The inactivation of bacteriophage by substances from susceptible bacteria has been studied by Burnet (1) and Freeman (2). Its inactivation by antisera has been studied by Andrewes and Elford (3) and Burnet, Keogh, and Lush (4). Recently Ashenburg et al. (5) reported that saline solutions

of gum arabic, starch, or glycogen inactivated an anti-Klebsiella pneumoniae phage. 



We have studied in more detail the rate of phage inactivation by culture filtrates of the susceptible bacteria in order to determine the dependence of the process on phage and inhibitor concentrations

Ellis, Emory L.

Spizizen, John

English

PubMed

Caltech Authors - Main

T H E  R A T E  OF B A C T E R I O P H A G E  I N A C T I V A T I O N  BY 
F I L T R A T E S  OF E S C H E R I C H I A  COLI  C U L T U R E S  
BY EMORY L. ELLIS AND JOHN SPIZIZEN 
(From the William G. Kerckhoff Laboratories of the Biological Sciences, California Institute 
of Technology, Pasadena) 
(Received for publication, October 18, 1940) 
]2ffTRODUCTION 
The  inactivation of bacteriophage by  substances f rom susceptible bac- 
teria has been studied by  Burne t  (1) and Freeman (2). I t s  inact ivat ion by  
antisera has been studied by  Andrewes and Elford (3) and Bumet ,  Keogh, 
and Lush (4). Recent ly  Ashenburg et al. (5) reported tha t  saline solutions 
of gum arabic, starch, or glycogen inact ivated an anti-KlebsieUa pneu- 
mon/ae phage. 
We have studied in more detail  the rate of phage inact ivat ion by  culture 
filtrates of the susceptible bacteria in order to determine the dependence of 
the process on phage and inhibitor concentrations. 
EXPE]~ ~ N T A L  
The phage inactivating solution was prepared from culture filtrates of the susceptible 
strain of co//(B1) previously used in this laboratory (6). The organisms were grown in 
synthetic medium of the composition given by DelbrUck (7). With adequate aeration, 
these cultures attained a maximum plate count at 48 hours of more than 5 × 109 organ- 
isms per cc. 16 liter cultures were incubated for 8 days with aeration. The cells were 
then spun out in a Sharples supercentrifuge and the clear supernatant concentrated in 
vacuo at 35°C. to about one-tenth its volume. This material was then dialyzed in 
cellophane sausage casing against running tap water for 48 hours. The remaining solu- 
tion was then clarified in the centrifuge and further concentrated in vacuo at 35°C. 
It was again dialyzed, clarified in the centrifuge, and passed through a Seitz filter. The 
final product had a pH of 6.5, contained 2.5 mg./cc, total solids, and represented a 35 to 
1 concentrate of the initial filtrate of the 8 day bacterial cultures. This concentrated, 
dialyzed bacterial filtrate is called "filtrate" or simply "F" below. 
Diluted bacteriophage suspensions were prepared by 100-fold dilution in distilled 
water of fresh filtered phage lysates of susceptible co//cultures. The suspension then 
contained 7 X 106 phage particles per cc. as determined by the plaque counting method 
previously described (6). 
Suitable dilutions in nutrient broth of the phage inactivating filtrate were brought to 
temperature. To 0.9 cc. of such a dilution of F was added 0.1 cc. of a dilution in broth 
of stock phage adjusted to contain about 3 X 106 phage particles per cc. This mixture 
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438 RATE OF BACTERIOPHAGE INACTIVATION 
was incubated at the selected temperature and 0.1 cc. samples removed from time to 
time, the amount of active phage remaining being determined by plaque counts. 
Each experimental determination of active phage corresponded usually to counts of 
two or four plates, containing a total of 100 to 400 plaques. Thus the sampling error 
lies between 5 and 10 per cent. The efficiency of plating (6) may change in the course of 
the inactivation. The data on the later stages of the inactivation reaction cannot be 
interpreted quantitatively until this point is determined. 
Rate of Phage Inactivation at O°C. 
A progressive decrease in phage assay was noted with all concentrations 
of F tried (Fig. 1). This decrease was logarithmic in all cases until 95 per 
cent of the phage had been inactivated, after which the reactions became 
slower. The reaction is therefore first order with respect to phage, as was 
indicated by Burnet (4) for several phages active against B. dysenteriae. 
The rate is not proportional to the concentration of inactivator but 
nearly proportional to the square root of this value. The rate of phage 
inactivation may thus be expressed by the equation 
log (P). ,  t~(F)~ 
dt 
where (P) is the phage concentration, (F) the concentration of filtrate 
preparation, the undiluted material being assigned the arbitrary value 
unity, and k0 is a constant. Table I shows the values of k0 calculated from 
this equation, using the rates obtained graphically, for inactivator concen- 
trations from 0.001 to 0.04. At concentrations lower than these, the re- 
sults become uncertain and variable. 
The slower rate of inactivation of the last fraction of phage is discussed 
below. That  this decrease in rate did not result from exhaustion of inacti- 
vator substance is shown in the experiment plotted in Fig. 2. The inactiva- 
tion of phage was permitted to proceed until 95 per cent inactivation had 
been accomplished. Then more phage was added to the reaction vessel 
and its rate of inactivation followed. As the figure shows, the second 
quantity of phage was inactivated at the same rate as the first batch, indi- 
cating that the concentration of inactivator had not appreciably decreased. 
Rate of Pkage Inactivation at 37°C. 
At this temperature phage inactivation followed the logarithmic curve 
for only about the first 50 per cent, after which the inactivation became 
slower. Furthermore, the relationship of initial rate to F concentration is 
different; the rate is nearly proportional to the concentration of F, not its 
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-0~ 
e~ 
- |  
0 
I I I I 
MIN. 60 120 180 240 
T I M F  
FIO. I. The influence of F concentration on the rate of phage inactivation at 0°in 
broth. The numbers adjacent to the curves refer to the concentration of filtrate in the 
reaction mixture in arbitrary units. 
TABLE I 
The Influence of F Concentration on the Rale of Phage Inacti~lion at O°C. in Broth 
~') 
0.04 
0.005 
0.002 
0.001 
- d l o g  (_P) 
0.042 
0.015 
0.009 
0.006 
0.21 
0.21 
0.20 
0.19 
-0~ 
-I.C 
¢i.o 
~J 
I t l l (  
, I I 
40  80 120 
TlUl: 
F~o. 2. The inactivation of a second portion of phage, after the inactivation of 95 
per cent of a first portion, demonstrating the presence of excess inhibitor. The second 
portion of phage was added at 65 minutes. 
439 
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440 RATE OF BACTERIOPHAGE INACTIVATION 
square root. Fig. 3 shows the course of the inactivation reaction for three 
F concentrations, and Table II  shows the corresponding values of the 
reaction rate constant, kaT, calculated from the equation 
d log (P) _- k~(F) 
dt 
- 0 . . =  o 
-I~} 
09 
.a 
I I I I I I 
MIN. I0 20 30 40 50 60 
TIME 
FZG. 3. The course of phage inactivation by filtrate at 37°C. in broth. The numbers 
adjacent to the curves refer to the concentration of filtrate in the reaction mixture in 
arbitrary units. 
TABLE II 
The Influence of F Concentratgon on the Rate of Phage Irugtinalion al 37°C. in Brotl~ 
~) 
0.05 
0.02 
0.005 
--  d log  (_P) 
d4 
0.15 
0.05 
0.016 
/m 
3.0 
2.5 
3.2 
Clearly, the rates are here proportional to the concentration of F, not F t 
as at 0°C. Similar experiments at intermediate temperatures showed that 
the initial rates were proportional to (F) at 20°C., but became proportional 
to (F) °.'e at 6°C. 
Effect of Salt on the Inactivation 
The inhibitor substance does not pass through a dialysis membrane and 
is probably a large molecule. Surface forces can be expected to play an 
important r61e in these reactions. These surface forces can be readily 
changed by changes in electrolyte concentration. The course of the phage 
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E. L. ELLIS AND J. SPIZIZEN 441 
inactivation in the absence of significant amounts of electrolytes was in- 
vestigated, by diluting both phage and inactivator in distilled water instead 
of broth. The effects of the addition of various concentrations of sodium 
chloride were also studied. 
The addition to the broth used as the diluting medium of 25 per cent 
NaC1 stopped the inactivation reaction. No significant decrease in phage 
occurred in 2 hours incubation at 37°C. in a filtrate concentration of 0.1. 
Without salt, at this (F) concentration, the phage would have been 90 per 
cent inactivated in less than 5 minutes. Similarly when tested at 0 °this 
concentration of salt practically stopped the inactivation. On the other 
hand, salt concentrations of 0.5 per cent were found to increase the rate of 
inactivation. A 2.5 per cent concentration slightly decreased the rate of 
inactivation. In distilled water dilutions, where the only electrolytes were 
the impurities in the filtrate and phage, the inactivation did not proceed at 
an appreciable rate. Incubation of the phage without (F) under the same 
conditions of temperature and salt concentration resulted in no change in 
the amount of phage. This is contrary to a statement by Gratia (8) who 
attributed an inactivation of phage in a lysate with added salt to a direct 
action of the salt on phage. We believe that this effect of moderate salt 
concentrations in his case may have been not on phage directly but due 
to salt accelerated inactivation of phage by substances from the lysed 
bacteria. 
The effects of salt concentration led us to test whether phage which had 
been inactivated by F could be regenerated by agents known to be effective 
dissociating agents. High concentrations of various salts, or low concen- 
trations of soaps, were added to solutions in which phage had been inacti- 
vated with F, but without the slightest indication of the reactivation of any 
of the inactivated phage. These reagents alone had no effect on active 
phage, in any concentrations used. 
Phage Inactivation by Common Polysaccharides 
The inactivation of phage by several common polysaccharides was 
recently reported by Ashenburg el al. (5). We have confirmed their results 
with gum arabic and starch, with our phage, and have found in addition 
that inulin and acetylated gum arabic also will inactivate our coli-phage. 
The concentrations of starch or gum arabic needed to attain reasonable 
rates of inactivation (50 to 100 nag. per cc.) are greatly in excess of those 
required in the case of the specific substance from the bacteria. The 
preparations of inhibitor substance (F) used by us contained 2.5 mg. total 
solids per cc. Additional chemical work on this substance, still in progress, 
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442 RATE OF BACTERIOPHAGE INACTIVATION 
shows that considerably more than half of this must be considered impurity~ 
Therefore, it may be safely concluded that concentrations of inhibitor of 
less than 0.002 rag. per cc. produce a rapid inactivation of phage (Fig. 1). 
At 0°C., the inactivation with starch is too slow to be measured. The 
course of the reaction at 37 ° with starch is similar to that with the bacterial 
inactivator. The decrease in phage is logarithmic at first, becoming slower 
in the later stages. This rate is proportional to the starch concentration. 
This is especially interesting in view of the high specificity of culture filtrates 
as inactivating agents, a specificity which parallels the adsorption of phage 
by the corresponding bacteria (1). 
DISCUSSION 
The interpretation of these data in terms of a reaction mechanism is 
complicated by several factors. The absolute concentration of inhibitor 
substance is lmbnown, and the relative molecular concentrations of phage 
and inactivator cannot be determined. Furthermore, this phage has not 
been isolated in high enough concentrations to make it feasible to study the 
course of the reaction in the presence of excess phage instead of excess 
inactivator. The change with temperature in the dependence of the rate 
on F concentration makes the consideration of temperature coefficients 
difficult. For example, at very low F concentrations, the inactivation at 0 ° 
is faster than that at 37 ° for the same concentration of inactivator. 
We shall consider first only the initial rates, reserving for later discussion 
the slower rates attained after inactivation of the main fraction of phage. 
The assumption of the following series of reactions accounts for the observa- 
tions in a simple way: 
b + b ~  ~b (1) 
P -{- b,-~-P:b (2) 
P:b --* X O) 
The symbol P:  b represents a complex of phage and inactivator in which the 
phage retains temporarily full activity, while X represents the inactivated 
complex. "b" is the molecular species of the inactivator substance which 
combines with phage and "bb" is a dimolecular form which is inert toward 
phage. Reactions (1) and (2) are equilibria, and are rapid and reversible, 
and reaction (3) is the rate controlling irreversible step leading to inactiva- 
tion of phage. Equilibrium (1), from our data, must be temperature 
sensitive, being pushed to the left by an increase in temperature. Accord- 
ing to these reactions, phage is monovalent with respect to "b" in this 
inactivation process. 
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E. L. ELLIS AND J. SPIZIZEN 443 
T~klug these reactions as descriptive of the mechanism and with the 
assumptions stipulated above, a rate expression can be derived. The 
equilibrium constants for reactions 1 and 2 are respectively 
(bb) 
K ~ = - -  (4) 
(b)' 
(P.b) 
Ks = -- (5) 
(P)(b) 
The total concentration (all forms) of inactivator is 
(F) = (b) + 2(bb) (6) 
The rate of phage inactivation is 
d(P) 
- -  - -  . .  k ' ( P . b )  (7) 
d# 
which by substitution from (5) becomes 
d(P) 
- -- =, k'K, CP)(b) (S) 
d# 
Substituting for (b) from (4) and (6) gives 
d ( P )  . .  k'K, p 
When the inactivator is chiefly in the "bb" form, K1 is large and the rate 
expression becomes 
d(P) ko(e)(F)t where k. - k'K,V'2 
d~ " 2K| 
which is the observed relationship at 0°C. When the inactivator is largely 
in the dissociated "b" form, the rate expression is substantially equation (8) 
d(P) 
- -  - k , ( P ) ( F )  w h e r e / ~  - k ' K i  
dt  
which is the observed relation at  37°C. 
Although the equations assumed above lead in this way to rate expres- 
sions which agree with the experimental findings, this does not prove their 
reality. However, they permit  discussion of the observations on definite 
terms. 
The influence of salt on the course of the reaction may be described as an 
influence on reaction (2) in which a mluimal electrolyte concentration is 
necessary to permit  the combination of P and b, and high concentration 
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444 RATE OF BACTERIOPHAGE INACTIVATION 
dissociates the P-b complex, preventing the inactivation by reaction (3). 
The precipitin reaction provides an analogy. Here, the presence of moder- 
ate amounts of electrolytes is necessary for the combination of antigen and 
antibody to proceed, whereas in strong electrolyte solutions, the combina- 
tion does not occur (9). 
After a part of the phage has been inactivated the rate decreased (see Fig. 
3), indicating that a fraction of the phage particles was more slowly inacti- 
vated than the remainder. Schlesinger (10), and Delbrtick (11) reported 
that in the adsorption on live and dead bacteria, there is a fraction of the 
phage which is less reactive than the remainder. This variation in adsorp- 
tion rate of different fractions of the phage cannot explain the results ob- 
tained in the filtrate inactivation. At 0°C. the phage inactivation was first 
order until after inactivation of more than 95 per cent of the phage (see 
Fig. 1) while at 37°C. the rate was first order during the inactivation of 
about 90 per cent when (F) = 0.05, and about 65 per cent when (F) = 0.02 
(see Fig. 3). Similar results were obtained in 2.5 per cent saline solutions. 
From these results, and those previously appearing in the literature (3 
and 4) it appears that there are two competing processes, one inactivating 
phage (the logarithmic part of the inactivation curve) and the other produc- 
ing "partly inactivated phage." The second process, if more temperature 
sensitive than the first, would result in logarithmic inactivation over a 
greater range at 0 ° than at 37°C. The inactivation of the "partially in- 
activated phage" might occur by (a) reversal of the second process to permit 
inactivation by the first mechanism, or (b) a direct inactivation of the 
partially inactivated phage. 
These considerations lead to the view that phage inactivation by filtrates 
is more than simply combination of phage with inhibitor substance, and 
that the extent of phage inactivation is not a direct measure of the extent 
of this combination. This view is supported by the observation of An- 
drewes and Elford (3) that phage ceases to pass through a membrane filter 
immediately after mixing with antiserum, indicating in this case the forma- 
tion of a phage-antibody complex still retaining phage activity. In this 
regard, phage is similar to catalase (12) and urease (13), where the combina- 
tion of the enzyme and anti-enzyme does not result in complete loss of 
activity. 
SUMM~RY 
1. The rate of inactivation of an anti-coli phage by filtrates of cultures of 
the homologous bacteria has been studied. 
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E. L. ELLIS AND ~. SPIZIZEN 445 
2. The inactivation rate at 37°C. is proportional to phage concentration 
and filtrate concentration. 
3. At 0°C. the rate of phage inactivation becomes proportional to the 
square root of the filtrate concentration. 
4. A reaction scheme to account for these observations is suggested and 
discussed. 
5. This coli-phage is also inactivated by relatively large concentrations of 
soluble starch, inulin, gum arabic, and acetylated gum arabic. 
6. The inactivation is markedly influenced by salt concentration, being 
rapid at moderate salt concentrations and slow at high or extremely low 
salt concentrations. 
7. The inactivated phage cannot be regenerated by high salt concentra- 
tions, or by soaps. 
One of us (E. L. E.) wishes to acknowledge a grant in aid from Mrs. 
Seeley W. Mudd. 
REFERENCES 
1. Burner, F. M., J. Path. and Bazt., 1934, 38, 285. 
2. Freeman, M., A~tra1~n J. Exp. Biol. and Med. So., 1937, 1§, 221. 
3. Andrewes, C. H., and Elford, W. J., Brit. J. Exp. Pa2h., 1933, 14, 367, 376. 
4. Burnet, F. M., Keogh, E. V., and Lush, D., AusCralmn J. Exp. Biol. and Med. So., 
1937, ].5~ 227. 
5. Ashenburg, N. J., Sandholzer, L. A., Scherp, H. W., and Berry, G. P., J. Ba~t., 
1940, 39, 71. 
6. Ellis, E. L., and Delbriick, M., J. Gen. Physiol., 1939, 29., 365. 
7. Delb~ck, M., J. Gen. Physiol., 1940, 23, 643. 
8. Gratia, A., Compt. rend. Soc. biol., 1939, 132, 62. 
9. Heidelberger, M., Kendall, F. E., and Teorell, T., J. Exp. Med., 1936, 63, 819. 
10. Schlesinger, M., Z. Hyg. u. Infectionskrankh., 1932, 1].4, 136. 
11. Delbrfick, M., J. Gen. Physiol., 1940, 9.3, 631. 
12. Campbell, D. H., and Fourt, L., J. Biol. Chem., 1939,129, 385. 
13. Kirk, J. S., and Sumner, J. B., J. Biol. Chem., 1931, ~p 21. 
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The rate of bacteriophage inactivation by filtrates of Eschericia coli cultures

THE RATE OF BACTERIOPHAGE INACTIVAT ION BY 
F ILTRATES OF ESCHERICHIA  COLI CULTURES 
BY EMORY L. ELLIS AND JOHN SPIZIZEN 
(From the William G. Kerckhoff Laboratories ofthe Biological Sciences, California Institute 
of Technology, Pasadena) 
(Received for publication, October 18, 1940) 
]2ffTRODUCTION 
The inactivation of bacteriophage by substances from susceptible bac- 
teria has been studied by Burnet (1) and Freeman (2). Its inactivation by 
antisera has been studied by Andrewes and Elford (3) and Bumet,  Keogh, 
and Lush (4). Recently Ashenburg et al. (5) reported that saline solutions 
of gum arabic, starch, or glycogen inactivated an anti-KlebsieUa pneu- 
mon/ae phage. 
We have studied in more detail the rate of phage inactivation by culture 
filtrates of the susceptible bacteria in order to determine the dependence of
the process on phage and inhibitor concentrations. 
EXPE]~ ~NTAL 
The phage inactivating solution was prepared from culture filtrates of the susceptible 
strain of co//(B1) previously used in this laboratory (6). The organisms were grown in 
synthetic medium of the composition given by DelbrUck (7). With adequate aeration, 
these cultures attained a maximum plate count at 48 hours of more than 5 × 109 organ- 
isms per cc. 16 liter cultures were incubated for 8 days with aeration. The cells were 
then spun out in a Sharples upercentrifuge and the clear supernatant concentrated in 
vacuo at 35°C. to about one-tenth its volume. This material was then dialyzed in 
cellophane sausage casing a ainst running tap water for 48 hours. The remaining solu- 
tion was then clarified in the centrifuge and further concentrated in vacuo at 35°C. 
It was again dialyzed, clarified in the centrifuge, and passed through a Seitz filter. The 
final product had a pH of 6.5, contained 2.5 mg./cc, total solids, and represented a 35 to 
1 concentrate of the initial filtrate of the 8 day bacterial cultures. This concentrated, 
dialyzed bacterial filtrate is called "filtrate" or simply "F" below. 
Diluted bacteriophage suspensions were prepared by 100-fold dilution in distilled 
water of fresh filtered phage lysates of susceptible co//cultures. The suspension then 
contained 7 X 106 phage particles per cc. as determined by the plaque counting method 
previously described (6). 
Suitable dilutions in nutrient broth of the phage inactivating filtrate were brought to 
temperature. To 0.9 cc. of such a dilution of F was added 0.1 cc. of a dilution in broth 
of stock phage adjusted to contain about 3 X 106 phage particles per cc. This mixture 
437 
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438 RATE OF BACTERIOPHAGE INACTIVATION 
was incubated at the selected temperature and 0.1 cc. samples removed from time to 
time, the amount of active phage remaining being determined by plaque counts. 
Each experimental determination of active phage corresponded usually to counts of 
two or four plates, containing a total of 100 to 400 plaques. Thus the sampling error 
lies between 5and 10 per cent. The efficiency of plating (6) may change in the course of 
the inactivation. The data on the later stages of the inactivation reaction cannot be 
interpreted quantitatively until this point is determined. 
Rate of Phage Inactivation at O°C. 
A progressive decrease in phage assay was noted with all concentrations 
of F tried (Fig. 1). This decrease was logarithmic in all cases until 95 per 
cent of the phage had been inactivated, after which the reactions became 
slower. The reaction is therefore first order with respect o phage, as was 
indicated by Burnet (4) for several phages active against B. dysenteriae. 
The rate is not proportional to the concentration of inactivator but 
nearly proportional to the square root of this value. The rate of phage 
inactivation may thus be expressed by the equation 
log (P)., t~(F)~ 
dt 
where (P) is the phage concentration, (F) the concentration of filtrate 
preparation, the undiluted material being assigned the arbitrary value 
unity, and k0 is a constant. Table I shows the values of k0 calculated from 
this equation, using the rates obtained graphically, for inactivator concen- 
trations from 0.001 to 0.04. At concentrations lower than these, the re- 
sults become uncertain and variable. 
The slower rate of inactivation of the last fraction of phage is discussed 
below. That this decrease in rate did not result from exhaustion of inacti- 
vator substance is shown in the experiment plotted in Fig. 2. The inactiva- 
tion of phage was permitted to proceed until 95 per cent inactivation had 
been accomplished. Then more phage was added to the reaction vessel 
and its rate of inactivation followed. As the figure shows, the second 
quantity of phage was inactivated at the same rate as the first batch, indi- 
cating that the concentration f inactivator had not appreciably decreased. 
Rate of Pkage Inactivation at 37°C. 
At this temperature phage inactivation followed the logarithmic urve 
for only about the first 50 per cent, after which the inactivation became 
slower. Furthermore, the relationship of initial rate to F concentration is 
different; the rate is nearly proportional to the concentration f F, not its 
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-0~ 
e~ 
- |  
0 
I I I I 
MIN. 60 120 180 240 
T IMF 
FIO. I. The influence of F concentration on the rate of phage inactivation at 0°in 
broth. The numbers adjacent to the curves refer to the concentration of filtrate in the 
reaction mixture in arbitrary units. 
TABLE I 
The Influence of F Concentration on the Rale of Phage Inacti~lion at O°C. in Broth 
~') 
0.04 
0.005 
0.002 
0.001 
- d l og  (_P) 
0.042 
0.015 
0.009 
0.006 
0.21 
0.21 
0.20 
0.19 
-0~ 
-I.C 
¢i.o 
~J 
It l l (  
, I I 
40 80 120 
TlUl: 
F~o. 2. The inactivation of a second portion of phage, after the inactivation of 95 
per cent of a first portion, demonstrating the presence of excess inhibitor. The second 
portion of phage was added at 65 minutes. 
439 
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440 RATE OF BACTERIOPHAGE INACTIVATION 
square root. Fig. 3 shows the course of the inactivation reaction for three 
F concentrations, and Table II shows the corresponding values of the 
reaction rate constant, kaT, calculated from the equation 
d log (P) _- k~(F) 
dt 
- 0 . . =  o 
-I~} 
09 
.a 
I I I I I I 
MIN. I0 20 30 40 50 60 
TIME 
FZG. 3. The course of phage inactivation by filtrate at 37°C. in broth. The numbers 
adjacent to the curves refer to the concentration f filtrate in the reaction mixture in 
arbitrary units. 
TABLE II 
The Influence of F Concentratgon the Rate of Phage Irugtinalion al 37°C. in Brotl~ 
~) 
0.05 
0.02 
0.005 
-- d log (_P) 
d4 
0.15 
0.05 
0.016 
/m 
3.0 
2.5 
3.2 
Clearly, the rates are here proportional to the concentration f F, not F t 
as at 0°C. Similar experiments at intermediate mperatures showed that 
the initial rates were proportional to (F) at 20°C., but became proportional 
to (F) °.'e at 6°C. 
Effect of Salt on the Inactivation 
The inhibitor substance does not pass through a dialysis membrane and 
is probably a large molecule. Surface forces can be expected to play an 
important r61e in these reactions. These surface forces can be readily 
changed by changes in electrolyte concentration. The course of the phage 
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E. L. ELLIS AND J. SPIZIZEN 441 
inactivation in the absence of significant amounts of electrolytes was in- 
vestigated, by diluting both phage and inactivator in distilled water instead 
of broth. The effects of the addition of various concentrations of sodium 
chloride were also studied. 
The addition to the broth used as the diluting medium of 25 per cent 
NaC1 stopped the inactivation reaction. No significant decrease in phage 
occurred in 2 hours incubation at 37°C. in a filtrate concentration f 0.1. 
Without salt, at this (F) concentration, the phage would have been 90 per 
cent inactivated in less than 5 minutes. Similarly when tested at 0 °this 
concentration f salt practically stopped the inactivation. On the other 
hand, salt concentrations of 0.5 per cent were found to increase the rate of 
inactivation. A 2.5 per cent concentration slightly decreased the rate of 
inactivation. In distilled water dilutions, where the only electrolytes were 
the impurities in the filtrate and phage, the inactivation did not proceed at 
an appreciable rate. Incubation of the phage without (F) under the same 
conditions of temperature and salt concentration resulted in no change in 
the amount of phage. This is contrary to a statement by Gratia (8) who 
attributed an inactivation of phage in a lysate with added salt to a direct 
action of the salt on phage. We believe that this effect of moderate salt 
concentrations in his case may have been not on phage directly but due 
to salt accelerated inactivation of phage by substances from the lysed 
bacteria. 
The effects of salt concentration led us to test whether phage which had 
been inactivated by F could be regenerated byagents known to be effective 
dissociating agents. High concentrations of various salts, or low concen- 
trations of soaps, were added to solutions in which phage had been inacti- 
vated with F, but without he slightest indication of the reactivation ofany 
of the inactivated phage. These reagents alone had no effect on active 
phage, in any concentrations used. 
Phage Inactivation by Common Polysaccharides 
The inactivation of phage by several common polysaccharides was 
recently reported by Ashenburg el al. (5). We have confirmed their results 
with gum arabic and starch, with our phage, and have found in addition 
that inulin and acetylated gum arabic also will inactivate our coli-phage. 
The concentrations of starch or gum arabic needed to attain reasonable 
rates of inactivation (50 to 100 nag. per cc.) are greatly in excess of those 
required in the case of the specific substance from the bacteria. The 
preparations of inhibitor substance (F) used by us contained 2.5 mg. total 
solids per cc. Additional chemical work on this substance, still in progress, 
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442 RATE OF BACTERIOPHAGE INACTIVATION 
shows that considerably more than half of this must be considered impurity~ 
Therefore, it may be safely concluded that concentrations of inhibitor of 
less than 0.002 rag. per cc. produce a rapid inactivation ofphage (Fig. 1). 
At 0°C., the inactivation with starch is too slow to be measured. The 
course of the reaction at 37 ° with starch is similar to that with the bacterial 
inactivator. The decrease in phage is logarithmic at first, becoming slower 
in the later stages. This rate is proportional to the starch concentration. 
This is especially interesting inview of the high specificity of culture filtrates 
as inactivating agents, a specificity which parallels the adsorption of phage 
by the corresponding bacteria (1). 
DISCUSSION 
The interpretation of these data in terms of a reaction mechanism is
complicated by several factors. The absolute concentration of inhibitor 
substance is lmbnown, and the relative molecular concentrations of phage 
and inactivator cannot be determined. Furthermore, this phage has not 
been isolated in high enough concentrations to make it feasible to study the 
course of the reaction in the presence of excess phage instead of excess 
inactivator. The change with temperature in the dependence of the rate 
on F concentration makes the consideration of temperature coefficients 
difficult. For example, at very low F concentrations, the inactivation at 0 ° 
is faster than that at 37 ° for the same concentration f inactivator. 
We shall consider first only the initial rates, reserving for later discussion 
the slower rates attained after inactivation of the main fraction of phage. 
The assumption of the following series of reactions accounts for the observa- 
tions in a simple way: 
b + b~ ~b (1) 
P -{- b,-~-P:b (2) 
P:b --* X O) 
The symbol P:  b represents a complex of phage and inactivator in which the 
phage retains temporarily full activity, while X represents he inactivated 
complex. "b" is the molecular species of the inactivator substance which 
combines with phage and "bb" is a dimolecular form which is inert toward 
phage. Reactions (1) and (2) are equilibria, and are rapid and reversible, 
and reaction (3) is the rate controlling irreversible step leading to inactiva- 
tion of phage. Equilibrium (1), from our data, must be temperature 
sensitive, being pushed to the left by an increase in temperature. Accord- 
ing to these reactions, phage is monovalent with respect o "b" in this 
inactivation process. 
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E. L. ELL IS  AND J. SP IZ IZEN 443 
T~klug these reactions as descriptive of the mechanism and with the 
assumptions tipulated above, a rate expression can be derived. The 
equilibrium constants for reactions 1and 2 are respectively 
(bb) 
K~=- -  (4) 
(b)' 
(P.b) 
Ks = - -  (5) 
(P)(b) 
The total concentration (all forms) of inactivator is 
(F) = (b) + 2(bb) (6) 
The rate of phage inactivation is 
d(P) 
- -  - -  . .  k ' (P .b )  (7) 
d# 
which by substitution from (5) becomes 
d(P) 
- - -  =, k'K, CP)(b) (S) 
d# 
Substituting for (b) from (4) and (6) gives 
d(P)  . .  k'K, p 
When the inactivator is chiefly in the "bb" form, K1 is large and the rate 
expression becomes 
d(P) ko(e)(F)t where k. - k'K,V'2 
d~ " 2K|  
which is the observed relationship at 0°C. When the inactivator is largely 
in the dissociated "b" form, the rate expression is substantially equation (8) 
d(P) 
- -  - k , (P ) (F )  where/~ - k 'K i  
dt 
which is the observed relation at 37°C. 
Although the equations assumed above lead in this way to rate expres- 
sions which agree with the experimental findings, this does not prove their 
reality. However, they permit discussion of the observations on definite 
terms. 
The influence of salt on the course of the reaction may be described as an 
influence on reaction (2) in which a mluimal electrolyte concentration is
necessary to permit the combination of P and b, and high concentration 
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444 RATE OF BACTERIOPHAGE INACTIVATION 
dissociates the P-b complex, preventing the inactivation by reaction (3). 
The precipitin reaction provides an analogy. Here, the presence of moder- 
ate amounts of electrolytes i  necessary for the combination of antigen and 
antibody to proceed, whereas in strong electrolyte solutions, the combina- 
tion does not occur (9). 
After a part of the phage has been inactivated the rate decreased (see Fig. 
3), indicating that a fraction of the phage particles was more slowly inacti- 
vated than the remainder. Schlesinger (10), and Delbrtick (11) reported 
that in the adsorption on live and dead bacteria, there is a fraction of the 
phage which is less reactive than the remainder. This variation in adsorp- 
tion rate of different fractions of the phage cannot explain the results ob- 
tained in the filtrate inactivation. At 0°C. the phage inactivation was first 
order until after inactivation of more than 95 per cent of the phage (see 
Fig. 1) while at 37°C. the rate was first order during the inactivation of 
about 90 per cent when (F) = 0.05, and about 65 per cent when (F) = 0.02 
(see Fig. 3). Similar results were obtained in 2.5 per cent saline solutions. 
From these results, and those previously appearing in the literature (3 
and 4) it appears that there are two competing processes, one inactivating 
phage (the logarithmic part of the inactivation curve) and the other produc- 
ing "partly inactivated phage." The second process, if more temperature 
sensitive than the first, would result in logarithmic inactivation over a 
greater ange at 0 ° than at 37°C. The inactivation of the "partially in- 
activated phage" might occur by (a) reversal of the second process to permit 
inactivation by the first mechanism, or (b) a direct inactivation of the 
partially inactivated phage. 
These considerations lead to the view that phage inactivation by filtrates 
is more than simply combination of phage with inhibitor substance, and 
that the extent of phage inactivation is not a direct measure of the extent 
of this combination. This view is supported by the observation of An- 
drewes and Elford (3) that phage ceases to pass through a membrane filter 
immediately after mixing with antiserum, indicating in this case the forma- 
tion of a phage-antibody complex still retaining phage activity. In this 
regard, phage is similar to catalase (12) and urease (13), where the combina- 
tion of the enzyme and anti-enzyme does not result in complete loss of 
activity. 
SUMM~RY 
1. The rate of inactivation of an anti-coli phage by filtrates of cultures of 
the homologous bacteria has been studied. 
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E. L. ELLIS AND ~. SPIZIZEN 445 
2. The inactivation rate at 37°C. is proportional to phage concentration 
and filtrate concentration. 
3. At 0°C. the rate of phage inactivation becomes proportional to the 
square root of the filtrate concentration. 
4. A reaction scheme to account for these observations is suggested and 
discussed. 
5. This coli-phage isalso inactivated by relatively large concentrations of 
soluble starch, inulin, gum arabic, and acetylated gum arabic. 
6. The inactivation is markedly influenced by salt concentration, being 
rapid at moderate salt concentrations and slow at high or extremely low 
salt concentrations. 
7. The inactivated phage cannot be regenerated by high salt concentra- 
tions, or by soaps. 
One of us (E. L. E.) wishes to acknowledge a grant in aid from Mrs. 
Seeley W. Mudd. 
REFERENCES 
1. Burner, F. M., J. Path. and Bazt., 1934, 38, 285. 
2. Freeman, M., A~tra1~n J. Exp. Biol. and Med. So., 1937, 1§, 221. 
3. Andrewes, C. H., and Elford, W. J., Brit. J. Exp. Pa2h., 1933, 14, 367, 376. 
4. Burnet, F. M., Keogh, E. V., and Lush, D., AusCralmn J. Exp. Biol. and Med. So., 
1937, ].5~ 227. 
5. Ashenburg, N. J., Sandholzer, L. A., Scherp, H. W., and Berry, G. P., J. Ba~t., 
1940, 39, 71. 
6. Ellis, E. L., and Delbriick, M., J. Gen. Physiol., 1939, 29., 365. 
7. Delb~ck, M., J. Gen. Physiol., 1940, 23, 643. 
8. Gratia, A., Compt. rend. Soc. biol., 1939, 132, 62. 
9. Heidelberger, M., Kendall, F. E., and Teorell, T., J. Exp. Med., 1936, 63, 819. 
10. Schlesinger, M., Z. Hyg. u. Infectionskrankh., 1932, 1].4, 136. 
11. Delbrfick, M., J. Gen. Physiol., 1940, 9.3, 631. 
12. Campbell, D. H., and Fourt, L., J. Biol. Chem., 1939,129, 385. 
13. Kirk, J. S., and Sumner, J. B., J. Biol. Chem., 1931, ~p 21. 
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p 21. o n

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The rate of bacteriophage inactivation by filtrates of Eschericia coli cultures

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