experiments and formalin suf-
ficient to prevent multiplication of the bacteria. The
approximate number of bacteria added to each tube was
determined by previous centrifugation of a.portion of
the suspension in a calibrated centrifuge tube. The
details of this method are described by Kreuger. (13)
After incubation of the flasks for 24 hours at room
temperature, their contents was passed through 3 cm.
Seitz filters and the filtrates titrated in order to
determine the quantity of the phage produced in these
various concentrations of peptone. Titrations were
performed according to the method first described by
Applemans (l) which consists of serial dilutions of
bacteriophage prepared by the addition of .lcc of
filtrate to 9.9 cc of broth and the transference of
.lcc of this mixture to a second tube containing 9.9 cc
of broth. This causes an initial dilution of 10"3 of
bacteriophage and increasing dilutions of 10-4, lO'eetc.
Culture suspension is added to each tube and the degree
of lysis read after 24 and 48 hours incubation at room

temperature.

Experiment 1.

Three peptones were used in the first experiment
two of which were products of the Difco Laboratories,
Bacto-Peptone and Bacto Proteose Peptone, the other
was the well known Witte's Peptone. Published analyses
(6) (14) of these peptones reveals the fact that they
are distinguished by the variation in non-protein
nitrogen with lesser variation in other nitrogen com»

pounds. The total nitrogen does not vary so markedly.

Bacto-Peptone:
Total N - about 14.0%
Non-protein.fl " 13.0%
Polypeptid n I .8%

8.2%

Van Slyke Amino N "
almost negligible

Free Ammonia

Proteose-Peptone:

Total n 12.5%
Non-protein N 6.9%
Protein N. 5. %
Polypeptid N 2.3%
Van Slyke Amino N. 4.8%

Free Ammonia

‘Witte's Peptone:

almost negligible

Total n 15.0%
Honpprotein N 6.0%
PrOtein Ne 8e0%
Polypeptid H. 2.5

Van Slyke Amino N. 8.0%

Free Ammonia

almost negligible

Turbidity standards were prepared for each con-
centration of peptone to be used, containing the fol-

lowing approximate numbers of bacteria per cc:

1. None
2. 1075 x 104
5. 2146 x 104
4. 4292 x 104
5. 8584 x 10%
5. 1073 x 10
7. 2145 x 103
8. 4292 x 105
9. 7438 x 106
10. 8584 x 10

12 flasks were prepared containing:

100.00 cc Peptone broth medium, pH 7.6. 4 flasks
each of Bacto, proteose, and Witte's
peptone with concentrations of .5%, 1.0%,
2.0%, an a 5 .5%.

1.00 cc Staphylococcus phage in each flask

.50 cc Culture suspension made by washing a 24
hour Staph. aureus culture (Lab. 30.1771)
grown in a Blake bottle (beef infusion
agar) with 20 cc of saline in each bottle.

The flasks were aerated by suction in order to keep the
liquid agitated. At irregular intervals about 2cc of
the content of each flask was removed and the turbidity
determined by comparison with the standards.

In Table I is found an analysis of the results of
experiment 1.

In Table II is found a graphic representation of
the results of this first experiment. In this graph
the log. of the concentration of bacteria per cc forms
the ordinate and the time in hours the abscissa.

Bacto 7
Proteose 7
Witte 7

Table I.

Logs. of Maximum Bacterial Growth

0.5% 1.0% 2.0% 5.5%

.95559 7.55255 7.95559 7.95559
.95559 8.55151 8.55151 8.55151
.95559 7.55151 7.95559 8.55255

Time Required for Maximum Growth of Bacteria

Bacto
Proteose
Witte

T

Bacto
Proteose
Witte

Bacto
Proteose
Hitte‘

4 hrs. 4 hrs. 5 hrs. 5.5 hrs.

5 5 5 5.5

5 5.5 8 8.0
ime Required for First Appearance of Lysis
5. hrs. 5. hrs. 5.5 hrs. 6. hrs.
5.5 5.5 5.5 6.
5.5 6.0 none in 6.

8 hrs

Time Required for Maximum Lysis

5.5 hrs. 5.75 hrs. 6.0 hrs. 7.0 hrs.
6.5 6.5 7.5 6.0
6.75 6.25 none in 6.0

8 hrs.

Lysates from the preceding were filtered at 24 hrs. and

filtrates ti

trated.
Titration Readings at 48 hrs.

10'2 10'4 10'5 lo"8 10"10 10"12

Bacto ,5% +++e ++++ ++++ - -
Protease .570 ++++ ++++ ++++ ++++ -
Witte ,5% ++++ ++++ ++++ ++++ -
Bacto 1% ++++ ++++ ++++ - -
Protease 1% ++++ ++++ ++++ ++++ -
Witt. 1% ++++ ++++ ++++ - -
Bacto 2% ++++ ++++ ++++ ++++ ++++
Protease 2% ++++ ++++ ++++ +++ -
Witte 2% ++++ - - - -
Bacto 3.5% ++++ ++++ ++++ ++++ ++++
Protease 3.5% ++++ ++++ ++++ - -
Witt. 3.5% ++++ ++++ - - -

10

Table II

                  

 

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Ordinate - Logs. of approx. numbers of bacteria present per cc.

Abeeisse - Pro

 

 

11

Table I reveals the fact that lysis was first eb-
servable after five hours in the flasks containing the
Bacto peptone in the lowest concentrations. The Witte's
peptone was distinctly inhibitory to the early appearance
of lysis. The completion of lysis also occurred more
rapidly when the Bacto peptone was used.) If these were
the only factors to be considered, one would conclude
from this single experiment that low percentages of Bacto
peptone were most suitable for the production of bacterio-
phage. It should, however, be born in mind that there
exists a relationship between the quantity of bacterio-
phage produced and the rate and degree of growth of
bacteria, and for the clinical use of bacteriophage it
is necessary that maximum bacterial growth occur before
lysis sets in. Referring to Table II it will be seen
that Proteose peptone in 2.0 and 3.5% concentrations
gave the greatest bacterial growth, but this was not
accompanied by complete lysis within 7 hours. Refer-
ring again to Table I it is obvious that slighly less
bacteriophage was produced in these concentrations of
proteose peptone. It would appear then that further
experiments were necessary in order to arrive at any

conclusions regarding the effects of these peptore s.

12

Experiment 2:

This experiment is essentially a repetition
of experiment 1 with the exceptions that Parke Davis
peptone with a total nitrogen content of about 14.%
was substituted for the Witte's peptone and the
concentrations of peptones changed to .1%, 1.0%,
5.0% and 10.0%.

Turbidity standards were prepared for each con-
centration as before.

The results are tabulated in Tables III and IT,
and are comparable to Tables I and II with respect

to the information given.

Table III. 15
Logs. of Maximum Bacterial Growth
0.1% 1.0% 5.0% 10.0%
Bacto 8.52956 8.50771 8.15152 7.85059
Proteose 7.83059 8.52955 8.52955 8.85059*
Parke Davis 8.50771 8.50771 8.13162 8.50771‘’

Time Required for maximum Growth of’Bacteria

Bacto 4.0 hrs. 4.0 hrs. 4.0 hrs. 1.0 hrs.
Proteose 2.0 3.5 3.5 5.5
Parke Davis 4.0 4.0 4.5 7.25

Time Required for'First Appearance of'Lysis

Bacto 4.75 hrs. 4.5 hrs. 6.0 hrs. 6.25 hrs.

Protease 6.0 5.5 6.5 5.25

Parke Davis 5.0 4.5 6.0 5.75
Time Required for Maximum Lysis

Bacto 6.0 hrs. 5.5 hrs. 7175 hrs. 7.75 hrs.

Proteose 6.25 6.25 6.5 6.75

Parke Davis 6.0 6.0 7.75 6.25

Lysates from preceding were filtered at 24 hrs. and
filtrate titrated.

Titration Readings at 48 Hrs.

-2 -4 -5 -8 -10 -12

10 10 10 10 10 10
Bacto .1% ++++ ++++ ++++ ++++ ++++ :-+
Protease .1% ++++ ++++ ++++ ++++ +t++ ++++
Pk.Davis .L% ++++ ++++ ++++ ++++ ++++ +
Bacto 1.% ++++ ++++ ++++ ++++ ++++ -
Proteose 1.% ++++ ++++ - - - -
Pk.Davis 1.% ++++ ++++ ++++ ++++ ++++ ++++
Bacto 5.% ++++ ++++ ++++ ++++ ++ -
Protease 5.% ++++ ++++ ++++ ++++ ++++ -
PIkDavis 5,% ++++ ++++ ++++ ++++ ++++ +
Bacto 10% ++++ ++++ ++++ ++++ ++++ .
Protease 10% ++++ ++++ ++++ ++++ ++++ ++++
Pk.Davis 10% ++++ ++++ ++++ ++++ +++ -

*A secondary growth occurred, more
profuse than the first.

Table IVe

Ordinate I Legs. of approx. numbers of bacteria present per cc.
MICHIGAN STATE COLLEGE

  
 
 
   
    

 

 

 

 

 

 

 

 

 

 

  

    

  

      
 
 
  

       
    

 

 

 

 

 

 

 

 

   
   
     
   
  
  
   
 
  
   
  
  
      

 

 

 

 

 

  

 

 

 

 
   
   
 
   
 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

    

 

     

 

 

     
   
 
    
     

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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2 DE ARTMEN F MAT6EMATIC8

15

As in the preceding experiment, the first
appearance of lysis occurred with the 1% Bacto peptone
although the Parke Davis peptone gave a similar result.
Completion of lysis was also first manifest in the 1.0%
Bacto peptone broth. 'Maximum growth again was present
with the Proteose peptone although the differences were
slight in all cases. The amount of bacteriophage pro-

duced was comparable throughout the series.

Experiment 3.

In experiment 3 the same peptones were used and
an attempt made to narrow the range of percentages,
and also to determine the effects of complete absence
of peptone. The results are tabulated in Tables V and.
VI.

16
. Table v.

Logs. af'Maximum Bacterial Growth

Na peptone .5% 2.5%
(7.83059)
Bacto 7.83059 7.52956
Protease 7.83059 7.83059

Parke Davis 8.13162 8.13162

Time Required for Maximum Growth of Bacteria

(4. hrs)
Bacto 4 0
Protease 5.0
Parke Davis .4 5

Time Required for First Appearance of Lysis

(5 hrs.)
Bacto 5.0 hrs. 5.75 hrs.
Protease 5.25 5.5
Parka Davis 5.0 5.0

Time Required for Maximum Lysis

(5.25 hrs.)
Bacto 5.25 hrs. 6.0 hrs.
Protease 5.5 7.5
Parke Davis 5.5 6.0

The lysates from the preceding were filtered at 24 hrs.
and the filtrates titrated.

Titration Readings at 48 hrs.

10'2 10"4 10"6 10"8 10‘10 10‘13
No peptone + + ++ ++++ ++++ -
Bacto .5% ++++ ++++ ++++ ++++ ++++ -
Protoos¢.5% ++++ ++++ ++++ ++++ ++++ -
Pk.Davia,a% ++++ ++++ ++++ ++++ ++++ ++++
Bacto 2.5% ++++ ++++ ++++ ++++ - -
Protease 2.5% ++ ++ ++++ ++++ - -
Pk.DaV18 2.5% ++++ ++++ ++++ ++++ ++ -

 

 

Table VI

MICHIGAN STATE COLLEGE

 

 

 

   

 

 

 

 

 

 

      

 

  
 

  

 

 

 

 

 

   

 

 

 

 

    

 

 

  
 
   

 

 

 

 

  

 

 

 

 

    
   

 

 

 

     
   

 

 

 

 

 

    

 

  

   

 

 

  
    
  

 

 

   

 

 

   

 

 

  

 

 

 

 

 

  
     

 
  
  
   

 

 

 

 

 

 

  

 

 

 

 

 

 

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‘ A __A AA_‘ A E L ii“ A A H-114; L A A A A4 ‘ AA A .A A_ _ __

 

 

 

DEF HTM EN réw‘ MAaH EMATICfi

18

In Table VI we again find that the first ap-
pearance of lysis and the shortest time required for
complete lysis occurred with the least concentration
of peptone, and that there is little difference with
respect to the different peptones used when low con-
centrations are employed. Titrations again reveal
that there is relatively little difference in the
amount cf bacteriophage produced under these condi-
tions. In this instance the Parke Davis peptone gave
a slightly greater concentration of bacteriophage.

Experiment 4.

Experiment 4 is in the nature of a check on
previous experiments. The procedure was identical.
The results are shown in Tables VII and VIII.

In Table VII we again find that the first appearance
of lysis occurs in the flasks containing the least amounts
of peptone. In the complete absence of peptone eight
hours were required for complete lysis as compared to
seven hours with the 1.0 and 2.0% peptone broths. The
titrations show relatively little difference in the

quantity of bacteriophage produced.

Table VII. 19
Logs. of Maximum Bacterial Growth

No Peptane 1% 2% 5%
(7.62325)
Bacto 7.40140 7.22531 7.52325
Protease 7.40140 7.92428 7.92428
Park Davis 7.40140 7.62325 7.62525

Time Required for Maximum.Growth of Bacteria

(4. hrs.)
Bacto 5.0 hrs. 4.0 hrs. 5 hrs.
Protease 5.5 5.0 5
Park Davis 4.0 5.0 5
Time Required for First Appearance of Lysis

(5 hrs.)
Bacto 5.5 hrs. 5.0 hrs. 6 hrs.
Protease 6.0 6.5 8
Park Davis 5 5.5 6

Time Required for Maximum Lysis

(8 hrs.)
Bac'to 7.0 ms. 700 hrs. 705 hrs.
Protease 7.5 7.5 8.0
Park Davis 7.5 8.0 8.0

The lysates from the preceding were filtered at 24 hrs.
and the filtrates titrated.

hrs.
10-10 10-12

Titration Readings at 48

-2 -4 -6 -8
No peptone 10 10 10 10

++++ ++++ +++ +++ -
Bacto 1% +++ - - - _ -
Protease 2% ++++ ++++ ++++ +++ - -
Pk. Davisl% ++++ ++++ t¢+ +++ a -
Bacto ++++ ++++ +++ +++ - -
Protease 2% ++++ ++++ ++++ ++++ - -
Pk.Davis 2% ++++ ++++ o + - -
Bacto 5% ++++ +¢++ - +++ - -
Protease 6% ++++ ++++ ++++ +++ - -
Pk.Dav15 5% ++++ ++++ +++ +++ - -

20

Table VIII.
MICHIGAN STATE COLLEGE

 

                 

          

 

 

       

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 
           

 
   

 

 

    
      

 

     
       

 

 

 

 

   

 

 

 

 

 

 

 

 

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21

Summarizing the results obtained in the preced-
ing four experiments, it is obvious that there is con-
siderable variation even when a single experiment is
repeated. Thus in the absence of peptone we found in
experiment 3 that complete lysis occurred in five and
a quarter hours,whereas in experiment 4 under simil-
ar conditions eight hours were required. Nevertheless,
within rather wide limits the results with the differ-
ent peptones in any one experiment are roughly compar-
able. There is no question but that high concentra-
tions of peptone retard the progress of lysis under the
influence of bacteriOphage. This apparently is a re-
sult of a slower growth of bacteria, maximum concen-
trations of bacteria in all cases reaching an ppparent
critical point numerically before lysis occurred. Our
experiments shed no light on the quality of phage pro-
duced through the use of different peptones. The quan-
tity was fairly uniform regardless of the type or con-
centration of peptone used. It would appear then that
one is Justified in using any peptone available in the
preparation of bacteriophage, and if,as in the case when

the product is to be used for clinical purposes,peptone

is undesirable, it may be omitted altogether.

22

But the problem of preparation of bacteriophage
involves more than the quantity produced. In order
to carry on successfully the propagation of this
principle the quality must be taken into considera-
tion, and the particular quality enabling the principle
to maintain its virulence throughout a number of ser-
ial passages is highly important. If peptones exert
no marked influence on the quantity of bacteriophage
produced, it remains to be determined to what extent
they influence the quality of the product. In Section

B are found experiments bearing upon this question.

23

Section B. Effect of'Peptone on Serial Transmission
of Bacteriophage.

General Procedure:

The experiments reported here followed a mat-
erially different method than the preceding. Tubes
were prepared containing 10 cc of medium. In one set
was beef extract broth containing no peptone, and in
the others Bacto, Proteose, and Parke Davis peptones
in concentrations ranging from .1% to 10%. (Specific
concentrations are listed on record of each experi-
ment.) To each of these were added 2 drops of culture
suspension obtained by washing one beef extract agar
slant bearing a 24 hour growth of organisms desired,
with 10 cc of saline; also .5cc of phage. After 48
hours the lysates were filtered through 50m.Seitz
filters and .5cc of the filtrate added to a new tube
containing the same peptone concentration, and the
series continued. These dilutions were carried through
as many contacts as results dictated. The incubation
was at room temperature. In all cases the degree of
lysis was checked by comparison with controls contain-
ing the same amount of culture suspension, but contain-
ing no bacteriophage.

A record of the results of individual experiments
appears on the following pages.

24

Experiment 1 e

In experiment 1, Staphylococcus aureus, culture
# 1771 which was shown to be susceptible to the
Staphylococcus phage was employed. Broth was used
containing Bacto, Proteose, and Parke Davis peptone
in concentrations of 0.1, 1.0, 5.0 and 10.0%. The
results are found in Table II. After the seventh
contact had occurred, a portion of the filtrate
was titrated in order to determine the quantity of
bacteriOphage produced.

Even a cursory examination of this table indi-
cates that the quality of the bacteriophage was a1-
tered in the low concentrations of peptone to such
an extent that it failed to propagate itself success-
fully after a few contacts. Differences resulting

from the brand of peptone used were insignificant.

25

++++
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.m

 

cosuapsoomac I o *

 

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++++

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on

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pompnoo

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pompsoo

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pompqoo

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2:: % .gohnu .smmpm

26

++++ ++++ o
++++ ++++ o
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e + o
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on.“ Oh om

 

s93

NH:

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++++ +++ ++++ o o o

On.“ 0" 0m On." C“ .m

986

mo;

cossapsoo : NH candy

++++
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*3 355.2
an armada
Rn omomponm

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at

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6.3 «a
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.mnn av
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penance m

27

Experiment 2.

In order to provide a basis for a little
more generalization, an entirely different
bacteriophage (Colon'bacillus) and its sus-
ceptible organism were employed in a procedure
the same as with Experiment 1. Table I shows
that the results here are likewise clear cut
in that they indicate that the low percentages
of peptone are less satisfactory for the serial
transmission of bacteriophage. No further dis-
cussion of these results is indicated at this

time o

28

 

+++ +++ ++++
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+++ ++ +++
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mo.a
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pecunoo

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29

 

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$0.0”

 

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ao.n

cessapsooman no *

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ma.a

counwpnoo u H candaq

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.ans we

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pospnoo

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.mne em
veepsoo

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¢H

9H

NH

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OH

m

penance o

30

0H:

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++

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penance m song moaunpaam Ho sofipmnpua

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359.3
omooponm

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oposm

Efifit ifit??? Sfi

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onopnom on

51

PART II. Work with Synthetic medium

Section A. Rate and Degree of Lysis in synthetic
medium.

In as much as high concentrations of peptone
seemed desirable fer the propagation of bacterio-
phage, and since they are undesirable in a product
to be used for clinical application, it would be of
material assistance to find some substitute for the
peptone which might enable one to carry on the prop-
agation of phage in mediums of low nitrogen content.
A recent publication by Hughes (9) concerning a
growth stimulating substance found in meat extract
suggested a solution to the problem. It was con-
sidered that the addition of this growth stimulat-
ing substance to a synthetic medium might provide
the elements necessary for the development of an
active bacteriophage capable of successful prOpaga-
tion.

Several investigators have reported the use of
synthetic mediums for the preparation of'bacterio-
phage for therapeutic use. Caldwell (5) found that

a medium described by Ayers gave satisfactory results

with the Colon bacillus, but that USchinsky's medium

52

was not satisfactory. macNeal prepared a synthetic
medium which he found suitable.

In view of these results it seemed advisable to
select one which gave the best growth of the organ-
ism to be used in the following'experiments. Accord-
ingly, four mediums, Uschinsky's glycerol ammonium '
lactate solution, Uschinsky's asparagin salt solu-
tion, Jordan's snythetic medium, MacNeal's medium,
(10) (15), and after inoculation with Staphylococcus
and with Colon bacillus, Uschinsky's Asparagin Salt
solution was selected. This medium has the follow;

ing formula:

Dist. water 1000 cc
Asparagin 3.4 g
NaCl 5.0 g
CaClg .1 g
g§P&1.0 g
044 Trace

The growth stimulating substance was contained
in the acetone soluble portion of beef extract,

which was added to the synthetic medium.

Kjehldal nitrOgen determination showed:

Uschinsky's asparagin salt solution plus activator
contains........84.50 mgs. N per 100 cc.

Beef extract broth (used as control).....218. 16
mgs. N per 100 cc.

53

Section A. Rate and Degree of’Lysis in
Synthetic Medium containing growth
stimulating substance.

Experiment 1.

The experimental methods employed were identi-
cal with those described under Section A of'Part I;
that is, noting the progress of'lysis in mechanical-
ly agitated media, upon introduction of phage and

susceptible culture.

Culture: Staph. aureus # 1963
Phage: #017873A Staphylococcus
Mediums: Beef Extract broth
Uschinsky's asparagin Salt sol. plus
growth stimulating substance.

Turbidity standards were prepared as usual.

The results appear in Tables II and III.

34
Table XI.

Logs. of Maximum Bacterial Growth

Beef Extract Broth Synthetic mediggf
7.50772 7.50772

Time Required for Maximum Growth of Bacteria
1.5 hrs. 5.0 hrs.
Time Required for First Appearance of Lysis
7.25 hrs. None at 10.5 hrs.
Complete at 24 hrs.
Time Required for Maximum Lysis
8.0 hrs. 10.5 - 24 hrs.

The lysates from the preceding were filtered at 24
hours and the filtrates titrated.

Titration Readings at 48 hrs.

- - -6 -7 -8 ' .9 -11
10 2 10 4 10 10 10 lO 10
Beef Ext.Broth ++++ ++++ ++++ - a - - -
Synthetic Media ++++ ++++ ++++ ++ - - -

* The term synthetic mediumrefers to
‘Uschinsky's asparagin salt solution
containing a constant amount of
activator.throughout the remainder
of experiments.

Table III.

 

 

 

 

   
 

 

 

 

   

 

 

 

 

 

 

  

 

   

   

 

 

 

 

 

 

 

 

 

      

 

 

       

 

 

        

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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4 6 8 DEPARTMENT OF MATHEMATICS

 

36

Table II shows that the progress of lysis
‘was slow in both mediums. However, the quantity
of bacteriOphage produced was esentially the same

as is shown by the titration results.

Experiment 2.

Experiment two is a repetition of experiment
1 with the exception of the fact that two dif-
ferent cultures of Staphylococcus were applied.
Results are shown in Tables XIII and XIV.

It is quite obvious in this case that the
beef extract broth permitted more rapid lysis
and that little difference resulted in respect.
to the organism employed. The titrations were

essentially identical.

57
Table XIII.

Legs. of Maximum Bacterial Growth

Beef Ext.Broth. §ynthetic Medium

Staph. aureus 1963 7.50772 7.50772
Staph. aureus 1961 7.50772 7.50772

Time Required for Maximum Growth of Bacteria
Staph. aureus 1965 5.5 hrs. 5.5 hrs.
Staph. aureus 1961 5.5 hrs. 5.5 hrs.

Time Required for First Appearance of'Lysis
Staph. aureus 1965 6.0 hrs. 6.5 hrs.
Staph. aureus 1961 6.0 hrs. 6.5 hrs.

Time Required for Maximum Lysis

Staph. aureus 1963 10. hrs. 14.0 hrs.
Staph. aureus 1961 8.5 hrs. 10.0 hrs.

The lysates from the preceding were filtered at 24
hours and the filtrates titrated.

Titration Readings at 48 hrs.

10'2 10" 10"6 10"7 10"8 10"9
Staph #1963
Beer Ext.Broth ++++ ++++ +49 - - -
Synthetic Medium ++++ ++++ +++ - - ..

Staph #1963 l _ _
Beef Ext, Broth ++++ ++++ ++++ - - -
Synthetic Medium_++++ ++++ ++++ - - -

38

Table XIV.
MICHIGAN STATE COLLEGE

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8
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DEP,9TMEN12F MATHEMATICS

6

12

59

Experiment 3.

Colon bacillus with two different bacterio-
phage strains were used instead of the Staphylo- ,
eooous employed in the previous two experiment.
Tables IV and XVI show no distinct differences
with the two methods employed.

Table X V.
Logs. of Maximum Bacterial Growth

Beef Ext.Broth §zpthetic Medium

 

 

E.coli ( " " 16 6.68124 6.68124

Time Required for Maximum Growth of Bacteria

E.coli (phage M.K.13) 2 hrs. 2 hrs.
E.coli ( " MgK.16 2 hrs. » 2 hrs.

Time Required for First Appearance of Lysis

E0 0011 (PhageM.K.13 3 hrs. 3 hrs.
E. coli ( " ' 16 3 hrs. 3 hrs.

Time Required for Maximum Lysis

E.coli (phage M.K.13) 5 hrs. 3 hrs.
E.coli (phage M.K.16) 3 hrs. 3 hrs.

The lysates from the preceding were filtered at 5 hrs.
and the filtrates titrated.

Titration Readings at 24 hours

102 10'4 10"6 10"8 10‘1° 10’13
E.coli
With phage #M;K,13 ++++ ++++ ++++ ++++ ++++ -

Beef Ext. Broth
Synthetic ++++ ++++ ++++ ++++ ++++ -

Beef Ext. Broth ++++ ++++ ++++ ++++ ++++ -
Synthetic ++++ ++++ ++++ ++++ ++++ -

Table XVI.

\ MICHIGAN STATE COLLEGE

 

         
 
       

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

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DEFJ‘RTM EN 7835- MATH EMATICS

 

 

42

The results of this group of experiments
indicate quite clearly that so far as the
quantity of phage produced in synthetic
mediums to which the growth stimulating
substances had been added is concerned, they
are preferable for the preparation or this
principle since their total nitrogen content
is far below that of the ordinary extract broth
with or without peptone. It remains to be
determined, however, Whether such mediums can
be used successfully in the propagation of
bacteriOphage. This problem is investigated
in Section B.

Section B. Transmission or Bacteriophage
in Synthetic Medium.

The procedure employed in these experi-
ments was similar to that used in Section B of
Part I; that is, .5 cc of phage was added to
a bacterial suspension to bring about lysis, the
lysed bacterial suspension was passed through a
‘Mandler filter and .5cc or this filtrate was
added to a second bacterial suspension. After
lysis had occurred the procedure was repeated
and the series continued in this manner.

Beef extract broth and the synthetic medium
broth with and without the growth stimulating
substance were used. Staph. aureus, and Colon
bacillus were the cultures employed.

There appears to be no occasion for consid-
ering these experiments separately since the pro-
oedures were essentially identical and the re-
sults are tabulated in Tables XVII to XIV in-
olusively. It is apparent from all of them that
the serial passage of bacteriophage in the synthe-
tic medium continued uninterrupted through vary-
ing numbers of contacts, and that the absence of
of the growth stimulating substance resulted in
the disappearance of the bacteriophage in all but

one instance.

Table XVII.

Exp, (1) Culture: Staph. aureus # 1910

Phage: # 017873A (Staph.)
Beef Extract Synthetic
Broth
lst contact ++++V ++++
2nd contact ++++ ++++
3rd contact ++++ ++++
4th contact ++++ ¢+++

(5th contact titrated)

Titration Readings at 48 hours
-2 -4 -6 -7 -8 -9
10 10 10 10 10 10
Beef Extract Broth ++++ ++++ ++ - - -

Synthetic ++++ ++++ + - - -

(2)

1st
2nd
3rd
4th
5th
6th
7th

9th
10th
11th
12th
13th
14th
15th

Culture:
Phage:

contact
contact
contact
contact
contact
contact
contact
contact
contact
contact
contact
contact
contact
contact

contact (titrated)

(titrated)

Beef Extract

Table XVIII

# 017873A Staph.

Broth
(Lysis)

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

++++.

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

Staph. aureus # 1963

Synthetic

(Lysis)

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

.++++

Titration Readings at 48 hours.

Beet Ext.Broth
Synthetic

.Beef ExtoBrOth
Synth etie

(Filtrate from 5th contact)

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

10‘2 10"4 10'6 10‘7 10"8

++++

++++

++++

+++4

++++

++++

Q

§

(Filtrate from 15th contact)

++++

++++

++++

++++

.+++

++++

++++

¢+++

++

+§

10'9

10‘10

45

Exp.

(3)

Culture:
Phage:

contact
contact
contact
contact
contact
contact
contact
contact
contact
contact
contact

Table XIX.

Staph. aureus # 1961

# 017875A (Staph)

Beef Extract

contact(titrated)

contact
contact
contact
contact
contact

contact(titrated)

Broth
(Lysis)

coco
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
+++¢
++++
++++

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

Titration Readings at 48 hours

Filtratc from 12th contact

10‘3 10'4 10"6 10’7 10"8 10'9

Beef Ext.Broth ++++ ++++

Synthetic

++++ ++++

Filtrate from 18th contact

Beef Ext. Broth++++ ++++

Synthetic

++++ ++++

++++

§+++

++++

++++

+

«p

(Lysis)

10

Synthetic

~10

46

Table II.

Exp. (4) Culture: Staph. aureus # 1963
Phage: # 90 (Stapho)
Beef Extract Synthetic
Broth

(LysiS) (Lysis)

1st contact ¢+++ ....
and contact ++++ ...+++
3rd contact +++§ 4.4.4....
4th contact ++++ ....
5th contact(titrated) ++++ ....
6th contact ++++ +4....
7th contact ++++ ....
8th contact ++++ ++++
9th contact +++¢ ++++
10th contact ++++ ++++
11th contact ++++ ++e+
,thh,contact ++++ ++++
13th contact ++++ ++¢+
14th contact ++++ 1.4.4....
15th contact ++++ ++++
16th contact ++++ ¢++¢
17th contact .... ....
18th contact ++++ ++++
19th contact , ++++ ....
20th contact(titrated) ....

++++

Titration Readings at 48 hours

Titration of filtrate from 5th contact

10'2 10" 10"6 10'7 10"8
Beef Ext. Broth ++++ ++ - - -
Synthetic ++++ ++ - - -
Titration of filtrate from 20th contact
Beet Ext. Broth ++++ ++++ - - -
Synthetic ++++ ++++ - - -

PM.

 

43
Table XII.

Experiments 5 and 6.

Note: In.5 and.6, synthetic media both with and
without the activator were used as well as
beef extract broth. Bacterial growth in“
the synthetic medhnlwithout the presence of
activator was so slight that the presence or
absence of lysis was hardly discernible by
observation. Therefore, the presence of
phage was tested for by adding 1 cc of the
filtrate to a tube of beef extract broth
containing 1 drop of culture suspension.

Exp.(5) Culture: Staph. aureus # 1963
Phage: #1078759. (Staph.)

Beef Extract Synthetic Synthetic
Broth (with activator) (without acti-
vator)
Lysis Lysis Lysis
1st contact ++++ ++++ .
2nd contact ++++ ++++ ?
3rd contact ++++ ++++ -

Exp.(6) Culture: Staph. aureus # 1961
Phage: E62B (Staph.)

Beef Ext. Synthetic' Synthetic
Broth (with activator) (without activator)
Lysis Lysis Lysis
lst contact ++++ ++++ ?
and contact ++++ ++++ -

5rd contact ++++ ++++ -

49

Table XXII
EprV) Culture: E. coli # 7c '
Phage: M.K. 1:5 (0011 phage)
Beef Extract Synthetic

Broth
(Lysis) (Lysis)
lst contact ++++ ....
and contact ++++ ' ++++
3rd contact ++++ ....
4th contact ++++ ....
5th contact ++++ ++++
6th contact ++++ ....
7th contact ++++ .++.
8th contact ++++ ++++
9th contact ++++ ++++
10th contact(titrated) ++++ ++++

10'3 10"4
Beef Extract Broth +++¢ ++++
++++ ++++

Synthetic

Titration Readings at 24 hours of
Filtrate from 10th contact

10'5 10'7
++++ ,++++
++++ ++++

10'8 10'°.10‘1°

++++

t++++

++++

++++

+§++

++++

 

(a

50

Table XXIII
E11149) Culture: E. coli # 70
Phage: M. K. 16 (e011)
Beef Extract Synthetic
Broth

(Lysis) (Lysis)

lst contact ++++ ....
2nd contact ++++ ++++
3rd contact ++++ ....
4th contact ++++ 4.4.4.4.
5th contact ++++ ++++
6th contact(titrated) ++++ ++++
7th contact ++++ ....
8th contact ++++ +4.4...
9th contact ++++ ++++
10th contact ++++ ++++
11th contact ++++ ¢+++
thhfcontact(titrated) ++++ ++++

++

Titration Readings at 24 Hours

Titration of filtrate from 6th contact:

.

10'2 10'4 10"6 10"8 10"10 10’12 10
Beef Extract Broth ++++ ++++ ++++ ++++ ++++ ++++ -
Synthetic ++++ ++++ ++++ ++++ ++++ ++++ -
Titration of filtrate from 12th contact:
Beef Extract Broth ++++ ++++ ++++ +¢++ ++++ - . -
Synthetic ++++ ++++ ++++ ++++ ++++ ++++ -

-14

Expl9)

Table XXIV.

Culture: E. coli,#7c
Phage: M.K. 13 (Colon phage)
Beef Extract Synthetic
Broth
(Lysis) (Lysis)

lst contact ++++ ....
and contact ++++ ++++
3rd contact ++++ ++++
éth contact ++++ ++++
5th contact ++++ ++++
6th contact ++++ ++++
7th contact ++++ ++++
3th contact ++++ ++++
9th contact ++++ ++++
10th contact(titrated) ++++ ++++
llth contact . ++++ ....
12th contact ++++ ....
13th contact ++++ ++++
14th contact * ++++ .+.+
15th contact(titrated) , ++++ ++++

.Titration Readings at 24 hours

Titration of filtrate from 18th contact

10-2 10-4 10"6 10-8
Beef.Ext.Broth ++++ ++++ ++++ ++++
Synthetic ++++ ++++ ++++ ++++

Titration of filtrate from 15th contact

++++
++++

++++
++++

++++
++++

++++
+¢++

BccreEXteBrOth
Synthetic

10-10 10-13
++++
++++

§+++
§+++

51

10-14
++ .
++++ -
++ —

52
Table XXV.

Experiments 10 and 11.

Note: Inlo and 1].,Synthetic media both with and
without activator were used. Lysis was
tested for as in 5 and 6; that is, by adding
one cc of the filtrate to beef extract broth
containing 1 drop of culture.

E23433 Culture: E. coli 70

Phage: M.K. 13 (Colon)
Beef Ext. Synthetic Synthetic
Broth (with activator) (without activator)
(lysis) (lysis) (lysis)
let contact ++++ ++++ +
and contact ++++ *+++ ?
3rd contact ++++ ++++ -

Exp.(ll) Culture: E. coli 70

Phage: M.K. 16 (Colon)
Beef Ext. Synthetic Synthetic
Broth (with activator) (without activator)
(Lysis) . (Lysis) (Lysis)
lst contact ++++ ++++ Bresent
2nd contact +++* +++* '
3rd contact *+++ *+++ ”
4th contact *+++ ++++ “
5th contact ++++ ++++ "
6th contact ++++ ++++ "
7th contact ++++ ++++ :

8th contact ++++

++++

53
Discussion

Contrary to expectations, the presence or absence

of peptone in culture medium in concentrations up to
10% had relatively little effect upon the ultimate
production of bacteriophage of moderately high titre.
Nor was there any marked advantage in one peptone over
another with the possible exception of Witte's which;
capecially in the higher concentrations, gave a low
yield. It would appear, then, that in the preparation
of bacteriOphage for clinical use, little or no peptone
need be used. In the presence of low concentrations of
peptone, however, bacteriOphage is apparently not success-
fully propagated.

There is, however, a very important consideration
that intervenes here which considerably modifies any
conclusions that may be drawn at the present time. Prop-
agation of bacteriOphage involves the serial transmission
of the principle from culture to culture with intervening
filtration. The filters customarily employed are made of
infusorial earth, asbestos or collodion. Those most comp
monly used are the Berkfeld, Mandler, Chamberland, and
Seitz. This introduces a conflicting factor in the trans-

mission of the principle for any loss of potency is as

54

conceivably occasioned by absorption to the filter as
by the action of any constituent of the medium. Thus,
although Larkum.and Semmes (12) reported that Seitz
filters exerted no appreciable effect on the bacter-
iophage, the mediums they employed were constant in
composition. In the work here reported, the Seitz
filter proved unsuited for serial transmission of
bacteriOphage when low concentrations of peptone were
employed, but no difficulty was encountered when the
higher concentrations were used. Although no further
attempt was made to investigate this phase of the
problem, it appears entirely warranted to to assume
that the loss of potency of bacteriOphage on serial
transmission was occasioned not so mmch by the effect
of the peptone upon the principle itself as by the ef-
fect of peptone on the filtration of the product. Al-
though this phase of the problem formed no part of'the
experiments planned, the observation could not be neg-
lected and is, therefore, presented here.

From the standpoint of practical use of the obserb
vation made, it might be said that in as much as we are
at present limited to certain types of filters in the

55

preparation of bacteriophage, care should be taken

to provide conditions enabling these filters to per-
form effectively; that is, to allow the passage of
bacteriophage. This is apparently assured in the
presence of moderately high concentrations of peptone.
In as much, however, as high concentrations of peptone
are undesirable in the material to be adapted to
clinical use, it is obvious that large lots of bacter-
iophage for therapeutic purposes must be made in a
medium containing little or no peptone, aid that
serial passages of bacteriophage to provide the 'start-
er' for each new lot of clinical material must be pre-
pared and carried along in.mediums containing higher

concentrations of peptone (5%-lo%).
Conclusions

The results of the comparative study of four
brands of commercial peptone added to beef extract
broth indicate that these peptones in concentrations
up to 10% exert no marked effect on the quantity of
bacteriOphage produced. High concentrations of peptones,
however, retard the pregress of lysis.

The propagation of bacteriophage through serial

transfers is materially affected by the presence of
high concentrations of peptone. A more active phage

56

results when higher concentrations of peptone are
incorporated in the medium.

Synthetic medium containing growth stimulating
substance is a suitable medium for the production
of bacteriOphage giving a product equal in amount
and potency to that produced in beef extract broth
with 1.0% peptone. The use of synthetic medium
without the growth stimulating substance is not
satisfactory for the production of bacteriophage.

1.

2.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

57

BibliOgraphy

Appelmans, R. Le bacteriOphage dans l'organisme.
Compt. rend. Soc. de biol., 1921, 85,722

Bail, 0. and S. Oineda. Der Abbaulebander Bakterien
dursch BakteriOphagen. Arch. F. Hyg., 1924,
xcii, 251.

Bordet, J. and M. Cuica. Autolyse microbienne et
Serum Antilytique. Compt. rend. soc. de Biol.,
1921. lxxxiv, 280

Burnet, F. Mt Recent Work on Biological Nature of
Bacteriophage. Trans. of Royal Soc. of Trop.
Med. and Hyg., Vol. xxvi, No. 5, March, 1933'

Caldwell, J. A. The Activity of an Anticolon
BacteriOphage in Synthetic Medium. Jour. Inf.
D180 VOle 39. Ange 19260 '

Difco Laboratory Publication, Peptones for Bacter-
iological Culture Media, May, 1932. .

Hadley, P. The Twort-d'Herelle phenomenon, Jour.
Info Dis. 42. 263-434, 1928.

D'Herelle, The BacteriOphage and its Behavior,
English Translation, Smith, Williams and
Wilkins Co.‘March, 1926.

Hughes, T. 2., Growth Requirement of Staphylococci.
Jour. of Bact. Vol. xxiii, No. 6, June, 1952.

Jordan, E. 0., General Bacteriology, W. B. Saunders 00.
Phil., 1926.

Kabeshima, T. Sur la nature du dit “Microbe Filtrait
BacteriOphage. dc d'Herelle. Compt. rend. Soc.
de Biol., 1921 lxxxiii, 219.

Larkum and Semmes, Filtration of BacteriOphage.
Joure BaOt.19 (3), Mare 19300 PP 213-2220

Krueger, A. P., A Method for the Quantitative Esti-
-mation of Bacteria in Suspensions. Jour. of
Gen. Phys. May 20, 1930, Vol. 13, No. 5, 555.

 

L

58

14. McAlpine, J. G. and Brigham, G. D., Some
Chemical Studies of Commercial
Bacteriological Peptones. Jour. of
Bact. Vol. 16, 1928, 251

15. Schoenlein and Levine, Compilation of
Culture Media. 1929

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