A COMPARATIVE STUDY OF THE EFFECT OF VARIOUS GERMICIDES ON THE VIABILITY AND INHIBITION OF CERTAIN RESPIRATORY ENZYMES OF GONOCOCCUS

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University Microfilms
300 N o rth Z e e b R oad
Ann A rb o r, M ic h ig a n 461C6
A X ero x E d u c a tio n C o m p a n y
LD3907
.G7
Bucca, Matthew Anthony, 19081942
A comparative study of the effect of
.B85
various germicides on the viability
and inhibition of certain respiratory
enzymes of gonococcus...
cNew Yorks
1942.
3p.l.,150 typewritten leaves.
tables
(2 fold.) diagrs. 29cm.
Thesis (Ph.D.) - New York university,
Graduate school, 1942.
Bibliography: p.141-150.
A84660
^
SMf ti-t r
Xerox University Microfilms,
T H IS D IS S E R T A T IO N
Ann A rbor, M ich ig an 48106
H A S B E E N M IC R O F IL M E D
E X A C T L Y A S R E C E IV E D .
A COMPAPATIVE STUDY OF THE EFFECT OF VARIOUS GERMICIDES
OK THE VIABILITY AND INHIBITION OF CERTAIN
RESPIRATORY ENZYMES OF GONOCOCCUS
/
^ t-
Matthew AV Bucca
A DISSERTATION
in the Department of Bacteriology
submitted to the faculty of the Graduate School of Arts and
Science in partial fulfillment of the requirements for the degree
of Doctor of Philosophy.
April, 1942
PLEASE NOTE:
Some pages may have
indistinct print.
F i 1m e d a s
University Microfilms,
r e c e iv e d .
A Xerox Education Company
ACKNOWLEDGMENT
The author takes this opportunity to express his
appreciation to all those who have assisted him in this
investigation.
Particular acknowledgment is made to Drs. C. Chester
Stock and Julius A. Klosterman, both formerly of New York
University Department of Bacteriology, and to Drs. Stacey
F. Howell and J. Durward Thayer of the United States
Public Health Service.
The author also wishes to acknowl­
edge the advice and assistance of Dr. Colin M. MacLeod in
the presentation of the material.
The author greatly appreciates the privileges extended
him by Dr. John F. Mahoney, director of the Venereal Disease
Research Laboratory, United States Marine Hospital, Staple­
ton, New York, in his generous provision of laboratory
facilities.
This work is dedicated to the author's wife, Helen
Bleniasz Bucca, because her constant Influence and inspira­
tion have proved Invaluable assets.
TABLE OF CONTENTS
I.
II.
III.
Introduction.........................................
1
Review of the Literature....................
3
Experimental.........................................
16
A. Methods...........................................
16
1. Glas sware......................................
16
2. Culture Media..................................
16
3. Suspending and Diluting Fluids................
19
4. Gonococcides Employed.........................
22
5. History of the Gonococcus Strain Used........
25
6. General Procedures Used for Conducting
Germicide and Enzyme Inhibition Tests......
25
7. The Germicidal Test...........................
27
8. Enzymatic Tests...............................
28
A. Dehydrogenases.............................
29
B. The Catalase Test..........................
37
C. The Peroxidase Test........................
40
D. The Indophenol Oxidase Test (Cytochrome
Oxidase)..................................
44
B. Presentation of Results..........................
50
1. The Germicidal Tests..........................
50
2. The Enzyme Inhibition Tests...................
58
C. Supplementary Studies............................
107
1. Supplementary Studies on the Nature of
Bacterial Peroxidase........................
107
2. Effect of Heat on GonococcalPeroxidase.......
114
3. Conditions necessary for the Quantitative
Indophenol Oxidase (Cytochrome Oxidase)
Test.........................................
118
P age
4. Effect of Potassium Permanganate on
the Indophenol Oxidase Test................ 123
5. The In Vitro Effect of sulfanilamide
on Gonococcus............................... 127
6. Studies on the Effect of Age of the
Culture on Glucose and Pyruvic
Dehydrogenases.............................. 129
IV.
General Discussion..................................
130
V.
Summary and Conclusions.............................
138
Bibliography
141
VI.
I. INTRODUCTION.
The mechanism of death of bacteria has been the subject of
numerous Investigations and a voluminous literature on the subject
has accumulated.
Various general theories have been advanced to
explain the process of physical and chemical disinfection.
One
such theory, the enzyme destruction theory of Isaacs (1932), re­
lates the cause of death of bacteria to the destruction or inhibi­
tion of the enzyme components of the cell.
Analogies based on the
known properties of enzymes are advanced by Isaacs to explain some
common observations of the prooess of disinfection (Gay, 1935).
That a definite relationship exists between disinfection and
the inhibition of metabolic activity of an organism as measured
by the manometric method, has been recognized by Bronfenbrenner,
Hershey and Doubly (1939).
In fact, the inhibition of carbon
dioxide production by yeast as a basis for the evaluation of cer­
tain germicides, is a procedure that has been advocated by several
investigators (Branham, 1929).
Increased interest in the relationship between respiratory
activity and viability of organisms in the course of disinfection
has been shown by the reoent appearance of several papers on the
subject (Casman and Rettger, 1933; Edwards and Rettger, 1937;
Hershey, 1939).
Evaluation of germicides by the manometric method
has also been carried out.
8uoh an evaluation is based on the
inhibition of the respiratory activity of the test suspension used.
Many therapeutic agents have been employed for the treatment
of gonorrhea and their selection has been based chiefly on the
toxicity and gonoooooldal behavior of the drug.
The speolflo
action of these drugs on the gonococcus is still largely a matter
of theory or conjecture.
The work of Barron and Miller (1932) has
shown that the gonococous oxidizes a relatively small number of
substrates and has no endogenous respiration.
The enzymatic functions of the gonococcus are relatively lim­
ited in comparison with many other bacteria.
It seemed, therefore,
that this organism would be well suited to the study of the action
of disinfectants.
As far as could be ascertained, a comprehensive
study of the relationship between the effect of various gonooocoides
upon enzyme activity and the viability of the gonococcus has not
been made previously.
The object of this investigation was to make a comparative
study of the effect of germicides on the viability and on the in­
hibition of various respiratory enzymes.
The germicides used were
silver nitrate, protargol, neo-sllvol, silver nuoleinate, argyrol,
merthiolate, potassium permanganate and sulfanilamide.
The enzymes
seleoted were lactic dehydrogenase, glyceric dehydrogenase, oatalase,
peroxidase and indophenol (cytochrome) oxidase.
The gonoooocides
seleoted represented some of the drugs generally used for the treat­
ment of gonorrhea at the time this investigation was commenced.
The enzyme systems studied were selected because of the consider­
able body of knowledge available concerning these systems, not
only in this and other speoies of bacteria, but also in plant and
animal tissues.
II. REVIEW OF THE LITERATURE.
Numerous investigations have been made on the subject of the
viability, resistance and susceptibility of the gonococcus to phys­
ical and chemical agents.
Pertaining to this broad subject, Thomas
and Bayne-Jones (1936), reporting on a survey of research on the
gonococcus and gonococcal infections, state in their review
(pg. 72):
"....that much of this work has been inadequately controlled, and
that the results of tests in laboratories have been too hastily
and uncritically applied to conditions in patients with gonococcal
infections.
This is especially true of the forms of therapy based
upon the results of test-tube experiments with disinfectants."
An abundant literature exists on the effect of various ohemlcal agents on the viability of gonococcus.
Silver salts and
organic mercurials particularly have been tested.
Muoh of this
work should be reacoessed in the light of our present knowledge
of the physiology of the gonococous.
Germicidal Tests.
Kolmer, Solis-Cohen and Heist (1917) undertook
to evaluate the various techniques then known for conducting the
germicidal tests.
One of the techniques used was the so-oalled
"centrifuge method" in which suspensions of organisms are exposed
to the germicide for variable periods of time, following which
they are preoipitated by centrifugation and washed free of the
germicide.
Following these manipulations, cultures of the washed
suspensions are made to determine whether or not the germicide
has oaused the death of the organisms.
Using the pneumooooous as
the test organism, these investigators oonoluded that the "centri­
fuge method" yields "sharp and definite" results.
The "centrifuge method" for testing the effect of various chem­
ical agents on the gonococcus has been used by other workers.
Davis
and Swartz (1920) studied a large number of germicides and made
various modifications in the technique.
They claimed several ad­
vantages for the "centrifuge method", particularly the opportunity
of using large quantities of gonococous suspension in the test,
and of washing the suspensions free of the germicide to avoid bac­
teriostatic action due to the presence of excess drug.
In the
studies of Davis and Swartz the gonococci were suspended in physio­
logical saline.
For this reason the bactericidal effect of com­
pounds which react with sodium chloride, such as various silver
salts, could not be determined.
Oxidative Enzymes.
Enzymatic Activities of Gonococcus.
The mo3t
extensive work on the enzymatic composition of the gonococcus has
been carried out by Barron and his collaborators (1932, 1933, 1934,
1936, 1939) with particular reference to the biological oxidations
of this organism.
The procedures, with occasional exceptions, were
conducted in the usual Barcroft-Warburg manometers.
A summary of
the oxidation of glucose as deduced by Barron and Miller (1932)
Is incorporated in the following equations:
c6h12°6 ■ff1y°0lytl0 enzyme ^ OHg.CHOH. C00H (fermentation)
(glucose)
(lactic acid)
C H 3.CHOH.COOH ♦ frog « - hy^°xy°xidase ,»CH3 .C0 .C00H ♦ H20 (first step
(lactic acid)
(pyruvic acid)
oxidation;
C H 3 .CO.COOH ♦ £ 0 g
(pyruvic acid)
gC-ketonoxldase ,^OH5 .COOH ♦ COo
(acetic aoid)
CflHioOg f 02------► 2CH3 .COOH + 2C02 ♦ 2H20.
(second step
oxidation)
(complete oxidation
equation for glucose)
Of the three enzymes, Barron and oo-workers found that alphahydroxyoxidase was the hardiest ferment.
The glycolytic enzyme
and alpha-ketonoxidase were easily destroyed when the gonococcus
suspension was permitted to stand or when the reaction of the
suspension was made alkaline.
It was suggested that all alpha-
hydroxy acids were oxidized by the same enzyme, alpha-hydroxyoxidase, thus indicating group specificity; the differences shown
in the rates of oxidation of the different alpha-hydroxy acids
were held to be due to various degrees of affinity between the
enzyme and substrates,
Barron and Hastings (1933) suggested
also that alpha-hydroxyoxidase consists of two separate enzyme
components: first, an activating coenzyme which activates the
substrate and is similar to dehydrogenase in the Wieland-Thunberg
terminology; and second, an oxidizing enzyme which oxidizes the
activated substrate and is similar to Warburg-Keilin'a cytochromecytochrorae oxidase system.
It was shown that in the oxidation of
lactic acid by alpha-hydroxyoxldase, the activated lactate does
not leave the enzyme surface until after collision with the
oxidizing catalyst has occurred and that oxidation is brought
about at the surface of the activating enzyme.
Green (1940) remarks that the behavior of Barron's oxidizing
enzyme component of alpha-hydroxyoxidase towards heat and res­
piratory inhibitors points to the identity of that system with
the Keilin cytochrome oxidase system.
The system laetate-
enzyme-pyruvate is a sluggish reversible redox system (Barron
and Hastings, 1934).
Alpha-ketonoxidase of the gonococcus oonsists also of two
components (Barron, 1936),
First, the activating enzyme
(dehydrogenase); and second, the oxidizing enzyme whose nature is
unknown and which is different from the alpha-hydroxyoxldase.
Dlphosphothiamine enhances the oxidation of pyruvic a d d by the
gonococous and may be a prosthetio group of this enzyme.
In
fact, Green (1940) classifies pyruvlo oxidase of the gonococcus
as a thiaminoprotein enzyme.
Krebs (1937) and Barron and Lyman
(1939) have shown that the gonococcus in the absence of oxygen
catalyzes the dlsmutation of pyruvate according to the equation:
2 CH3 .OO.COOH + H20 ---------- ► C H 3 .CHOH.COOH + CH3 .COOH + C02 .
(pyruvic acid)
(lactic acid)
(acetic acid)
The oxygen tension of the system determines which reaction will
occur, dlsmutation or direct oxidation.
Some of the respiratory enzymes of the gonococous have been
studied by various workers although in most cases not as extensive­
ly as in the work of Barron.
Schumacher (1915) examined the ef­
fect of various chemical reagents upon the oxidation of a gon­
orrheal pus slide preparation, stained according to the method
of Unna.
This stain consisted of an acidified methylene blue
solution which was kept reduced by the addition of "Rongalit,"
a trade name presumably for a product containing formaldehyde
and sodium sulfite (Drury, 1914).
When slide preparations of
gonorrheal pus were immersed in this almost d e a r solution and
rinsed in boiling water, the protoplasm of the leucocytes stained
a weak blue color while the nuclei were darker blue.
ocoool stained an intense blue.
The gon-
Schumacher interpreted this to
mean that whereas the leuoocytlo nuclei were rloh in "oxygen
content," the gonococci were more so.
Furthermore, any reducing
agent whioh prevented the appearance of the intense blue color
in the gonococci when Unna's RW (rongalit white) stain was ap­
plied, indicated a deprivation of oxygen from the organisms.
Sal-
varsan and pyrogallol-carboxylic acid were reported as agents
which had the strongest reducing action on the gonococcus, while
metallic salts like silver nitrate and mercuric chloride had a
distinctly catalytic effect on the organism, turning It blue
instantly.
KIrchner and Nagell (1926) studied the factors affecting the
quantitative estimation of catalase and peroxidase of the gon­
ococcus, Staphylococcus aureus and B. coll.
They observed that
the gonococcus has powerful catalase activity; in comparison
B.
coll catalase activity is one-tenth that of Staphylococcus
aureus which in turn is one-third that of the gonococcus.
Gon­
ococcus catalase is sensitive to changes in temperature above
0° C. and to variations in pH.
Results of the quantitative
peroxidase test using pyrogallol were also markedly influenced
by the temperature and variations in pH.
The oxygen content af­
fected chiefly the spontaneous oxidation of pyrogallol.
They
found the peroxidase activity of Staph, aureus to be ten times
that of B. coll and the gonococcus.
The temperature effect was
greater on peroxidase than on catalase.
While the peroxidase
values of B. coll and Staph, aureus were parallel to the catalase
values, such was not the case with the gonococcus.
This indicated
that peroxldative purpurogallin was produced in addition to the
spontaneous purpurogallin formed as a result of oxygen liberated
from hydrogen peroxide in the experimental fluid by the action
of catalase.
Experiments substituting monoethyl hydrogen peroxide
for hydrogen peroxide in the peroxidase system showed that no
decomposition of the organic peroxide took place either by per­
oxidase or by catalase.
In the case of B. coll. the results of
the catalase studies by Kirchner and Nagell are in harmony with
those of Virtanen and Karstrom (1925).
Derivatives of para-phenylenediamine as reagents for the demon­
stration of "oxidase" have been used extensively in bacteriology.
When alpha-naphthoi and a diamine are used as substrates, the test
is commonly called the Indophenol reaction and the enzyme produc­
ing this reaction is known as Indophenol oxidase.
These two re­
agents are sometimes known as the "nadi" reagents, the word coined
from the first two letters of the two chemicals used.
Confusion
still exists in the classification of the enzyme catalyzing the
reaction, both as to its nature and its relationship to the par­
ticular substrate used.
As early as 1902, Dietrich and Liebermeister
used p-phenylenedlamlne and alpha-naphthol in solution as a stain­
ing agent to bring out certain granules in B. anthracls.
They in­
terpreted these granules as being centers of oxidation in the cell.
Kramer (1912) employed and greatly extended the color method of
Schultze (1910) for demonstrating bacterial oxidase.
Both of these
workers used the nadi reagents incorporated in nutrient agar.
When
bacteria were streaked on the surface of this medium, oxidase posi­
tive organisms rapidly produced a blue color along the streak.
Potassium ferrlcyanide was used as a control.
Over 100 different
strains and species were studied, but no observations were made on
gonococcus.
Gordon and McLeod (1928) were the first to propose the
use of a one per cent aqueous solution of dlmethyl-p-phenylenedlamine
hydrochloride to differentiate between members of the Neisseria
group and oxidase negative organisms.
The diamine solution is
freshly prepared and when flooded over the surface of a plate
containing colonies of gonococcus, the colonies become pink,
then red and finally blaok, demonstrating the presence of an
oxidase.
Ellingworth, McLeod and Gordon (1929) demonstrated
that diamines are toxic to bacteria, including the gonococcus.
Monomethyl-p-phenylenedlamlne is most toxic while tetramethyl-pphenylenediamlne is the least.
Loele (1929) compared the re­
activity of a large number of organisms to the Indophenol reagent,
p-phenylenediamine, and to a peroxidase reagent consisting of pphenylenediamlne and hydrogen peroxide.
He found that the gonococcus
contained enzymes oxidizing all three reagents.
Happold (1930)
reported that dimethyl-p-phenylenedlamlne was oxidized more rapid­
ly than catechol by the gonococcus,
Kellin and Hartree (1938) indicated that the correct name
for indophenol oxidase is cytochrome oxidase.
This was substan­
tiated by the fact that the oxidation of such compounds as diamine
derivatives, hydroquin one, catechol and others, by cells and their
extracts, is not catalyzed directly by Indophenol oxidase but
with the cooperation of cytochrome.
The oxidase is specific only
for the oxidation of reduced cytochrome.
This work has been con­
firmed and extended by Stotz, Sldwell, and Hogness (1938).
Relation of Enzyme Inhibition to Viability.
Reports of compara­
tive studies on the effect of various ohemioal and physical agents
on the respiratory enzymes and the viability of bacteria have been
of recent date.
This may be accounted for (1) by the failure of
investigators to appreciate the significance of such a relation­
ship,
(2) by the Incompleteness of our knowledge of the respira­
tory enzymes of bacteria, and (3) by the arbitrary definitions
advanced by various workers for the criteria of death of micro­
organisms when acted upon by physloal and chemical agents.
Much
of the work reported on the effect of chemicals on bacterial
metabolism has been on inhibitors of certain enzyme reactions,
without concurrent observations on viability.
Dietrich and Llebermelster (1902) extended their studies to
Include the effect of various chemioals on the granules.
Potassium
hydroxide, sodium hydroxide, aoetlc aoid, strong mineral acids,
and ammonia, did not dissolve the granules.
Exposing suspensions
of B. anthracis for one hour at 100° 0. did not change the appear­
ance nor the reaction of the granules while the bacilli themselves
were killed.
They concluded that these heat-stable, oxygen ac­
tivating granules were not related to the viability or virulence
of the organisms, nor could they be considered as spores.
Rettger and his co-workers have reported the results of a
number of studies relating to the effect of heat on the inactiva­
tion of various respiratory enzymes of bacteria and on their
growth.
Gasman and Rettger (1933) investigated the relationship
between the maximum temperature at which growth would occur and
the thermolablllty of succinic dehydrogenase,"paraphenylenediamine
oxidase," catalase and peroxidase, in members of the genus Baolllus,
as well as some strict thermophiles.
They found, in general, that
in most of the organisms studied, succinio dehydrogenase shows
distinct inhibition at the maximum temperature of growth while
oxidase and catalase vary considerably in this respect.
A heat-
stable peroxidase was demonstrated and examples are given to show
that the presence of catalase and succinic dehydrogenase may ac­
tually inhibit the benzidine peroxidase test.
Edwards and Rettger (1937) studied the effect of heat on
growth and inhibition of suoolnic dehydrogenase, indophenol ox­
idase, catalase and peroxidase, of a number of thermophiles and
members of the genus Bacillus.
By treating their data in a sta­
tistical manner, it was revealed that a high degree of correlation
exists for the inactivation of each enzyme individually and for
all three collectively, when oompared with the maximum temperature
of growth.
The relationship between the inactivation of indophenol
oxidase and catalase to the maximum growth temperature of the
members of the genus Bacillus was so correlated that the maximum
growth temperature of an unknown species of the genus may be
estimated mathematically, when the inactivation temperature of
either one of these enzymes is given.
On the basis of the exper­
imental data, these investigators indicated that when the respira­
tory enzymes are destroyed all growth ceases.
A study of their
tables reveals that at the temperature of maximum growth of some
organisms, one or more of the enzymes which they studied may still
be active.
Vedberg and Rettger (1941) investigated the maximum growth
temperature and the minimum temperatures destructive for catalase,
Indophenol oxidase and succinic dehydrogenase, of several classes
of organisms.
They employed the same general techniques as had
been used by Casman and Rettger (1933) and Edwards and Rettger
(1937).
The results show a relationship between maximum growth
temperature and the minimum heat inactivation of the enzymes,
with the exception of peroxidase.
The peroxidases studied were
all found to be heat-etable, as has also been noted by Edwards
and Rettger.
In the case of some bacteria the minimum destructive
temperature of catalase was much higher than the maximum growth
temperatures.
Quastel and Woddrldge (1927) studied the effect of various
physical and chemical agents on the growth and Inhibition of
numerous dehydrogenases of B. coll.
It was found that only cer­
tain of the dehydrogenases are Inactivated by any single agent
and that the order of inactivation depends upon the agent used.
With a decrease in the pH of the suspension or an increase in con­
centration of sodium chloride or sodium nitrate, the dehydrogenases
for glycerol, glutamic acid and the sugars were most labile, while
the dehydrogenases for formic, acetic, butyric and tf(-glycerophosphoric acids were most resistant.
When B. coll was treated with
toluol, benzene, phenol, ether, chloroform, or acetone, the most
labile enzymes were the sugar dehydrogenases.
Subcultures of the
treated organisms showed that either no growth occurred or only a
few discrete colonies appeared; the control subcultures showed
prolific growth.
Sykes (1939) studied in great detail the effect of alcohols,
phenol and phenol derivatives upon the succinic dehydrogenase of
B. coll.
The Inhibitors were serially diluted, the endpoint chosen
being the highest dilution completely inhibiting the dehydrogenase
activity.
He found that at concentrations of the germicides whloh
were Just lethal, suoolnic dehydrogenase was either considerably
or completely inhibited.
In all cases, at a slightly higher con­
centration of the germicide than was necessary to kill B. coll.
suoolnic dehydrogenase was completely inhibited.
Yudkln (1937) Investigated the action of silver sulphate on
several enzymes of B. coll.
Glucose, succinic and lactic dehydro­
genases, hydrogenase and hydrogenlyase, were all inhibited com­
pletely by approximately the same concentration of silver, whereas
formic dehydrogenase began to be affected only when the concentra­
tion of silver was tenfold higher.
The concentration of silver
which caused the death of most of the cells produced a negligibly
small effect on the enzymes studied; the lethal action of silver
was shown to be muoh greater than its effect on the enzymes.
Stapp (1924) studied the effect of various chemicals (NaOH,
HC1, CSg, etc.) on catalase, peroxidase and the viability of a
number of species of bacteria.
In every case peroxidase was shown
to be unaffected by the agent.
Moreover, Stapp came to the con­
clusion that catalase Inhibition is Independent of the viability
of the organisms.
Kramer (1912) investigated the action of various
chemicals (organio solvents, HC1, ammonia) upon "dimethyl-pphenylenedlamlne oxidase" and the viability of various micro­
organisms.
He concluded that the destruction of "oxidase" does
not occur ooincldently with the death of the organism.
Effect of Sulfonamides on Gonococcus.
A number of investigations
have been carried out in vitro on the effect of p-aminobenzenesulfonamide (sulfanilamide) and its derivatives on the gonococcus.
Wengatz, Boak and Carpenter (1938) reported that sulfanilamide
in a concentration of 0.01# in Douglas' broth, markedly inhibited
the growth of several recently Isolated strains when exposed at
36° C. for 20 hours.
Complete inhibition occurred after the
organisms were exposed to the drug for 50 hours or longer.
Cohn
(1938)
demonstrated that when gonooocoi are exposed to dilutions
of sulfanilamide of 1-100 or 1-1000 in 20 per cent ascitic broth
at 37.5° C. for 5 hours, no growth oocurred upon lnoubatlon.
In
concentrations of the drug from 1-1000 to 1-100,000 no growth
occurred after exposing the organisms for 24 hours.
Carpenter
and Wingate (1941) observed in a study of 106 strains of the
gonococcus that all of the strains were killed after 8 to 48 hours
exposure to 1-10,000 sulfanilamide.
Approximately half of the
strains were killed within 12 to 24 hours.
Burton, McLeod,
McLeod, and Mayr-Harting (1940) Investigated the response of many
species of bacteria inoculated on the surface of agar plates con­
taining graded dilutions of sulfanilamide, p-hydroxylamino-benzenesulphonamide and p-nitro-benzenesulphonamide.
The organisms were
divided into five groups depending upon their reaction to these
three compounds.
The gonococous, as well as other members of the
Neisseria group, were remarkably sensitive to sulfanilamide but
more so to p-nitro-benzenesulphonamide.
These authors concluded
that since blood was used as an enrichment for growing the
gonococcus, and since blood counteracts the bacteriostatic effect
of hydroxylamino-sulfonamide, there is a probability that the
lesser activity of this compound is more apparent than real.
Barron and Jacobs (1937) measured manometrically the effeot of
sulfanilamide and "prontosil" upon the glucose and pyruvate
dehyrogenation of gonooooous.
tration of 0,01 mole per liter.
took place.
The drugs were used in a concen­
No inhibition of respiration
Chu and Hastings (1938) studied manometrically the
effect of various concentrations of sulfanilamide on the utlllza-
tlon of dextrose by the gonococous.
They found that concentrations
of the drug between 0.01 and 0.1 gram per cent had no inhibitory
effect, while 0.66 gram per cent inhibited the oxygen uptake by
approximately 34#.
In this study the observations were carried
out over a period of one hour.
III. EXPERIMENTAL.
A. Methods.
1) Glassware.
With the exception of pipettes and burettes, all glassware
used was made of Pyrex or equivalent brands.
Whenever possible,
transfer and Ostwald-Folln pipettes were employed.
Measuring
pipettes used for oarrylng out dilutions of the germicides were
re-calibrated.
All glassware was chemically cleaned using concentrated
sulphuric acid-sodlum dlchrornate cleaning mixture, and rinsed
with distilled water after washing with tap water.
With the exception of the reaction vessels used for the quan­
titative enzyme tests, all glassware was plugged with cotton and
sterilized.
2) Culture Media.
In the course of the work several culture media were used.
For carrying the stock cultures of gonococcus, a modified semisolid, beef heart "hormone" agar, equivalent to medium "C" of
Torrey and Buckell (1922) was used.
Because Dlfco Proteose No. 3
Peptone has high nutritive properties for gonoooccus, it was used
throughout the work.
The reaction of the medium was adjusted so
that the final pH was 7.2, which is within the optimum zone for
growth.
The medium was dispensed in small test tubes and stop­
pered with corks previously dipped in hot paraffin to prevent
evaporation.
Stock transplants of the strains were made monthly
and stored in the inoubator at 35° to 36° C.
In addition to the
stock cultures, several vials of lyophlllzed oultures were stored
in the refrigerator (-5° C.) for reference.
The suspensions for the germicidal and enzyme Inhibition
tests were prepared from mass cultures on a beef heart-egg digest
agar.
The base for this medium was Torrey and Buckell1s salt-free
"hormone" agar (medium "B") with the following modifications:
Proteose Peptone No. 3 was used; a two per cent oonoentration of
agar was employed in order to minimize the carrying over of agar
particles in the final suspension of organisms; egg digest was
substituted for ascitic or hydrocoele fluids as a source of enrich­
ment.
The egg digest was prepared according to the method of Price
(1935) and the medium contained twenty per cent of this enrichment.
The pH was 7.2.
The medium was sterilized in Pyrex bottles, and
when needed was melted and dispensed aseptlcally into test tubes
and Kolle flasks.
The beef heart-egg digest medium was chosen
because it supports the luxuriant growth of gonococcus without
the addition of blood.
In addition this medium may be sterilized
in the autoolave.
To determine the viability of the drug-treated organisms
in the germicidal tests, two types of broth were used, the base
for both being Torrey and Buckell's beef heart "B" broth.
When
ready for use, the first medium contained five per oent egg di­
gest, while the second contained twenty per oent hydroooele fluid.
Broth stored for more than 6 weeks was considered too old
to use.
The enriched media were prepared the day before the
germicidal tests were carried out.
Ten oo. of broth containing
5 per oent egg digest was dispensed into 20 x 150 mm. test tubes,
and 8 cc. of broth base was dispensed into similar tubes.
After
sterilization in the autoclave, 2 oo. of hydrocoele fluid, ster­
ilized by Seitz filtration, was added aseptlcally to the second
set of tubes. (Hydrocoele fluid, after sterilization by Seitz
filtration and stored in the refrigerator, has a tendency to
change in pH.
Therefore, when preparing hydrocoele broth, it
was neoessary to run a preliminary test to determine the altera­
tion in pH upon the addition of the hydrocoele fluid.
As in the
case of egg digest broth, the reaction of the broth base to be
used with hydrocoele fluid, must be adjusted to compensate for
the fall in pH after sterilization.)
According to Torrey and
Buckell (1922) the optimum pH for growth of the gonococcus is
close to 7.2; according to Tulloch (1929), and Thomas and BayneJones (1936), the optimum pH zone is between 7.3 and 7.6.
The
egg digest broth was adjusted so that the final pH was 7.4, and
in the case of the hydroooele broth pH 7.3.
Preliminary tests re­
vealed that when sterile broths were incubated between 35° and
36° C. for one week, a slight change in hydrogen ion concentration
occurred.
This shift in the reaction of the media was noticed
even if 0.5 per cent KHg PO4 was added.
A maximum fall of 0.1 pH
unit occurred In the egg digest broth, while the hydroooele broth
showed a maximum Increase of 0.25 pH unit.
The pH of both media
was therefore so adjusted that it was within the optimum growth of
the gonococcus, even when stored in the incubator for one week.
In order to determine whether growth had occurred in the
broth tubes, samples of 0.2 cc. were taken at appropriate inter­
vale and streaked on the surface of Douglas' agar prepared accord­
ing to the formula of Carpenter (1937).
This medium is excellent
for the primary Isolation of the gonococcus.
It consists of a
pancreatlo digest of lean beef which is made into a "chooolate
blood agar'1 by the addition of 5 per cent sterile deflbrinated or
citrated sheep blood and heating at 80-85° C.
3) Suspending and Diluting Fluids.
Solutions of sodium chloride cannot be used as a diluent in
the study of silver germicides, because of the formation of in­
soluble salts.
Accordingly, it was necessary to choose a menstruum
which would not form a precipitate in the presence of silver salts
and at the same time be non-toxic to the gonococcus.
In a consideration of autolysis, Tulloch (1929) has stated
that there are two separate factors to be considered: viability
and autolysis, neither of which is necessarily an index of the
other.
Viability is partially dependent upon the antagonistic
action of ions and the tonicity of the menstruum, while true
autolysis, to which the gonococcus is very susceptible, appears
to be an intracellular enzymic process as shown by Wollstein
(190?).
Thomson (1923) found that acid suspensions favored
autolysis of gonococcus, while alkaline suspensions inhibited it.
Miller, Hastings and Castles (1932) studied the influence of the
addition of inorganic salts to media on the multiplication of
gonococcus.
They came to the conclusion that osmolar salt concen­
trations below 150 and above 550 mlllimols per liter inhibited
growth.
Acetate was not included in their studies.
A study was made of substitutes for physiological saline.
The silver salts of acetate and nitrate are soluble in water at
3 7 ° C. in the maximum concentrations used for the enzyme and
germicidal studies, and for this reason isotonic solutions of
sodium acetate and sodium nitrate were tested for their effect on
viability under standard conditions.
cerning the solutions used.
Table 1. showB the data con­
A 2.04 per cent solution of sodium
acetate in distilled water has a pH of 7.58.
At this reaction
autolysis of the gonococcus is enhanced; therefore, solutions of
the salt were adjusted to a slightly acid reaction, pH 6.60 to
6.70, by the addition of 0.075 ml. glacial acetlo acid per liter.
The final concentration of acetate in the solution was 0,15 molar.
Physiological NaCl solution (0,9 per cent) and distilled water
were used as controls.
All solutions were prepared in double
distilled water and sterilized in the autoclave.
TABLE 1.
Characteristics of Solutions Used in Viability Test.
Solution
NaCl
NaCoH302.3H20
S a i l e d water
Quantity
ifsed
gms./ L.
Molarity
M.
9.00
20.41*
0.154
0.150
1S:16
°--150
25
5.38
6.62
I'M
* 0.075 ml. glacial acetic acid added per liter solution.
The organisms were suspended in the respective solutions,
centrifuged at approximately 3700 r.p.m. and standardized with
the aid of a photoelectric colorimeter to a turbidity correspond­
ing to the No. 5 barium sulfate tube as described by McFarland
(1907),
Immediately after standardization of the suspensions,
aliquots were taken, diluted serially in the test solutions, and
plated on Douglas chocolate agar for colony enumeration.
The time
of the first sampling was designated "initial time."
On the
average, a period of one hour ensued between the time when the
organisms were collected, until the "initial time."
The tubes
containing the suspensions were placed in a water bath at 37° C.,
and sampled every hour for five hours.
TABLE 2.
Effect of Isotonic Solutions on the Viability
of the flonococcua
Number of Viable Gonococci per ml.
Time in
Hours 0.154 M NaCl
Initial
1.
2.
3.
4.
5.
506,500,000
355,250,000
252,500,000
54,350,000
50,250,000
29,950,000
0.150 M NaCgHgOg
585,000,000
220,000,000
153,000,000
100,000,000
51,750,000
47,500,000
0.150 M NaN03
Distilled
H20
675,000,000
100,000,000
18,500,000
260,000
127,500
90,000
750,000
10,000
0
0
0
0
* Organisms suspended in and washed with each respective solution,
the turbidity standardized, and aliquot quantities from each stand­
ardized suspension diluted and plated on Douglas chocolate blood
agar at stated Intervals and incubated for 48 hours in an atmos­
phere of 10# CO2 .
From the results shown in Table 2, It can be seen that the
sodium acetate solution is as satisfactory a suspending medium for
the gonococcus as physiological sodium chloride.
In order to check the isotonicity of the sodium acetate solu­
tion further, a tonicity test was made as described by Kolmer
(1925) for use In serological tests.
Normal rabbit and sheep
erythrocytes were washed three times with physiologloal NaCl, and
0.05 oc. of packed erythrocytes was added to three ml. of test
solution.
As shown in Table 3., 0.15 molar sodium acetate solution
did not cause hemolysis of the erythrocytes of either species
under the experimental conditions.
TABUS 3 .
The Effect of Various Solution
on Rabbit and Sheep ferythrooytes.»
Solutions used
0.150 M NaCgH302
0.150 M N&NO3
0.154 M NaCl
Distilled water
Normal Rabbit Erythrocytes Normal Sheep Erythrocytes
A 1***
B#*#
A*#
B***
No hemolysis No hemolysis No hemolysis No hemolysis
No hemolysis No hemolysis No hemolysis No hemolysis
No hemolysis No hemolysis No hemolysis No hemolysis
Hemolysis
Hemolysis
Hemolysis
Hemolysis
* Blood was taken from normal animals, centrifuged and the red
blood cells washed three times with normal NaCl. 3 ml. test
solution added to 0.05 co. packed erythrocytes.
A**: Solutions and erythrocytes thoroughly mixed, placed in a
water bath at 37® C. for one hour, left at room temperature
for two hours, then oentrlfuged and observations made.
B***: Solutions and erythrocytes mixed, placed in refrigerator at
6 ° C. for twenty hours, then oentrlfuged and observations
made.
4) Qonococcldes Employed.
The germicides chosen for this work were those drugs commonly
used in the treatment of gonorrhea at the time the experimental
work was begun.
The Cyclopedia of Medicine (1932) lists a number
of commonly used drugs in the treatment of this venereal disease.
With the advent of sulfanilamide and its numerous derivatives,
these older and local medications have been generally supplanted.
The gonoooccides used are as follows: silver nitrate, c.p.;
protargol; argyrol; silver nuclelnate; neo-silvol; merthiolate;
potassium permanganate, c.p.; and sulfanilamide.
Protargol (or silver albumose) is a protein silver compound
of the "strong" variety having the specifications according to
the Pharmacopoeia of the United States of America (1935) for argentum
protelnlcum forte; It contains about eight per cent silver.
Argyrol (or silver vitellin) is a silver protein compound of
the "mild" variety according to the properties for argentum protelnicum mite (U. S. Fharm&oopoeia, 1935).
20 to 25 per cent silver.
Argyrol contains from
According to Pilcher and Sollmann (1923),
the antiseptic efficiency of silver compounds and their content of
silver ions may be correlated with their restraining effect on gas
production by yeast.
Argyrol has less "active" or "ionic" silver
than protargol.
Silver nuclelnate (or silver nucleate) is a silver protein
compound of the "mild" variety according to the properties given
for argentum protelnlcum mite (U. S. Pharmacopoeia, 1935), and con­
tains about 20 per cent silver.
Neo-silvol is a colloidal silver iodide stabilized by means
of a protein (Hamilton, 1924).
It contains about 20 per cent silver
iodide, equivalent to 8.5 to 10.3 per cent silver; the protective
colloid is an oxidized form of gelatin.
With the exception of silver nitrate, none of the silver-con­
taining materials used can be considered as chemloal compounds of
fixed composition.
Some variation in the amount of silver occurs
in different lots of the same compound.
Except for neo-silvol,
the silver content of each of the various germicides used has been
determined by Lehmann's permanganate oxidation method (Deutsches
Arzneibuch, 1926, pg. 77; Dragenesco and Weinberg-Saohetti, 1930);
the silver iodide content of neo-silvol was determined gravlmetrically after the organic oompound had been treated with hydro­
chloric acid (New and Non-Official Remedies, 1941, pg. 498).
In
the various tables in this dissertation which show the effect of
germicides upon gonococci, calculations of molarity are expressed
in terms of the amount of silver In the lot of drug used.
Merthlolate (sodium ethyl mercuri thiosalicylate), donated by
Ell Lilly and Company, was obtained as a powder.
The commercial,
aqueous solution of merthlolate (1 :1000) was not used since it al­
so contains 0.1 per cent monoethanolamine.
Merthlolate contains
49.15 to 49.65 per cent mercury (The Cyclopedia of Medicine, Surgery
and Specialties, 1940).
Sulfanilamide (prontylln or para-aminobenzenesulfonamide, re­
purified for injection) was donated by the Wlnthrop Chemical
Company.
Two per cent stock solutions of the various compounds used
in the germicidal tests, with the exception of sulfanilamide, were
made in distilled water.
Because of the low solubility of sul­
fanilamide in water, a 1.5 per cent solution was prepared.
It was
dissolved by heating in a water bath and maintaining the tempera­
ture at 370 c. until used in the tests.
For the enzyme inhibition
tests it was sometimes necessary to use stock solutions of some
of the drugs as high as 20 per cent.
All stock solutions were fresh­
ly prepared every two days and stored in the ice box.
Dilutions
of the 8took solutions were made in sterile 0.15 molar sodium
acetate solution.
The stock solutions themselves, with the ex­
ception of sulfanilamide, were found to be sterile.
Sulfanilamide
was sterilized by one or other of three methods: a) Seitz filtra­
tion; b) autoolaving at 15 pounds for 20 minutes; c) heating In
the Arnold at 100° C. for one hour on three successive days.
The
results obtained with sulfanilamide solutions sterilized by any
of the aforementioned, means were identical.
5) History of the Gonococcus Strain Used.
The strain of gonococcus used throughout this investigation,
designated # 1111, was isolated from a female patient suffering
from chronic gonorrheal endocervioltis.
The oervical specimen was
obtained on a sterile swab whioh was placed in a tube containing
Torrey and Buokell's semi-solid agar.
A oervical smear made at
the same time showed extracellular and intracellular gram-negative
diplooocol.
The colonies on the Douglas plates were transluoent,
gray-white in color, not perceptibly mucoid, and gave a positive
oxidase reaction using a one per cent solution of dlmethyl-pphenylenediamine monohydrochloride (Gordon and McLeod, 1928).
organism fermented glucose, but not maltose, sucrose
The
or lactose.
The patient responded to treatment with sulfapyrldlne and
cure resulted.
The strain used in this investigation was isolated
before chemotherapy was used.
The patient's blood gave a positive
complement fixation test for the gonococcus.
Routinely, strain #1111 was transplanted monthly on semi­
solid agar which served as a stock or reference culture.
For use
in the germicidal or enzyme Inhibition tests, the organism was
grown on beef heart-egg digest agar slants, and transplanted daily
for at least two days on this medium before inoculating into Kolle
flasks.
In the preliminary dehydrogenase studies, strains of the
gonococcus obtained from 3 male and 2 female patients were used
in addition to #1111.
6 ) general Procedures Used for Conducting Germicidal and Enzyme
Inhibition Test's^
The conditions employed in this investigation for the germ-
icldal and enzyme inhibition tests were essentially those used
by Davis and Swartz (1920) for testing the action of gerraioides
upon the gonococcus by the centrifuge method.
Tests of both types
were conducted in the same way up to and including the stage where
the drug treated suspensions were standardized for turbidity.
The suspensions of organisms were standardized to a turbidity
equivalent to the No. 5 tube of McFarland's barium sulfate sus­
pension.
This turbidity represents a total oount of approximately
3,000,000,000 gonococci per ml. as determined in a Petroff-Hauser
bacterial counter.
Turbidity measurements were made in an Analco-
Dlller photoelectric colorimeter (Diller, 1936).
The turbidometrically standardized acetate suspensions of
gonococci were further checked from time to time by determination
of the total nitrogen by the method of Koch and MoMeekln (1924).
Nitrogen determinations for the standard suspensions of gonocooci
were reasonably constant; they varied from 0.103 to 0.118 mg. per
ml. of gonococcus suspension.
Kolle flasks were inoculated from slant cultures and lnoubated for 48 hours in a moist Incubator at 35 - 36° C. in atmospherlo air.
The organisms were washed off with sterile 0.15 molar
sodium acetate solution, placed in a 50 ml. centrifuge tube, and
centrifuged for 20 minutes at about 3700 r.p.m.
Care was taken
that no media particles were included in the acetate suspension.
The supernatant fluid was discarded and enough sterile sodium
acetate solution added to the sediment to ensure homogeneous sus­
pension of the organisms.
The turbidity was determined and the
organisms placed in a water bath at 37° C.
The water bath used
in these experiments had a temperature tolerance of ±0.05° 0.
and was equipped with a stirrer.
Serial dilutions of the germicides
were made in aoetate solution and 3 ml. amounts placed in 18 x 150
mm. tubes for sedimentation in the angle centrifuge.
An angle type oentrifuge (Aktiebolaget Winkelcentrifug,
Stockholm, Sweden; Type SP) with a speed of 6000 r.p.m. was used
to collect the organisms following exposure to the germicides.
Relatively little loss occurred, even though centrifugation was
carried on for only a short time.
This centrifuge took one minute
to reach full speed, 2.5 minutes to sediment the organisms oompactly, and five minutes to come to a full stop.
The tubes containing the germicides were placed in the water
bath for five minutes after which the organisms were added rapidly
and the mixture left at 37° C. for 20 minutes.
At the end of this
time, the tubes were centrifuged for 2,5 minutes, the supernatant
was discarded and 10 ml. of aoetate solution added.
were then washed twice with aoetate solution.
The organisms
Finally, the sus­
pension was transferred to smaller tubes and the turbidity
standardized as described.
The viability of the cells and the
activity of various enzyme systems were then tested.
7) The Germicidal Test.
Preliminary tests showed that the use of slants as employed
by Davis and Swartz to determine the viability of the organisms
is unsatisfactory.
Growth on agar slants was barely visible when
only a few viable organisms were present, and moreover the inoculum
of dead organisms which collected at the bottom of the slant made
the observations difficult to interpret.
By the use of hydroooele
and egg digest broths for preliminary cultivation, and subcultur-
ing to plates at appropriate intervals, it was possible to obtain
a roughly quantitative idea of the number of viable organisms pres­
ent in the original inoculum.
One
ml. of the test suspension was added respectively to
10 ml. of 5 per cent egg dlgest-beef heart broth and to 10 ml. of
20 per cent hydrocele-beef heart broth previously warmed to 36° C.
The inoculated broths were mixed thoroughly, 0.2 ml. of the mix­
ture streaked immediately to prewarmed, Douglas chocolate agar
plates, and lnoubated for 48 hours at 35-36° C. in a CO2 tension
of 10 per cent.
The broth cultures were then incubated at 35-36° C.
and plated serially on Douglas chooolate agar after 48, 96, and
168 hours.
All final tests were repeated three times.
A control
suspension of untreated organisms was subjected to the same man­
ipulations as the drug treated suspensions.
The
endpoint of the germicidal test was taken as the highest
dilution
of the compound which caused the death of all the organ­
isms in the test suspensions, as determined by culturing in
hydrocele and egg digest broth for one week, and finally suboulttir­
ing on agar plates.
8 ) Enzymatic Teats.
In addition to studying the lethal action of various drugs
on suspensions of gonocoooi, determinations were also made on the
effect of the same compounds upon certain enzyme systems.
Of the
dehydrogenase systems, those for lactlo and glyceric acid were
studied.
The effect of the drugs upon catalase, peroxidase and
lndophenol oxidase (cytoohrome oxidase) was also investigated.
All enzyme inhibition tests were repeated at least three times.
A) Dehydrogenases.
Preliminary Observations:
The use of methylene blue as an indicator for estimating
dehydrogenase activity is a well established procedure, having
been used extensively with both animal tissues and bacteria.
Several methods have been employed to produce and maintain
anaeroblosls during the test.
The classical vacuum type tube
suggested by Thunberg (1917, 1930), in his dehydrogenase experiments
with animal tissues, has had wide use in demonstrating bacterial
dehydrogenase activity.
Braun and Wordehoff (1933) as well as
MacLeod (1939) used a vaseline seal to prevent absorption of atmos­
pheric oxygen.
Bach (1937) used a method of evacuation and nitro­
gen replacement while the liquid was under a layer of neutral
paraffin oil, while Callow (1926) resorted to olive oil to prevent
absorption of oxygen.
Still (1941) measured pyruvic dehydrogenase
activity of Esch. coll man©metrically.
For the preliminary experiments on the dehydrogenases of the
gonococcus, the vaseline tube method as employed by MacLeod was
used.
An effort was made to determine the dehydrogenase activity
of gonococcus on various substrates, and to establish the conditions
for the test.
Into Pyrex tubes measuring 12 x 100 mm.the following materials
were introduced:
1.0 ml. of standard suspension of organisms;
0.5 ml. of 0.02 per cent (1-5000) solution of methylene blue in
M/20 phosphate buffer at pH 7.4; 0.5 ml. substrate, adjusted to
p H 7.4 with NaOH; 1.9 ml. M/20 phosphate buffer, pH 7.4 (accord­
ing to Clark and Lube); 0.1 ml. broth as a source of "ooenzymes."
The final volume was 4.0 ml.
Eaoh substrate was tested with and
without the presence of 0.1 ml. of broth.
In addition, control
tubes from which the suspension of organisms and the substrate
respectively had been omitted, were always included in the test.
The final volume was also made to 4.0 ml. by the addition of phos­
phate buffer.
A layer of melted vaseline 2.5 cm. deep was then
pipetted into each tube, following which they were incubated at
37° 0.
The vaseline tube method was found useful in preliminary
tests of the dehydrogenase activity of gonococcus toward various
substrates.
Barron and Miller (1932) reported that glucose and pyruvate
are oxidized by the gonococcus, and Barron (1936) showed that
methylene blue is reduced when pyruvate is used as a substrate.
In the present experiments, no dehydrogenase activity for either
glucose or pyruvate could be demonstrated in suspension of organ­
isms made from cultures which had been incubated for 48 hours.
However, methylene blue reduction took place when Esoh. coll communlor and these two substrates were used.
Certain observations
on the effect of the age of the culture on dextrose and pyruvic
dehydrogenase aotlvlty will be discussed subsequently.
Beef infusion broth, Douglas (Hartley) broth, and Torrey broth
were used in 0.1 ml. quantities in the dehydrogenase tests as a
source of "coenzymes."
In the oontrol tubes complete reduction
of methylene blue occurred in from 1 to 24 hours, without the
addition of substrate when these broths were used.
A suitable
source of "coenzymes" was found to be a solution containing 2
per cent proteose peptone No. 3 and 0.5 per cent NaCl, sterilized
by autoclaving, and having a final pH of 7.2.
With this peptone
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solution the time of reduction of the methylene blue with the oxidizable substrates and organisms was shorter than when the solu­
tion was omitted; moreover, the controls which did not contain
substrate showed no reduction within twenty-four hours.
These
results are shown In Table 4.
As may be seen from Table 4, the presence or absence of
ascorbic acid dehydrogenase could not be determined, slnoe the
period for the deoolorizatlon of methylene blue was the same in
the control tube containing no bacterial suspension as in the test
proper.
TAB IE 5 .
Tests for Dehydrogenase Activities of the Gonococcus*
48 hour cultures
Dehydrogenase Activity
Substrate
None.###
Glucose (1 and 5#)
None.
Lactose (1#)
None.
Maltose (1#)
None.
Sucrose (1#)
Present.
lactic Acid (1 and 5#)
None.
Pyruvic Acid (1 and 5$)
None.
Formic Acid (1#)
None.
Fructose (1#)
Present.
Glyceric Acid (1 and 5$)
None.
Succinic Acid {1#)
None.
Sodium beta glycerophosphate (1#)
None.
Sodium glycerophosphate (1#)
None.
Glycerol (1#)
None.
Ethyl alcohol (1#)
None.
Acetaldehyde (1#)
None.
Sodium citrate (1#)
None.
Sodium nitrate (l#j
Sodium acetate (1#)**
None.
Ascorbic acid (2#)
Not determinable,
* Each tube contained 0.5 cc. 1-5000 methylene blue solution, 0.5
ml. substrate, 0.1 ml. peptone-NaCl solution as source of
coenzymes, ana 1.0 ml. M/20 phosphate buffer, pH 7.4. 1.0 ml.
of a standard suspension of several strains of gonocooci in
aoetate were used. A layer of melted vaseline 2.5 cms. deep was
applied. The tubes were placed in water bath at 370 c. and read
up to 2 hours with a final reading after 24 hours.
Controls as
stated in the text were included.
*» When sodium acetate was used as a substrate, both
suspension of gonococci and a suspension in pnysli0lSg?glia!8.85#)
saline were tested.
*«• one strain dehydrogenated glucose.
It should be noted that the time required for 90 per cent re­
duction of methylene blue was taken as the endpoint.
Previous
workers have demonstrated that the rate of methylene blue reduction
is approximately constant up to 90 per cent, after which it de­
creases irregularly (Sykes, 1939).
A list of the substrates tested for the presence of the re­
spective gonococcus dehydrogenases in 48 hour cultures is shown
in Table 5.
One strain dehydrogenated glucose but unfortunately
it was lost.
None of five other strains of gonococci, including
# 1111, showed a glucose dehydrogenase or a pyruvic dehydrogenase
when tested in quadruplicate in four separate experiments.
In
addition to the vaseline tube method, the Thunberg tube was also
used to test these two substrates.
Negative results were obtained.
Both lactic and glyceric dehydrogenases gave a shorter
methylene blue reduction time when a 5 per cent rather than when
a 1 per cent solution of substrate was used, as shown in Table 6 .
TABLE 6 .
Effect of Varying The Concentration of the Substrates On the
Methylene Blue Reduction Time of Gonococcus (Strain ^lllj.).
Concentration of Substrate*
Lactic Acid, 5
Lactic Acid, 4
Lactic Acid, 3
Lactic Acid, 2
Lactic Acid, 1
Glyceric Acid,
Glyceric Acid,
Glyceric Acid,
Glyoerio Acid,
Glyceric Acid,
per cent
per cent
per cent
per cent
per cent
5 per cent
4 per cent
3 per cent
2 per cent
1 per cent
Reduction Time
(minutes)
11
11
11.5
14
14.5
43.5
43.5
45
51
75
• Substrates neutralized with NaOH to pH 7.4. The conditions
were the same as for the series of experiments recorded in
Table 5.
This is In conformity with the results obtained, by Quastel and
Whetham (1925) who obtained shorter reduction times of methylene
blue when higher concentrations of lactic and glyceric acids were
used, Esoh. coll being the test organism.
It was of interest to find out what relationship exists be­
tween the number of gonococci present in the suspension and the
reduction time of methylene blue.
Table 7 shows this relationship.
TABLE 7 .
Effect of Varying The Number of Organisms (Gonococcus #1111)
on Lactic Dehydrogenase Reduction Time.*
Dilution of Standard
Suspension of Organisms
Reduction Time
(minutes)
0
6
10
20
30
40
50
60
70
80
90
10.5
10.5
11.5
12.5
16
17.5
22
26.5
37
52
114
* Conditions for this experiment were the same as for the experi­
ments recorded in Table 5.
From the results shown in Table 7, it can be seen that there
is an inverse relationship between the reduction time of methylene
blue and the number of gonococoi present in the dehydrogenation
experiments using lactate as a substrate.
These findings agree in
general with those of Sykes (1939), using the succinlo dehydrogenase
of Esoh. coll.
Sykes found that as the numoer of organisms was de­
creased to 60 per cent of the original number, the reduction time
was proportional to the dilution of the bacterial suspension, but
with lesser numbers of organisms the reduotlon time became propor-
tlonal to the square of the dilution.
In the present investigation, the experiments Involving the
effect of the various germicides on gonococcal dehydrogenases
were carried out In Thunberg tubes.
For this reason a comparison
of the methylene blue reduction time using the vaseline seal method
and evacuated Thunberg tubes seemed desirable.
The results are
shown in Table 8 .
TABLE 8 .
Comparison of the Vaseline Seal Tube and Thunberg Tube Methods
for Determining Dehydrogenase Activity.*
Substrate
Reduction Times (minutes)
Vaseline Seal
Thunberg~Tube
Method
Method
Lactio Acid (5#)
Glyceric Acid (5$)
11
43.5
6.5
38
* The conditions for these two methods were the same, except that
a vaseline seal was used in one case and evacuation of the air
was employed in the other to prevent re-oxidation of the methy­
lene blue.
Gonococcus #1111 was used. The final pH of both
lactic and glyceric dehydrogenase test mixtures was 7.3.
The results shown in Table 8 Indicate that when the Thunberg
tube is employed to determine dehydrogenase activity, the reduction
time of methylene blue is shorter with both substrates.
Lactic Dehydrogenase Test;
The gonococcus lactic dehydrogenase was one of the two de­
hydrogenases selected for testing the inhibitory effect of the
germicides.
Kellln's modification of the Thunberg tube (Kellin, 1929) was
used.
This tube is equipped with an Inverted U-type bulb stopper
holding about 2 ml. liquid when held in an upright position.
The following materials were pipetted into the Thunberg tube:
0.5 ml. 1-5000 (0.0005 M) methylene blue monoohloride (La Motte
Special, zinc-free) dissolved in M/20 pH 7.4 phosphate buffer.
0.5 ml. 5 per cent (0.555 M) lactic acid (neutralized with NaOH to
pH 7.4.)
0.1 ml. peptone-sodium chloride solution.
1.9 ml. M/20 pH 7.4 phosphate buffer (Clark and Luba.)
In the hollow ground stopper was placed one ml. of standard­
ized drug-treated suspension of gonococci.
evacuated by water pump.
The tubes were then
For any one group of tests in a series,
all of the Thunberg tubes were connected to the same water pump.
Ebullition of the fluid contents took place and evacuation was con­
tinued for 10 minutes after which the stoppers were closed.
The
tubes were then placed in a constant temperature water bath at 37° C.
f o r five minutes until they reached the temperature of the bath.
The suspension in the stopper was then tipped Into the tube, mixed
thoroughly and the reduotlon of methylene blue was timed from this
point.
At the end of the 120 minutes, all methylene blue tubes were
observed for complete, partial, or no reduction.
To determine the
degree of reduction, standards containing 10, 30, 50, 70, and 90
per cent of the total quantity of methylene blue were prepared.
Each group of tests was accompanied by the following controls:
a) system control: which contained the same materials as the ex­
periment, except that the gonococcal suspension was not treated
with a drug before being added;
b) heated enzyme oontrol: the same as the system control except
that the suspension of gonococci was heated to boiling for 15
minutes before being added;
c) normal oontrol: as in system oontrol except for omission of the
substrate;
d) substrate control: as in system control except for omission of
gonococcal suspension.
The normal, substrate, and heated enzyme controls accompany­
ing each group of tests were always negative; that Is, no reduc­
tion of methylene blue took place within the 120 minute observation
period.
The system control was usually reduced within seven min­
utes.
The endpoint cnosen for the Inactivation of lactic dehydrogenase
activity was the highest dilution of the germicide which completely
inhibited the reduction of methylene blue in the lactic dehydrogenase
test.
Q-lycerlo Dehydrogenase Test:
The glyceric dehydrogenase of the gonococcus was the seoond
of the two dehydrogenases selected for testing the enzyme inhibitory
properties of the germicides used.
All of the conditions and precautions observed for testing
lactic dehydrogenase activity were used in testing for the presence
or absence of glyceric dehydrogenase.
0.5 ml. of a 6 per cent
(0.471 M) glyceric acid solution at pH 7.4 was used as the substrate.
The endpoint originally chosen for the inactivation of glyceric
dehydrogenase activity was the highest dilution of the germicide
w h i c h completely inhibited the reduction of methylene blue.
As
will be pointed out later, 65 per cent inhibition of glyceric de­
hydrogenase was finally selected as the endpoint.
B) The Catalase Test:
In the literature bacterial catalase activity is often desig­
nated in such terms as "weak," "considerable," and "strong," as
Judged by the amount of oxygen bubbling through the test liquid.
Such designations obviously have little quantitative value.
Catalase activity has been quantitatively determined by three
general methods
(Waksman and Davison, 1926):
(1) Permanganate Method. This method is based on the observa­
tion that in the presence of sulphuric acid, hydrogen peroxide
decomposes permanganate stolchiometrically.
The residual peroxide
present after the catalase has been allowed to act for a definite
period of time is titrated with standard potassium permanganate
in an acid menstruum.
This method was used in the present in­
vestigation.
(2) Iodometrlc Titration. In this method the peroxide liber­
ates free iodine from potassium iodide.
In general, the catalase
solution is allowed to act upon an excess of hydrogen peroxide.
After an appropriate period sulphuric acid Is added to stop enzyme
activity.
The undecomposed hydrogen peroxide is then determined
indirectly by the addition of potassium iodide, and the iodine
which has been liberated by the interaction with hydrogen peroxide
is titrated with standard sodium thlosulphate.
This method is
unsuitable for measuring bacterial catalase since the bacteria
themselves liberate iodine from potassium iodide.
(3) Volumetric Determination. This method is based on the
direct manometrlc measurement of the oxygen liberated from H2O2
by the action of catalase.
This method as well as the permanganate
method have been used extensively for measuring baoterial and
animal catalase activity.
As mentioned above the volumetric determination of catalase
activity was used in the present study.
The permanganate method
used by Vlrtanen and Karstrom (1925) and by Klrchner and Nagell
(1926) was utilized with slight modification.
Vlrtanen and
Karstrom expressed their results as Katalase Fahigheit (Kat. F.)
after the work of von Euler and his collaborators.
In the present
investigation, catalase activity is expressed In terms of N/lo
potassium permanganate titration values of the residual hydrogen
peroxide.
Into a 125 ml. Erlenmeyer flask were pipetted 26.0 ml. of
M/150 phosphate buffer at pH 6.5 and 3.0 ml. of N HgOg, diluted from
Merck's Superoxol.
The flask was placed in an ice bath.
When
the mixture in the flask had reached the temperature of the ice bath
(10 to 2o C.), which took about 15 minutes, one ml. of the standard
drug-treated suspension of organisms was added, the vessels shaken
and the reaction was timed for exactly one hour.
At the end of one
hour, 2 ml. of 25 per cent sulphuric acid were added to each vessel
to stop the enzyme action, and the contents mixed.
The reaction
mixture was then titrated for the presence of residual hydrogen
peroxide with N/10 potassium permanganate.
The titration endpoint
was taken as the appearance in the mixture of a pink color which
persisted for at least one minute.
With each set of tests, the following controls were
a)
system control: which contained
included:
the same materials as the ex­
periment, except that the gonococcal suspension was not treated
with a drug before being added;
b)
heated enzyme control: the same
as the system oontrol except that
the suspension of gonococci was
heated to boiling for 15 minutes
before being added;
c) normal oontrol: contained the same materials as the system control
except the untreated organisms were added after the buffer and
hydrogen peroxide were incubated in the ice bath for one hour
and the H2SO4 added;
d) blank: contained the same quantity of HgOg as the tests and
above controls; buffer was added to give a total volume of 30 ml.
The blank, normal control and heated enzyme control always
gave comparable titration values while the system control invariably
exhausted the entire quantity of hydrogen peroxide present.
As indicated above, the whole test mixture was titrated In­
stead of an aliquot quantity thus giving a more accurate titration
value, especially when small quantities of residual hydrogen
peroxide were present.
The endpoint chosen for the inactivation of catalase was the
highest dilution of the germicide which completely inhibited
catalase activity.
C.) The Peroxidase Test:
Both oxidases and peroxidases may act upon the same substrates
essentially, but peroxidase requires the presence of hydrogen
peroxide, whereas oxidase does not (Waksman and Davison, 1929).
Among the substrates which have been used for measuring the activity
of these enzymes are included o<-naphthol, p-aminophenol and its
derivatives, leucomalachlte green, hydroquinone, gualacum, ben­
zidine and pyrogallol (Waksman and Davison, 1929; Ka6tle, 1910).
Quantitative methods for estimating peroxidase activity have been
described by Willstatter and his associates.
Pyrogallol (a trlphenol)
is oxidized by peroxidase in the presence of hydrogen peroxide to
purpurogallin (a cyclopentane derivative), and leucomalachite
green (a trlphenylmethane derivative) is oxidized to malachite
green (a trlphenylhydroxymethane derivative).
Waldschmldt-Leltz
(1929) states that since the results using leucomalachite green
and pyrogallol are in close agreement the validity of the
purpurogallin method may be considered completely established.
The pyrogallol test for the quantitative estimation of
peroxidase activity was devised by Bach and Ohodat, but because
of certain undesirable features, namely long reaction time and
the gravimetric determination of the purpurogallin formed,
Willstatter and Stoll (1918) modified the method.
The unit
for peroxidase activity of a substance has been designated by
Willstatter as the Purpurogallin Zahl (P. Z.) which indicates the
milligrams of purpurogallin formed under standard conditions
(one mg. dry weight of enzyme preparation exposed for five minutes
at 20°).
Klrchner and Nagell (1926) adopted Willstatter and Stoll's
pyrogallol method in a quantitative study of the peroxidase
activity of bacteria.
An attempt was made to correlate catalase
and peroxidase activities of Staphyloooocus aureus. B. coll
and gonococcus, using essentially the same conditions for both
tests: temperature, pH and time.
Correlation between the two
enzyme activities was not observed.
The irregularities ap­
parently came from the spontaneous oxidation of the pyrogallol,
due to the oxygen liberated through the activity of the catalase
which was also present.
In the present investigation the
pyrogallol method was satisfactory, and the controls checked from
day to day.
As will be demonstrated later in hydroxylamine inhibi­
tion experiments, the presence or absence of catalase did not
apparently affect the peroxidase values obtained.
In this connec­
tion it should be noted that a mlcropyrogallol method was employed
by Masamune and Kodama (1932) for the determination of blood
peroxidase.
These investigators used blood taken from the ear vein
of rabbits which was diluted 100 to 150 times.
The peroxidase
activity was determined Dy a method similar to that of Willstatter
and Stoll.
In the present investigation the method of Klrohner and
Nagell (1926) was followed with the modification that a smaller
volume of test mixture was used.
Into a 125 ml. Erlenmeyer flask
were pipetted 21 ml. M/150 phosphate buffer pH 7.2 (Clark and
Lubs) and 3 ml. N/l hydrogen peroxide (diluted from Merck's Superoxol).
The mixture was placed in a water bath at 20° C. and when
the temperature in the flask had reached that of the bath, 5 ml.
of a freshly prepared 1.56 per cent solution of resublimated
pyrogallol in pH 7.2 M/150 phosphate buffer were added.
It is
Important that the pyrogallol solution be prepared Just before
use to keep the value of the blank as low as possible, since at
pH 7.2 purpurogallin is formed spontaneously during the course of
the experiment.
One ml. of drug-treated standardized suspension
of organisms is added, the mixture stirred and timed for exactly
16 minutes.
Two ml, of 25 per cent sulphuric acid are then added
to stop the reaction.
The flask is allowed to stand at room
temperature for five minutes after which the purpurogallin formed
is extracted with purified ether in a separatory funnel.
The
ether extract of purpurogallin is dispensed into volumetric flasks
(50, 25, or 10 ml.) and brought to volume by the addition of
purified ether.
The purpurogallin content of the unknown is then
determined in a Duboscq type colorimeter using a purpurogallin
ether solution as a standard.
Ten readings were made for each
test mixture and an average obtained.
With each group of tests,
the following controls were included:
a) system control: contained the same materials as the experiment,
except that the gonococcal suspension was not treated with a
drug before being added;
b) heated enzyme control: the same as the system control except
that the suspension of gonococci was heated to boiling for
15 minutes before being added;
c) normal control: contained the same materials as the system con­
trol except the untreated organisms were added after the sub­
strates were incubated at 20° C. for 15 minutes and the sulphuric
acid had been added;
d) blank: contained substrates only and no organisms.
The value of the blank was subtracted from the values obtained
in the test.
It was always low and varied slightly from day to day.
The ethyl ether used was freed of aldehyde and peroxide by
treating with sodium sulphite, washing free of the sulphite, de­
hydrating with anhydrous calcium chloride several times and finally
distilling at 35° C.
The purpurogallin used for the standard was as reoommended by
Kirohner and Nagell, and was made according to the method of Graebe
(1914).
Briefly, pyrogallol was subjected to the action of nitrous
acid produced by the addition of formic acid and sodium nitrate.
This process was carried out in the complete absence of air and
necessitated continuous bubbling of carbon dioxide through the
mixture.
The purpurogallin formed was recrystallized by the method
of Sumner (1938).
This was accomplished by filtering and washing
the water-insoluble purpurogallin, dissolving the residue in
boiling 95 per cent ethyl alcohol, refiltering and recovering the
crystals by the addition of six volumes of distilled water to the
filtrate.
The purpurogallin was repurlfled three times by the
above method, dried in vacuo over anhydrous oaloium chloride and
stored in amber glass stoppered bottles.
The product prepared by
this method compared favorably with a preparation supplied by
Drs. Sumner and Howell which had been prepared by the biological
method.
Purpurogallin standards for the test were made up in
20, 10, 5, and 2.5 mg. per cent concentrations; these standards
were kept in amber glass stoppered bottles in the refrigerator.
The endpoint selected for the effect of drugs on gonococcal
peroxidase was the highest dilution of the germicide which com­
pletely inhibited the peroxidase activity of the standardized
suspension of organisms used.
D . ) The Indophenol Oxidase Test (Cytochrome Oxidase);
Tests for oxidase activity have been carried out with many
plant and animal tissues as well as on bacteria.
Indophenol
oxidase has been perhaps the most extensively studied system
(Meldrum 1934).
A quantitative method for the determination of
indophenol oxidase was first described by Vernon in 1911.
The
great majority of quantitative procedures employed have been
colorimetric, using as substrate
^(-naphthol and a diamine.
Quantitative determinations have also been made by manometrio
techniques by measuring the 03 uptake of animal tissues in the
presence of p-phenylenediamine or hydroquinone (Stotz, Sidwell
& Hogness, 1938).
Yamagutchi (1935) measured bacterial "in-
dophenolaseH manometrically using as substrates neutral solutions
of either p-phenylenediamine or dimethyl-p-phenylenediamine.
One of the chief drawbacks of the colorimetric methods de­
scribed in the literature for the measurement of indophenol or
p-phenylenedlamine oxidase activity appears to have been the lack
of adequate standards.
Battelll and Stern (1912) tested the
oxidase activity of animal tissues using aqueous solutions of
p-phenylenedlamine.
used as substrate
carbonate.
Vernon (1911) and Steamraler and Sanders (1925)
o(-naphthol, p-phenylenedlamine and sodium
Dye (1927), Laskowski (1928), and Slmola and Noro
(1937) employed
sodium carbonate.
o^-naphthol, dimethyl-p-phenylenediamine, and
In the above techniques, the reaction was
carried out in a petrl dish in which was plaoed the substrate and
tissue; after a certain time interval the dye formed was compared
directly with a standard without being extracted.
The standard
was prepared in several ways: the substrate was oxidized by the
use of bleaching powder (Vernon), by prolonged exposure to the
atmosphere (Staeramler and Sanders; Dye), and by the use of tissues
(Battelll and Stern; Laskowski).
Slmola and Noro used the step
photometer for estimating oxidase activity.
In this investigation a new quantitative Indophenol oxidase
test for the gonococcus was used.
As will be pointed out later,
the preparation of an adequate standard was suggested by the work
of Guthrie (1931) on the quantitative test for plant tissues.
It was thought that if a purified standard could be prepared, a
simple quantitative test for bacterial indophenol (cytochrome)
oxidase could be worked out employing the generally available
Duboscq type colorimeter; the results could then t>e expressed in
terms of the actual quantity of the pure oxidation product of
the substrate formed.
The method evolved and finally adopted
for the indophenol oxidase inhibition studies is outlined below.
In a subsequent section the validity of the procedure is more
fully discussed.
The substrate consisted of the following mixture which was
prepared Immediately before use In order to reduce the value of
the blank:
0.144# (0.01 M)
c<-naphthol (Eastman Kodak Company) in 50 per cent
alcohol;
0.109# (0.006 M) dimethyl-p-phenylenedlamlne hydrochloride* in
distilled water;
0.044# (0.004 M) sodium carbonate In distilled water.
Redistilled water from a Pyrex glass still was used throughout
this work since Wertheimer (1926) has shown that the presence of
traoes of heavy metals accelerates the oxidation of
o(-naphthol
and p-phenylenedlamine.
Into 25 x 100 ml. Pyrex test tubes were pipetted 5.0 ml. of
the substrate and 5.0 ml. M/20 pH 6.6 phosphate buffer (Clark and
Lubs).
The tubes were placed in a 37° water bath and when the liquid
had reached the temperature of the bath (five minutes), one ml. of
a standard suspension of the drug-treated organisms was added, the
tubes whirled to mix the contents and the reaction allowed to
• The Eastman Kodak Company lists this chemical as p-amldodlmethylanlllne HC1.
continue for exactly 15 minutes.
At the end of this time, 2 ml.
of a 2 per cent solution of potassium cyanide were added to stop
the reaction.
The dyestuff formed was extracted in a 125 ml.
separatory funnel with a solvent consisting of equal parts of
chloroform and absolute alcohol.
The extracted dye compound was
placed into appropriate volumetric flasks and the level of the
liquid brought to the mark with the ohloroform-aloohol solvent.
Depending upon the quantity of dye formed, 50, 25 and 10 ml,
volumetric flasks were used.
The following controls were always
included:
a) system control: which contained the same materials as the ex­
periment, except that the gonococcal suspension was not treated
with a drug before being added;
b) heated enzyme control: the same as the system control except
that the suspension of gonococci was heated to boiling for 15
minutes before being added;
o) normal control: contained the same materials as the system con­
trol except the untreated organisms were added after the buffer
and Hnadl" reagents were incubated at 37° C. for 15 minutes and
the KCN added;
d) blank: contained buffer and wnadi" reagents only.
The standard was prepared according to the method of Koechlln
and Witt (1881) and Witt (1882), oxidation being accelerated by
the addition of ferric chloride, as advocated by Guthrie (1931).
The method finally used was as follows: To 24 gm. of c<-naphthol
was added a solution consisting of 24 gm. of sodium hydrox­
ide in 300 ml. of distilled water.
After continuous stirring
of the mixture, a olear brown solution was obtained.
The diamine
solution was prepared by dissolving 20 gm. of dimethyl-pphenylenediamlne hydrochloride in 2000 ml. of distilled water.
The alkaline naphthol solution was added slowly to the diamine
solution with the development of an intense dark blue color.
The solution was opaque.
Twenty gm. of FeCl3 .6H20 were dissolved
in 100 ml. of distilled water and added gradually to the naphtholdlaraine mixture with constant stirring.
solution resulted.
room temperature.
An opaque bluish-green
This mixture was left standing overnight at
By the next day the oolor of the liquid had
changed to violet-brown and the dye compound was present as a
finely divided suspension.
The dyestuff was then separated by
filtration through a No. 12 Whatman filter paper, and the filtrate
discarded.
The precipitate was washed six times with distilled
water at 37° c. so that the final washings were a weakly violet
color.
The crude dye was then dissolved in absolute ethyl alcohol
with the aid of gentle warming.
a coppery sheen.
The resulting blue solution had
It was filtered while hot through a Whatman
No. 12 filter paper and the alcohol insoluble residue which was
brownish-black in color was discarded.
trated in vacuo at 40° C.
The filtrate was concen­
When the liquid had evaporated to a
relatively small volume, it was transferred to a beaker which was
placed in a dessicatlng Jar.
The dyestuff was concentrated further
in vacuo until crystallization took place at room temperature.
The crystals were collected in a fluted filter paper and the mother
liquor discarded.
The crystals were then washed with small
quantities of ice-cold absolute alcohol.
For recrystallization,
the dye wae again dissolved in warm (37°) absolute alcohol, fil­
tered through a Whatman No. 12 filter paper, and finally concen­
trated in vaouo as described above until crystal formation took
place.
The dye was recrystallized three times, and after being
dried was stored in amber glass-stoppered bottles.
0.187 gm. of
o^-naphthol blue was obtained.
A yield of
Macroscopioally, a
dark blue-violet dye compound having a bronze sheen was obtained,
as described by Mbhlau (1885); microscopically, the compound
appeared as Jagged particles of various sizes.
ard,
the
For use as a stand­
c^-naphthol blue was weighed out and dissolved in 1:1
chloroform-alcohol.
The dye dissolved quickly.
made on a Duboscq type colorimeter.
Readings were
Ten readings were made for
each test mixture and an average obtained.
The endpoint of the oxidase-inhlbition test was taken as the
highest dilution of the germicide which completely inhibited the
indophenol (cytochrome) oxidase activity of the standardized sus­
pension of gonococci.
B. Presentation of Results.
1, The Germicidal Tests.
TABLE 9
The Effect of Sliver Nitrate on the Viability of the Gonococcus.*
Experiment I.
Degree of d-rowth In Subculture.
Concentration
of AgNOs
1-700,000
1-800,000
1-900,000
1-1,000,000
1-2,000,000
Start
ED HYD
Moles
8.35
7.35
6.53
5.88
2.94
x
x
X
x
x
10“6
10-6
10-6
10-6
10"6
Culture Control.
Broth Sterility Test.
0
0
45
0
0
31
44
444
44
4444
0
0
2 Days
n r HYP
4 Days
ED h YB
7 Days
I F HYP
0
0
0
0
0
0
4+
44
4+44
0
0
0
44
44
4444
44
4+4
+4+4
0
0
444
+44
4+44
0
0
4+44 +444
4+4+ 44+4
4+4 + 4+44
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
44
4+
44
44
4+
4+4
4+
4+44
44
+++
++♦+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+4
4+4
+4
4+4
Experiment II.
1-600,000
1-700,000
1-800,000
1-900,000
1-1,000,000
9.77
8.35
7.35
6.53
5.88
x
x
x
X
X
10-6
10-6
10~6
10-6
10-6
Culture Control.
Broth Sterility Test.
0
0
0
170
++
0
0
0
0
182
+4+4
44+4
Experiment III.
1-600,000
1-700,000
1-800,000
1-900,000
1-1,000,000
9.77
8.35
7.35
6.53
5.88
x
x
X
x
x
10“6
10-6
10“6
10“6
10-6
Culture Control.
Broth Sterility Test.
0
0
0
103
0
0
0
125
44
44
44
444
0
0
0
0
0
444
44+4
0
444
4444
0
+4+4
+444
0
* Conditions of e^eriments: Egg digest broth (ED) and hydroooele
broth (HYD) Inoculated with 1.0 ml. standardized suspension of
drug-treated gonococci; broth cultures incubated at 35-36° C.
for period of one week under atmospheric conditions; at start of
experiment and at end of the 2nd, 4th, and 7th days, 0.20 cc. of
Inoculated broth subcultured onto Douglas chocolate plates; inoc­
ulated plates Incubated 48 hours at 35-36° C. under 10 per cent
C02 .
Legend: Numbers indicate colony counts up to 200 colonies; 4+
Indicates 201 to 500 colonies per plate; +++ Indicates 500 to
1000 oolonies per plate; ++++ indicates more than 1000 colonies
per plate. 0 indicates no growth.
TABLE IQ.
The Effect of Protargol on the Viability of the Conococcus.*
Experiment I.
Degree of Growth In Subculture.
Concentration
of Protargol Moles (Ag)
1-90,000
1- 100,000
1- 200,000
1-300,000
1-400,000
6.41
7.76
3.76
2.52
1.89
X
X
X
X
X
10-6
10-6
10-6
10-6
10-6
Start
ED
HYD
0
0
0
24
66
0
0
0
29
58
2 Days
ED
HYD
0
0
0
0
0
0
0
0
0
4+
444
44+
444
4444
+++
++++ ++++ ++++
0
0
0
Culture Control.
Broth Sterility Test
4 Days
to
ED
0
0
0
7 ;
Days
t o
ED
0
0
0
0
0
0
++++
4444
444
4444
4444
4444
4+44
4444
4+44
4444
+ 44+
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment II.
1- 100,000
1- 200,000
1-300,000
1-400,000
1-500,000
7.76
3.7Q
2.52
1.89
1.49
X
X
X
X
X
0
10~6
10“6
0
10-6 16
10-6 43
10“6 106
Culture Control.
Broth Sterility Testi.
++44
0
0
0
20
48
101
0
0
0
0
44
+4+
44 +
44
444
444
4+
444
44+4
444
4444
4444
+ 44
4444
4 + 44
4444
4+4+
4+44
++4+
44+4
4444
44+4
4444
+44+
444+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment III.
1- 100,000
1- 200,000
1-300,000
1-400,000
1-500,000
7.76
3.78
2.52
1.89
1.49
X
X
X
X
X
10-6
0
10-6
0
10-6 42
10-6 54
10-6 121
Culture Control.
Broth Sterility Test .
++++
0
0
0
45
58
138
0
0
0
0
+4
444
444
444
4444
4444
0
0
44+
+4+
4+4
444
4++4
4444
+ 444
444
+ 4+4
4 + 44
44++
+44+
4 + 4+
+444
4444
+++ 4
++++
4444
444
0
0
0
0
* The conditions of the experiments and legend used is same as
shown In Table 9.
0
TABUS 11.
The Effect of Neo-311vol On the Viability of the Gonococcus.»
Experiment I,
Degree of Q-rowth In Subculture.
Concentration
of Neo-Sllvol
1-20,000
1-30,000
1-40,000
1-50,000
1-60,000
1-70,000
Moles .(Ak I
4.09 x 10-5
2.72 x 10-5
2.04 x 10-5
1.63 X 10-5
1.36 X 10-5
1.16 X 10-5
Culture Control.
Broth Sterility Test.
Start
ED HYP
2 Days
ED HYP
0
0
0
0
0
3
0
0
0
0
0
5
44+
0
0
0
0
0
0
0
52
0
0
0
0
89
+44
4 Daye
ED HYP
0
0
0
0
+++
4 + 4+
0
0
0
0
4+4
4+4+
7 Days
ED HYP
0
0
0
0
444+
+ 4+4
0
0
0
0
4+++
++++
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment II.
1-40,000
1-50,000
1-60,000
1-70,000
1-80,000
2.04
1.63
1.36
1.16
1.02
x
x
x
x
x
10-5
10-5
10-5
10-5
10-5
0
0
i
12
42
0
0
3
12
38
0
0
++++ ++++ ++++ ++++
0
0
0
0
Culture Control.
Broth Sterility Test
44+4
44+4
0
0
0
0
0
0
++++
0
444+
0
Experiment III.
1-40,000
1-50,000
1-60,000
1-70,000
1-80,000
2.04
1.63
1.36
1.16
1.02
x
x
x
x
x
Culture Control.
Broth Sterility Test,
10-5
10-5
10-5
10-5
10-5
0
0
10
34
58
0
0
4
30
61
+♦+
44+
+++
+++
+++
+ 4+
0
0
0
0
0
0
0
0
+ 44
444+
444 +
0
+ 44
44+4
444+
0
0
0
+44
+ 44+
4+44
0
0
0
444+
+44+
+44+
0
» The conditions of the experiments and legend used are the same
as shown In Table 9.
TABLE 12.
The Effeot of Silver Nuolelnate On the Viability of the Qonoooccus.
Experiment I .
Degree of Growth In Subculture.
Concentration
of Silver
Moles
Nuolelnate
1-70,000
1-80,000
1-90,000
1 -100,000
1 -200,000
2.55
2.22
1.99
1.79
8.96
x
x
x
x
x
(Ag)
10-5
10-5
10-5
10-5
10~6
Culture Control.
Broth Sterility Test.
Start
is m b
0
0
5
14
0
0
3
18
0
0
2 Dava
E£ gYB
4 Days
12 M 2
7 Days
12 HYP
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A
Experiment II.
1-70,000
1-80,000
1-90,000
1- 100,000
1- 200,000
2,55
2.22
1.99
1.79
8.96
X
x
x
x
x
10-5
10-5
10-5
10-5
10-5
Culture Control.
Broth Sterility Test.
0
0
1
43
0
0
6
46
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment III.
1-70,000
1-80,000
1-90,000
1 -100,000
1- 200,000
2.55
2.22
1.99
1.79
8.96
x
x
x
x
x
10“5
10-5
10-5
10-5
10“e
Culture Control.
Broth Sterility Test.
0
0
5
22
0
0
7
22
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
• Conditions of the experiments and legend used are the same as
shown In Table 9.
TABLE 15.
The Effect of Argyrol On the Viability of the Gonococcus.*
Experiment I .
Degree of Growth In Subculture.
Concentration
of Argyrol
Moles .(Ml
1-40,000
1-50,000
1-60,000
1-70,000
1-80,000
4.65
3.71
3.09
2.64
2.31
Start
El? HYP
0
x 10-5
10-5
0
x
x 10-5 18
X 10“5 121
x 10-5 4 +
Culture Control.
Broth Sterility Test.
0
0
0
9
147
+++
2 Days
HYP
e £j
0
0
4+
0
0
+4
++
44 +4
+ 4+
4+44
0
0
4 Days
ED HYP
7 Days
ED "HYP
0
0
0
0
+ 44
4 4+4
+4+4
0
0
+4+4
+4+4
44+4
0
0
4+44
4444
4+4+
444+
4444
+4+4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment II.
1-40,000
1-50,000
1-60,000
1-70,000
1-80,000
4.65
3.71
3.09
2.64
2.31
x
x
x
x
X
10-5
0
10“5
0
10-5 10
10-5 111
10“5 4 +
Culture Control,
Broth Sterility Test.
0
0
13
129
4+
4 +4 + ++44
0
0
0
++
4+4
444
+44
4+4
4 + 44
+4+
4+4+
4+44
4+4+
+ 44+
+4+4
4444
444+
+4+4
44+4
4+44
+4++
44+4
+44 +
444+
+4+4
4444
4+44
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4+
+4
4+
4+4
0
Experiment III.
1-40,000
1-50,000
1-60,000
1-70,000
1-80,000
4.65
3.71
3.09
2.64
2.31
x
x
x
x
X
10-5
0
10-5
0
10-5
2
10-5 22
10“5 195
Culture Control.
Broth Sterility Test.
+4+4
0
0
0
2
24
186
+++
++++ ++++ ++++ ++++
+444
+4+4
+444
+4++
4+ + +
4+44
444 +
0
0
0
4+4
+ 44+
+ 4+4
0
4444
4444
4+44
4 + 4+
+ 4+4 44+4
0
0
0
* Conditions of the experiments and legend used are the same as
shown in Table 9.
TABLE 14.
The Effect of Kertblolate on the Viability of the Gonococcus.*
Experiment I .
Degree of G-rowth In Subculture.
Concentration
of Merthiolate
1-700,000
1-800,000
1-900,000
1- 1 ,000,000
1-2 ,000,000
Start
ED t&D
Moles
3.51 X 10-6
0
3.07 x 10-6
0
2.73 X 10-6
0
2.46 X 10-6
1
1.23 x 10-6 180
Culture Control
Broth Sterility Test,
44 44
0
2 Days
ED 5 y d
4 Days
ED HYP
7 Days
ED m
0
0
0
0
0
0
0
0
0
0
0
0
1
162
44
44
444
4444
4444
444
444
4444
4444
4444
4444
4+44
4444
4444
4444
4444
4444
4444
0
0
0
0
0
44
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment II.
1-700,000
1-800,000
1-900,000
1- 1 ,000,000
1-2 ,000,000
3.51 x 10-6
3.07 x 10-6
2.73 X 10-6
2.46 X 10-6
1.23 x 10-6
Culture Control
Broth Sterility Test.►
0
0
0
1
0
0
0
4
44
44
4+ + +
4444
0
0
0
0
0
44
444
44
4444
444
444
4444
4444
4444
4444
4444
4444
444+
4444
44 44
4444
4444
4444
0
0
0
0
0
0
Experiment III.
1-700,000
1-800,000
1-900,000
1-1 ,000,000
1-2 ,000,000
3.51
3.07
2.73
2.46
1.23
x
x
x
x
x
10-6
10-6
10-6
10-6
10-6
Culture Control.
Broth Sterility Test.i
0
0
0
10
0
0
0
18
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
* The Conditions of the experiments and the legend used are the
same as shown in Table 9.
56 ,
TABLE 15.
The Sffeot of Potassium Permanganate on the Viability of the
Gonococcus.#
Experiment I .
Degree of Growth In Subculture.
Concentration
of KMnCU
1-100,000
1-200,000
1-300,000
1-400,000
1-500,000
Moles
Start
ED H¥D
6.32 x 10-5
0
3.16 x 10~5 4 4
2.10 x 10"5 + 4 4
1.57 X 10“5 4 + 4 4
1.26 X 10“5 4 4 4 4
Culture
Control.
B roth Sterility Test.
4444
0
0
++
44+
+444
44+4
4444
0
2 Days
ED " HYP
0
+4+4
++++
+4+4
4444
0
0
++4
+++
444+
4444
44+4
0
4 Days
ED HYD
+4+4
++44
+44+
4444
44 44 4444
0
7 Days
ED HYP
0
0
4444
4444
4444
4444
4+44
+4++
4444
4444
444+
+44+
4444
4444
4444
444+
4444
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment II.
1-90,000
1-100,000
1-200,000
1-300,000
1-400,000
7.03 X 10“5
0
6.32 x 10-5 0
3.16 x 10-5
1
2.10 X 10“5 70
1.57 x 10“® 103
Culture
Control.
Broth Sterility Test.
0
0
5
75
110
0
0
44
444
444
444
444
444
4444
+444
4444
444-4
444
444+
+ 44 +
++4+
+444
4444
4444
4444
4444
4444 44 44
44+4
4444
4444
0
0
0
44
444
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment III.
1-90,000
1 - 100,000
1-200,000
1-300,000
1-400,000
1-500,000
7.03 x
6.32 x
3.16 x
2.10 x
1.57 X
1.26 X
10-5
0
10-5
0
10“5 11
10-5 85
10"5 1Q4
10-5 4 4
Culture
Control.
Broth Sterility Test.
++++
0
0
0
20
80
190
44
+444
0
0
0
0
0
44
44
444
44+
++++
444
444
4444
4444
4444
4444
444
4444
444
4444
4444
+44+
+444
44+4
44+4
4+4+
4444
4+44
4-44 +
4444
4444
0
+ 4 4 + 4+44
0
0
4444
0
0
44 44
0
• Conditions of the experiments and legend used same as shown in
Table 9.
TABLE 16.
The Effect of Sulfanilamide On the Viability of the gonococcus.#
Experiment I.*#
Degree of Growth In Subculture,
Concentration of
Sulfanilamide
Moles
1-133
1-1,333
1-13,333
1-133,333
4.36
4.36
4.36
4.36
x
X
x
x
Start
ED HYD
2. Days
ED HYD
4 Davs
ED HYD
7 Days
ED HYD
0
0
0
0
10-2
10-3
10“*
10"5
Culture Control.
Broth Sterility Test.
0
0
0
0
Experiment II.###
Degree of Growth In Subculture.
Concentration of
Sulfanilamide
Moles
1-133
1-1,333
1-13,333
1-133,333
4.36
4.36
4.36
4.36
x
x
X
x
Start
ED HYD
2 Davs
ED HYD
4 Days
ED HYD
0
0
0
7 Days
ED HYD
10-2
10-3
10“4
10~5
Culture Control,
Broth Sterility Test.
0
0
0
0
0
Experiment III.*##*
Degree of Growth In Subculture.
Concentration of
Sulfanilamide
Moles
1-133
1-1,333
1-13,333
1-133,333
4.36
4.36
4.36
4.36
x
x
x
X
Culture Control.
Broth Sterility Test.
10“2
10-3
10"4
10-5
Start
I E HYP
4444
4444
4444
4444
0
4444
4444
4444
4444
0
2 Days
£
126
4444
4+44
4444
4444
4444
4444
4444
4444
e
0
0
4 Days
IS HYP
4444
4444
4444
4444
0
4444
4444
4444
4444
0
7 Days
El" HYD
4444
4444
4444
4444
0
4444
4444
4444
4444
0
* Conditions of the experiments and legend used are the same as
shown In Table 9.
*# Sulfanilamide sterilized by passing through a Seitz filter.
### Sulfanilamide sterilized in the autoclave at 15 pounds for 20
minutes.
#### Sulfanilamide sterilized by Intermittent sterilization in the
Arnold sterilizer for one hour on three successive days.
Results of the Germicidal Testa.
Under the conditions of the experiments the following end­
points were obtained in the germicidal tests:
Silver nitrate in a concentration of 1-800,000 (7.35 x 10"® M)
destroys completely the viability of gonococcus, as shown in Table
9.
Table 10 shows that protargol in a concentration of 1-200,000
(3.78 x 10-6 m Ag) destroys completely the viability of gonococcus.
Neo-silvol in a concentration of 1-50,000 (1.63 x 10”® M Ag)
destroys completely the viability of gonococcus, as shown in
Table 11.
Silver nuolelnate 1-80,000 (2.22 x 10“® M Ag) destroys com­
pletely the viability of gonococcus as shown in Table 12.
Table 13 shows that argyrol in concentrations of 1-50,000
(3.71 x 10“5 M Ag) destroys completely the viability of gonococcus.
Merthiolate in a concentration of 1-900,000 (2.73 x 10“® M)
destroys completely the viability of gonococcus as shown in
Table 14.
Potassium permanganate, as shown in Table 15, completely
destroys the viability of gonococcus in concentrations of 1-100,000
(6.32 x 10-® M).
Sulfanilamide in concentrations as great as 1-133 (4.36 x
10-2
m
) did not affect the viability of the gonococcus (Table 16).
58.
2. The Enzyme Inhibition Teats.
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Results of the Laotlo Dehydrogenase Inhibition Tests.
Under the conditions of the experiments the following end­
points for the Inhibition of lactic dehydrogenase were obtained:
A concentration of silver nitrate 1-60,000 (9.7? x 10”5 M),
as shown in Table 17, completely Inhibits the lactic dehydrogenase
of gonococcus.
Table 18 shows that protargol in a concentration of 1-4,000
(1.89 x 10-4 M Ag) completely inhibits the lactic dehydrogenase
of gonococcus.
Neo-silvol in a concentration of 1-100 (8.1? x 10-3 M Ag)
completely inhibits lactic dehydrogenase of gonococcus as shown
in Table 19.
From Tables 20, 21 and 22, it can be seen that silver nuolelnate
(1,79 x 10_1 M Ag), argyrol (1.85 x 10-1 M Ag) and merthlolate
(2.46 x 10**1 M) respectively do not completely inhibit the lactic
dehydrogenase of N. gonorrhoeae even in concentrations of the drug
as high as 1-10, although at that concentration very marked in­
hibition of the enzyme is obtained.
Potassium permanganate in a concentration of 1-10,000
(6.32 x 10-4
m
) completely inhibits the lactic dehydrogenase of
gonococcus as shown in Table 23.
From the results shown in Table 24 it can be seen that
sulfanilamide in a concentration of 1-133 (4.36 x 10“2 M) causes
partial inhibition of the lactic dehydrogenase.
In the control
tubes which did not contain sulfanilamide the methylene blue was
reduced in 6.5 minutes, whereas In the tubes containing sulfan­
ilamide 1-133, the reduction time was increased to 9 minutes in two
experiments and to 8.5 minutes in a third.
This increase in
reduction time in the presence of sulfanilamide represents an
average of 26.3 per cent Inhibition of lactic dehydrogenase.
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Results of the Glyceric Dehydrogenase Inhibition Tests.
The length of time taken for reduction of methylene blue In
control tubes In the glyceric dehydrogenase test makes determina­
tion of the 100 per cent inhibition endpoint very difficult to
obtain.
Accordingly, the results of the inhibition of this enzyme
have been expressed using 65 per cent inhibition as the endpoint.
Under the conditions of the experiments, the following endpoints
for the inhibition of glyceric dehydrogenase of gonococcus were
obtained:
Silver nitrate in a concentration of 1-600,000 (9.77 x 10-6 M ) ,
causes 66.5# inhibition of the glyceric dehydrogenase of gonococcus
as shown in Table 25.
Protargol in a concentration of 1-70,000 (1.0Q x 10“5 M Ag),
as shown in Table 26, inhibits glyceric dehydrogenase by 68.9#.
Neo-silvol in a concentration of 1-500 (1.63 x 10"3 M Ag),
causes 64.4# inhibition of glyceric dehydrogenase as shown in
Table 27.
Table 28 shows that a concentration of 1-10,000 silver
nucleinate (1.79 x 10“4 M Ag) inhibits 70,# of the glyceric de­
hydrogenase of gonococcus.
Argyrol in a concentration of 1-10,000 (1.85 x 10“4 M Ag)
inhibits glyceric dehydrogenase of gonococcus by 65.5# (Table 29).
Merthlolate in a concentration of 1-30 (8.24 x 10“2 M) in­
hibits glyceric dehydrogenase by 64.8# as shown in Table 30.
Table 31 shows that potassium permanganate in a concentra­
tion of 1-100,000 (6.32 x 10“5 M) inhibits 64.5# of the glycerio
dehydrogenase of gonococcus.
75a
From Table 32, it is seen that sulfanilamide In a concentra­
tion of 1-133 (4.35 x 10-2
m)
dehydrogenase of gonococcus.
exerts no effect on the glyceric
76.
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Results of the Catalase Inhibition Tests.
Under the conditions of the experiments, the following end­
points for the Inhibition of gonococcus catalase were obtained:
As shown In Table 33, silver nitrate completely Inhibits
gonococcus catalase In a concentration of 1-2,000 (2.94 x 10-3
m ).
Protargol completely Inhibits gonoooccus catalase In a con­
centration of 1-200 (3,78 x 10-3 M Ag), as shown In Table 34.
Table 35 shows that neo-sllvol, in a concentration of 1-10
(8.17 x 10-2 M Ag), causes only 3.3 per cent Inhibition of catalase.
Table 36 shows that silver nucleinate 1-10 (1.79 x 10-1
m
Ag)
causes no inhibition of gonoooccus oatalase.
Merthlolate In a concentration of 1-10 (2.46 x 10-1 M), as
shown in Table 38, causes only 4.6 per cent inhibition; similarly
argyrol 1-10 (1.85 x 10-1 M Ag), as shown In Table 37, inhibits
catalase to the extent of 7.9 per cent.
Table 39 shows that concentrations of potassium permanganate
greater than 1-10,000 (6.32 x 10-4 M) Inhibit almost completely
gonococcus catalase.
Sulfanilamide, as shown In Table 40, does not oause any In­
hibition of catalase in a concentration of 1-133 (4.36 x 10~2 M).
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Results of the Peroxidase Inhibition Tests.
Under the conditions of the experiments, the following end­
points were obtained for the Inhibition of peroxidase:
Silver nitrate completely Inhibits gonococcus peroxidase in
a concentration of 1-3,000 (1.96 x 10-3 M) as shown in Table 41.
As shown in Table 42, protargol completely inhibits gonococcus
peroxidase In a concentration of 1-200 (3.78 x 10-3 M Ag).
Neo-silvol in a 1-10 (8.17 x 10-2 M Ag) concentration
71.6 per cent inhibition of peroxidase
Silver nucleinate in a l-lo (1.79
causeB
as shown in Table 43.
x 10“ 1 M Ag) concentration,
as shown in Table 44, causes 49.4 per cent inhibition.
Argyrol 1-10 (1.85 x 10-1 M Ag), inhibits the peroxidase
activity of gonococcus by 45.7 per cent, as shown in Table 45.
Merthiolate in a 1-10 (2,46 x 10-1 M) concentration causes
77.5 per cent
Table 47
inhibition of peroxidase
as shown in Table 46.
shows that potassium permanganate
completely inhibits
peroxidase in a concentration of 1-10,000 (6.32 x 10“4 M ) .
Sulfanilamide in concentrations as great as 1-133 (4.36 x
10-2 M), causes no significant inhibition of peroxidase, as shown
in Table 48.
94.
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Results of the Indophenol Oxidase (Cytochrome Oxidase) Inhibition
Tests.
--------------- ----------Under the conditions of the experiments, the following end­
points for the inhibition of indophenol (cytochrome) oxidase were
obtained:
Silver nitrate in a concentration of 1-2,000 (2.94 x 10-3 M)
causes virtually complete inhibition of gonococcus indophenol
oxidase as shown in Table 49.
Table 50 shows that protargol completely inhibits indophenol
oxidase of the gonococcus in a concentration of 1-100 (7.76 x
10“3 M Ag).
Neo-silvol, as shown in Table 51, inhibits the indophenol
oxidase of gonococcus 78.9 per cent in a concentration of 1-10
(8.17 x lO"2 M Ag).
Silver nuclelnate 1-10 (1.79 x 10"! M Ag) causes 51.4 per
cent inhibition of indophenol oxidase, as shown in Table 52.
Argyrol in a concentration of 1-10 (1,85 x 10~1 M Ag) inhibits
the oxidase by 50.4 per cent (Table 53).
Merthiolate completely inhibits Indophenol oxidase in a con­
centration of 1-10 (2.46 x 10-1 M), as shown in Table 54.
The effect of potassium permanganate on indophenol oxidase
cannot be measured by the present technique, as the results in
Table 55 show.
Larger amounts of KMn(>4 apparently caused less
inhibition of the enzyme system than did smaller amounts.
As shown
in a subsequent portion of this dissertation, this paradoxical
effect is due to direct oxidation of the nadi reagents by potassium
permanganate which is bound to the organisms and cannot be removed
even with 4 successive washings.
Indophenol oxidase is partially inhibited by sulfanilamide
as can be seen from Table 56.
Sulfanilamide 1-133 (4.36 x 10”2
causes 55.1 per cent inhibition.
m
)
Even in a dilution of 1-1,333
(4.36 x 10“3 M) the oxidase is still inhibited to the extent of
12.7 per cent.
The significance of this partial inhibition of
the indophenol oxidase of gonococcus is not known.
However, it
is possible that inhibition of this enzyme system may be concerned
in the bacteriostatic effect of the sulfonamide drugs.
TABLE 57.,
Compilation of the Results of the Germicidal and Enzyme Inhibition
Viability
Teat.
Compound.
Silver
Nitrate.
Protargol.
Neo-sllvol.
^ Inhibition.
Concentration
of Drug.**
1-300,000
Molarity.***
7.35 x 10-6
Inhibition.
Concentration
of Drug.**
Lethal
1-200,000
Molarity.***
3.78 x 10-6
&
Inhibition.
Concentration
of Drug.**
%
Molarity.***
Silver
Nuolelnate.
Inhibition.
Concentration
of Drug.**
%
Molarity.***
Argyrol.
Merthlolate.
Lethal
Dehydrogenases
Lactic
0-lyceric
100
1-60,000
9.77 x 10-5
1-600,000
9.77 x 10-5
100
68.9
1-4,000
1-70,000
1.89 x 10-4
Lethal
100
1-50,000
1-100
1.63 x 10-5
66.5
8.17 x 10-3
1.08 x 10“5
64.4
1-500
1.63 x 10-3
Lethal
94.0
70.0
1-80,000
1-10
1-10,000
2.22 x 10-5
1.79 x 10-1
1.79 x 10-4
# Inhibition.
Concentration
of Drug.**
Lethal
92.1
65.5
1-50,000
1-10
1-10,000
Molarity.***
3.71 x 10-5
1.85 x 10-1
^ Inhibition.
Concentration
of Drug.**
1-900,000
Molarity.***
2.73 x 10-6
PotnSfil^iq
.iQJU.'b.UiQ.a.
Permanganate. Concentration
of Dr.ng.**
Molarity.***
Lethal
Lethal
1.85 x 10-4
85.1
64.8
1-10
1-30
2.46 x 10-1
100
8.24 x 10-2
64.5
1-100,000
1-10,000
1-100,000
6.32 x 10-5
6.32 x 10-4
6.32 x 10-5
104
DY,
he Germicidal and Enzyme Inhibition Tests on Gonococcus
,11 ty
Jc.
tal
000
: 10-6
Lai
000
: 10-6
Lai
000
: 10-5
Dehydrogenases
lactic
Glyceric
1-60,000
9.77 x 10-5
100
100
1-600,000
1-2,000
1-3,000
1-2,000
9.77 x 10-6
2.94 x 10-3
1.96 x 10-3
2.94 x 10“ 3
100
68.9
100
100
100
1-4,000
1-70,000
1-200
1-200
1-100
3.73 x 10“3
3.78 X lO-3
7.76 x 10-3
64.4
3.3
71.6
78.9
1-500
1-10
1-10
1-10
1.89 x 10"4
100
1-100
8.17 x 10-3
1.08 x lO"5
1.63 x 10'3
tal
94.0
70.0
000
1-10
1- 10,000
: 10-5
1.79
X
10-1
1,79 x 10-4
tal
92.1
65.5
000
1-10
1-10,000
: 10-5
Peroxidase
Indoohenol
(Cytochrome)
Oxidase.
100
66.5
100
Catalase
(#1111)
1.85 x 10-1
1.85 x 10-4
Q.17 x 10*2
0
1-10
1.79 x 10-1
8.17 x 10-2
8.17 x 10-2
49.4
51.4
1-10
1-10
1.79 x lO"1
1.79 x 10-1
7.9
45.7
50.4
1-10
1-10
1-10
1.85 x 10-1
1.85 x 10-1
1.85 x 10-1
Lai
85.1
64.8
4. 6
77.5
100
000
1-10
1-30
1-10
1-10
1-10
: 10“6
lal
2.46 x 10-1
100
8.24 x 10-2
64.5
2.46 x 10-1
100
2.46 x 10-1
100
2.46 X 10”!
*###
000
1- 10,000
1-100,000
1 -10,000
1-10,000
*#*#
: 10-5
6.32 x 10- 4
6.32 x 10-5
6.32 x 10-4
6.32 X lO"4
##*#
TABLE 57.
Viability
Test.
Compound.
S ulfanilamide. % Inhibition.
Concentration
of Drug.**
Molarity.»»»
Not lethal
(Continued)
Dehydrogenases
kfto&U
26.3
1-133
1-133
4.36 x 10-2
4.36 x 10“2
1-133
4.36 x 10-2
* In the case of neo-silvol, merthiolate, sliver nucleinate and
argyrol, the drugs could not be used in a concentration higher
than 1-10 for reasons of solubility.
Similarly, concentrations
of sulfanilamide above 1-133 (4.36 x 10“2 M) could not be prepared,
*# Expressed in terms of dilution of the drugs.
### Expressed in terms of moles of germicide, except for the sllverprotein compounds, causing death or inhibition as indicated.
In
the case of the silver-proteln compounds the molarity is calcu­
lated on the basis of the silver content.
##*# Could not oe determined.
Llity
*t.
105.
TABLE 57.
Dehydr Ofrenas e s
Lactic
Glyceric
ithal
26.3
L33
1-133
c lO-2
(Continued)
4.36 x 10-2
Catalase
Peroxidase
Indophenol
(Cytochrome^
Oxidase
55.1
2.1
0
2.3
1-133
1-133
1-133
1-133
4,36 x 10-2
4.36 x 10-2
4.36 x 10-2
4.36 x 10”
>late, sliver nucleinate and
jed In a concentration higher
iy.
Similarly, concentrations
5 x 10“ 2 M) could not be prepared.
? the drugs.
germicide, except for the silveri or inhibition as Indicated.
In
jorapounds the molarity is calcu? content.
106
Analysis of Tabulated Endpoint Data of Germicidal and Enzyme
Inhibition Tests.
In Table 5 7 ,
the results of the viability tests and enzyme
inhibition tests using the various drugs are summarized.
In no
instance is there an obvious correlation between the concentra­
tion of a drug which is lethal for gonococci and that causing
partial or complete inhibition of the various enzymes tested.
Sulfanilamide is a notable exception.
Within the limits of
solubility of this drug no effect on viability could be observed
during the test period.
Moreover, there was relatively little
effect on the activity of the enzymes, although it should be
pointed out that partial inhibition of Indophenol oxidase (cyto­
chrome oxidase)
(55.1#) and of lactic dehydrogenase (26.3#) occurred
at the highest concentration of sulfanilamide.
It is possible that were an endpoint selected other than
complete inhibition of the enzyme, a correlation might be observed
between the concentration of a particular drug causing death and
the partial inhibition of one or other enzyme system.
The results of the enzyme inhibition and viability tests with
the various germicides are also depicted graphically in figures
1 to 8.
FICURE 1.
Effect of Silver Nitrate on Viability mid Ijjngvme Aetlrlty
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FIGURE 7.
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^
C. Supplementary Studies.
1) Supplementary Studies on the Nature of Bacterial Peroxidase.
Additional work was done in connection with the peroxidase
test with the object of clarifying certain obscure questions con­
cerning the nature of the reaction.
It has been stated that hydrogen peroxide is the only known
substrate acted on normally by catalase (Kellln and Hartree, 1936),
and for this reason catalase has been considered as the prototype
of enzymes exhibiting absolute specificity (Stern, 1936).
In view
of earlier work which showed that catalase does not decompose
monoethyl hydrogen peroxide if this compound is used in the
peroxidase test, Kirchner and Nagell (1926) substituted this alkyl
peroxide for hydrogen peroxide in their quantitative peroxidase
and catalase tests.
This was done to determine what Influence
the oxygen liberated had on the coexisting catalase.
Their re­
sults were unsatisfactory since no decomposition of monoethyl
hydrogen peroxide took place either by catalase or peroxidase.
They concluded that the bacteria they tested were probably peroxidase-free.
In the present investigation, a search was made for substances
which would selectively inhibit catalase.
It was believed that
if gonococcus catalase could be totally inhibited by the use of
a selective inhibitor without affecting or only partially in­
hibiting peroxidase activity, then the validity of the peroxidase
reaction employed in the present investigation might be established
and the objections raised by Kirchner and Nagell set aside.
It was observed that when certain catalase inhibitors
(formalin and hydroxylamine) were added to the catalase test
mixtures, the controls showed that these inhibitors interfered with
the potassium permanganate titration of the residual peroxide.
The lodometric method for determining catalase activity could not
be utilized in this work, therefore the volumetric method was
employed.
Because gonococcus contains a powerful catalase it was un­
necessary to use a microrespirometer to measure the oxygen liber­
ated.
In order to insure proper results two volumetric methods
were followed: one by Bailey (1917), who determined the catalase
activity of wheat flours, and the other by Morgulis (1921), who
worked with a crude catalase preparation from liver.
Bailey used
a closed vessel, the stopper of which had three openings: one
through which a separatory funnel could be introduced, another
was connected to a gas burette, while in the third was placed a
stop-cock to equilibrate the air present in the vessel when the
reagents were added.
The gas burette was connected to an over­
flow bulb so that the water displaced by the gas in the burette
accumulated in the overflow bulb.
In the present investigation
the vessel employed was a 250 ml. suction flask shaken mechan­
ically to mix the contents thoroughly.
of 20° C. was maintained.
took place.
A uniform temperature
Rapid liberation of the oxygen formed
The method of Morgulis makes use of an Erlenmeyer
flask agitated by a shaking device; the oxygen liberated Is col­
lected by water displacement into an eudiometer.
The same pH and time was used for the catalase inhibition
tests as for the peroxidase tests sinoe the Inhibitor to be
selected was intended for use in the peroxidase inhibition test.
No attempt was made to correct the figures obtained for the
evolved oxygen to standard temperature and pressure.
In one set
of experiments, using Bailey's method, the oxygen produced was
16.9 ml., whereas Morgulis1 method gave only 14.5 ml. oxygen.
This discrepancy is undoubtedly due to the fact that Morgulis'
water displacement method for collecting the oxygen evolved does
not measure all the gas produced by the action of catalase; some
gas remains dissolved in water and does not reach the eudiometer.
When Bailey's method is used, negative tension may be produced
in the reaction vessel by manipulating the position of the over­
flow bulb, thus withdrawing from solution much of the dissolved
gas.
The catalase inhibitors selected were those suggested by
various workers, and are listed in Table 58.
The catalase test
mixture consisted of the following reagents for both the volumetric
and permanganate methods:
3 ml. N/l hydrogen peroxide; varying amounts of in­
hibitors as indicated in Table 58; 1 ml. standard
suspension of untreated organisms (#1111); M/150
phosphate buffer, pH 7.2 to give a final volume of
30 ml.
The organisms were added last and the re­
action was timed for 15 minutes after wnloh the
mixture was acidified to stop any activity.
The
peroxidase test mixture consisted of the following
reagents:
3 ml. N/l hydrogen peroxide; varying amounts of in­
hibitors as Indicated in Table 58; 5 ml. 1.56#
pyrogallol dissolved in M/150 phosphate buffer, pH 7.2;
1 ml. standard suspension of normal organisms (#1111);
M/150 phosphate buffer, pH 7.2, to give total volume of
30 ml.
The organisms were always added last and the re­
action timed for 15 minutes at 20° G. after which sulphuric
acid was added.
One of the inhibitors used was M/20 citrate buffer, pH 4.5;
in tills case citrate buffer was substituted for phosphate buffer
in the reaction mixtures.
All inhibitors which could be adjusted
to pH 7.2 were so neutralized either with HC1 or NaOH.
From Table 58 it can be seen that none of the inhibitors used
gave 100 per cent Inhibition of catalase activity without peroxidase
being inhibited to some extent also.
Hydroxylamine seemed most
satisfactory for the present study.
Azlde, potassium cyanide and hydroxylamine are known to be
potent respiratory poisons (Keilln and Hartree, 1936; Keilln, 1936),
hydroxylamine being one of the most powerful inhibitors of catalase.
Keilin and Hartree (1936) separated the catalase inhibitors into
two categories:
(a) those, such as KCN and HgS, which prevent the
formation of an Intermediate reduced catalase compound, and (b)
those like azide, hydroxylamine and hydrazine which stabilize the
reduced intermediate compound and thereby inhibit the catalase re­
action.
The hydroxylamine hydrochloride (Merck) used in this in­
vestigation waB acid, and was neutralized with normal NaOH to
pH 7.2.
Blaschko (1935) studied manometrlcally the effect of
several Inhibitors on a purified horse liver catalase, among which
0.0001 M hydroxylamine hydrochloride completely inhibited the re-
action.
In the present Investigation final concentrations of
0.000099, 0.00099 and 0.00997 M hydroxylamine hydrochloride were
tested in the catalase (volumetric) and peroxidase tests.
TABLE 58.
The Effect of Various Inhibitors on Gonococcus Catalase
and Peroxidase.*
Inhibitor Used.
Moles
Remarks
10-4
lO-4
10-4
lO"2
10-4
10-3
NagS.9HgO
KCIO3
Resorcinol
Sodium formate
Cysteine
NaN02
6.88
4.83
NaN02
KCN
9.66 x 10-3
5.26 x 10-3
Ethyl alcohol
1.82
9.79
9.99
1.22
3.16
Methyl alcohol
Hydroxylamine*#
4.98
9.97 x 10"5
Hydroxylamine*#
9.97 x 10-4
Kydroxylami ne **
9.97 x 10-3
NaN03
1.33 x 10"1
NaN3
NaF
2.97 x lO"3
7.93 x 10-2
Pyridine
4.17 x 10-1
Chloroform
2.09
Toluol
1.56
Sodium barbital
4.85 x 10-3
Sulfanilamide
3.87 x 10-3
Sulfapyrldlne
1.98 x 10-4
Citrate buffer (pH 4.5)
0.02
Formalin*#
1.97 x 10-2
Catalase hardly affected.
Catalase hardly affected.
Catalase weakly affected.
Catalase hardly affected.
Catalase hardly affected.
Marked peroxidase and
slight catalase inhibition.
Slight catalase inhibition.
Total peroxidase and virtual­
ly total catalase inhi­
bition.
Half of catalase activity
inhibited.
Catalase hardly affected.
32# catalase inhibited and
1.9# peroxidase.
All catalase and 56#
peroxidase inhibited.
Catalase and peroxidase
totally inhibited.
Half of catalase and
peroxidase Inhibited.
All peroxidase inhibited.
Half peroxidase and 4/5
catalase inhibited.
Catalase hardly Inhibited.
Catalase hardly inhibited.
Catalase not affected.
Catalase not affeoted.
Catalase hardly affected.
Catalase hardly affeoted.
100# peroxidase and 50#
catalase inhibition.
Catalase and peroxidase
markedly affected.
# Conditions for catalase and peroxidase tests: total volume 30 ml.,
pH 7.2, time 15 minutes at 20° C. Proper controls were used.
#* The volumetric method was used for determination of catalase
activity.
Table 59 shows that 0.000997 M hydroxylamine completely in-
hlblts catalase activity while peroxidase activity was Inhibited
by 56 per cent.
Therefore, when catalase activity is totally
TABLE 59.
The Effect of Various Concentrations of Hydroxylamine on Qonococcal
Catalase and Peroxidase. «
Final Concentration
of Hydroxylamine. HC1
(Moles)
9.97 x 10-5
9.97 x lO'4
9.97 x 10“3
Catalase
Inhibition
(oer cent)
32.0
100
100
Peroxidase
Inhibition
(per cent)
1.9
56.0
100
♦ T h e conditions for the tests were the same as given for Table 58.
The catalase test was measured manometrloally and the hydroxylamii
HC1 was neutralized before use.
Inhibited in gonococcus, peroxidase activity can still be demon­
strated although somewhat reduced.
TABLE 60.
Silver Nitrate Inactivation of Peroxidase With and Without
hydroxylamine Added.*
Concentration
Moles
of AgNOs
1-2,000
2.94 x lO'3
1.96 x 10-3
1-3,000
1-4,000
1.46 x lO"3
1.17 x lO'3
1-5,000
9.77
x 10-4
1-6,000
5.88 x 10“4
1-10,000
System Control.
Normal Control.
Blank.
Heated Enzyme Control.
Mg % Purpurogallln Formed.
No NHpOH added
NH p OH added***
Cor­
Cor­
% Inhi
Found rected Found rected bition
0.07
0
0
0.20
0
0
0.20
0.07
0.04## 0.04##
0.49
0.62
2.27
1.00
1.00
2.40
56.0
3.87
4.00
1.50
1.50
61.3
8.47
59.9
8.60
3.40
3.40
10.34
0.13
0.13
0.14
10.21
0
0.01
4.60
0
0
0
4.60
0
55.0
0
* The conditions for this experiment are the same as those de­
scribed In the text.
♦♦ This value has a large error and may be omitted for practical
purposes since the colorimetric readings are Inaccurate in this
range.
♦ ♦♦ 9.97 x 10-4 M NH2OH added.
*♦♦♦ This value represents the per cent inhibition of the peroxidase
inhibition test with AgN03 caused by the addition of NHjjOH
to the test mixtures.
TABLE 61.
Potassium Permanganate Inactivation of Peroxidase With and Without
the Addition of HydroxylamlneT*
Concentration
of KMnOa.
Moles
7.03 x 10-4
1-9,000
6.32 X 10-4
1 - 10,000
3.16 X l O " 4
1 - 20,000
2.10 X 10“ 4
1-30,000
1.57 x lO”4
1-40,000
System Control.
Normal Control.
Blank.
Heated Enzyme Control.
Mg % Purpurogallln Formed,
No NHgOH added
NljpOri
"
added***
CorCor% InhlFound rected Found rected bltipn****
0
0.11 -0.01
0
0.07** 0.07**
0.23
0.11
47.7
0.42
0.22
0.54
0.22
0.97
0.85
0.31
0.31
64.0
1.08
1.08
2.86
2.74
60.1
10.18
0.12
0.12
0.12
10.06
0
0
4.16
0
0
0
4.16
0
58.7
0
* The conditions for this experiment are as described in the text.
** This value has a large error and may be omitted for practical
purposes since the colorimetric readings observed were not con­
sidered accurate.
*** 9.97 x 10-4 m NHgOH added.
**** This value represents the per cent inhibition of the peroxidase
Inhibition test with KMn04 caused by the addition of NHgOH
to the test mixtures.
In order to determine what effecthydroxylamine would
have on
drug-treated organisms, peroxidase inhibition tests with silver
nitrate and potassium permanganate were studied with and without
the presence of NHgOH.
The results are shown
From the data presented in Tables
in
Tables60
and 61.
60 and 61
it canbe
seen
that the endpoints of the peroxidase inhibition tests using silver
nitrate and potassium permanganate were the same whether or not
9.97 x 10”4
m
hydroxylamine was also present in the test mixture.
For all practical purposes hydroxylamine may therefore be omitted
from the regular peroxidase inhibition tests with the various
germicides, since gonococcal catalase apparently does not inter­
fere with the peroxidase reaotion.
2) The Effect of Heat on Gonococcal Peroxidase.
As with other enzyme tests studied In this investigation, the
quantitative peroxidase tests were always controlled with a sus­
pension of organisms previously exposed to 100° C.
In all oases,
heating the suspension of organisms for 15 minutes at 100° C.
totally inaotivated the peroxidase activity of gonococcus when
tested by the pyrogallol method.
In a study of the peroxidases of a number of species of micro­
organisms, Callow (1926) found them heat-stable In all instances,
since they withstood boiling for one hour.
The tests were carried
out with benzidine or guaiao; the pyrogallol method was not used.
Callow showed that some organisms having weak peroxidase reactions
give stronger and more lasting colors when boiled.
Also, some
organisms showing peroxidase activity gave rise to a colored com­
pound which faded upon standing; however, when all suspensions were
boiled the color remained permanently.
Callow attributed the fading
to a heat—labile dehydrogenase which was destroyed by boiling.
The
gonococcus was not Included in the list of organisms tested.
Upon repeating the observations of Callow on the effect of heat
on bacterial peroxidases, peroxidase reagents were prepared con­
sisting of one per cent benzidine base (Pfanstiehl, for blood tests)
in 50 per cent alcohol, and 20 volumes per cent of hydrogen peroxide.
The standard suspensions of gonococci were taken up both in dis­
tilled water and in acetate solution.
To one ml. of the gonococcus
suspensions were added 0.1 ml. of benzidine solution and 0.5 ml.
hydrogen peroxide.
A weak blue color was obtained with both sus­
pensions; the color of the aqueous suspension of organisms faded
in three minutes, while the acetate suspension faded in half a
minute.
When heated for 15 minutes at boiling temperature, the
peroxidase tests were stronger than in the unheated suspensions.
In this case, however, the acetate suspension of organisms dis­
colored in six minutes while the water suspension kept the original
olue color for at least ten minutes.
When a one per cent aqueous
solution of pyrogallol and hydrogen peroxide were used as peroxidase
reagents, a red-brown color of purpurogallin was formed in the un­
heated water and acetate suspensions of gonococci, whereas the
heated suspensions produced no color.
One ml. of the acetate and aqueous suspensions of gonococci
respectively were added to 0,1 ml. of 1-5000 (0.0005 M) methylene
blue and placed in the 60° C. water bath.
blue occurred in ten minutes.
No reduction of methylene
Callow observed reduction of methylene
blue within two or three minutes at 600 C. by those bacteria show­
ing a fading of color of the peroxidase tests.
From the above data it may be concluded that the peroxidase
system of gonococcus responsible for the production of purpurogallin
from pyragallol is heat-labile, although the cells contain also a
heat stable system which gives a positive peroxidase test when
benzidine is used as the test reagent.
In other experiments, when
KCN was used as an inhibitor of peroxidase, the heated suspensions
of gonococcus no longer gave a positive benzidine test.
The benz­
idine test with heated cells was likewise negative when hydrogen
peroxide was omitted from the test system.
These observations
indicate that the positive benzidine reactions obtained with heated
cells are due to a heat-stable peroxidase system.
It may be of
Interest to point out that stronger positive benzidine tests were
obtained with boiled suspensions of gonococci than with the un­
heated cells, and that the color obtained with the heated cells
lasted longer than when unheated cells were used.
As indicated above the suspending fluid affects the rate of
disappearance of the color obtained in the benzidine test; when
cell suspensions made in distilled water were tested the color per­
sists longer than when an acetate solution was used as a suspending
medium, both in tests using heated and unheated cells.
The pyrogallol test was also compared in the presence and
absence of hydrogen peroxide.
The object was to determine whether
or not a significant amount of pyrogallol is oxidized when hydrogen
peroxide is omitted.
For this experiment the usual pyrogallol
peroxidase test was carried out uBlng heated and unheated acetate
suspensions of the gonococcus.
The results are shown in Table 62.
TABI£ 62.
The Effect of Using Heated and Unheated Suspensions of Gonococci
on Pyrogallol VIth and Without Presence of Hydrogen Peroxide.
Normal Suspension
Heated Suspension
Blank
Normal Suspension
Heated Suspension
Blank
Normal Control
Mgs. $_ Purpurogallin Formed.
In the Presenoe of HpQg.»
Found
Corrected»»
10.36
10.18
0.18
0
0.18
No H p O p. Added»»»
Found
Corrected
0.41
0.23
.18
0
.18
0.18
0
« The test mixture consisted of 21.0 ml. M/150 phosphate buffer
pH 7.2, 3.0 ml. N/l H2O2 , 5.0 ml. 1.56# pyrogallol dissolved in
pH 7.2 M/150 buffer, and 1.0 ml. standard suspension of gonococci
in acetate solution heated at 100° C. for 15 minutes or unheated.
*« Corrected figures represent the values obtained by subtracting
the amount of purpurogallin formed in the blank from that obtained
in the experiment.
»** The composition of the test mixture was the same as desoribed
for (#), except that the hydrogen peroxide omitted was replaced
with the same volume of burrer.
v
From the results shown In Table 62 it can be seen that when
hydrogen peroxide is omitted from the system a negligible amount
of oxidation of pyrogallol occurs.
3) Conditions Necessary for the Quantitative Indophenol Oxidase
(Cytochrome Oxidase) Test.
~"~*
In the development of the quantitative indophenol oxidase
test, a method had to be selected which was applicable to the
gonococcus.
The use of the petri dish as a container for the ex­
perimental mixture is impractical for quantitative experiments.
The larger the surface of the test mixture exposed to the air, the
higher the value of the blank becomes.
The best results were ob­
tained when 25 x 100 ml. pyrex test tubes were used.
It was noted early In the course of the work that various lots
of dlmethyl-p-phenylenediamine HC1 differed In color when made in­
to solutions of known strengths.
Since black or brown colored
diamine crystals gave dark colored solutions and increased the value
of the blank, the grey-white product was chosen in preference.
Somewhat better results were obtained using M/20 phosphate
buffer at pH 6.6 than when M/20 acetate solution at the same re­
action or distilled water were used.
Distilled water cell suspen­
sions were found to give slightly higher values In the test than
acetate suspensions.
TABLE 65.
A Study of the Relation of Time and Quantity of Dye Formed by
Gonococcus and the Blank at Room Temperature.*
Intensity of Color formed.
Time
(minutes):
5
10
15
Testi
Moderate color Marked color Marked color
Blank:
Almost colorTrace of
Darker tint
less
color
20
Marked color
Distinct color
# The test consisted of 5 ml. substrate mixture, 5 ml, M/20 phos­
phate buffer, pH 6.6 and 1.0 ml. of standard suspension of
organisms.
The blank consisted of 5 ml. substrate mixture, 5 ml. M/20 phos­
phate buffer pH 6.6 and 1.0 ml. of 0.15 M. acetate solution.
Table 63 shows the results obtained at room temperature and
the Intensity of color formed In the blank and test mixtures at
various time Intervals.
When the same experiment was repeated at
37° C., the quantity of dye formed in each tube was increased.
The experimental time adopted for the routine oxidase test was
fifteen minutes since it was found that the values for the blanks
remained low over this period.
The addition of an equal quantity of phosphate buffer to the
substrate mixture altered the final pH of the experimental mixtures.
The final pH of the mixture was not necessarily the same as the
of the buffer tested.
pH
This fact is shown in a study of the optimum
pH of indophenol (cytochrome) oxidase activity of the gonococcus,
shown in Table 64.
TAB IE 64.
Effect of Buffers on the Final pH of the Experimental Mixture
and the Indophenol Oxidase of the Gonococcus.*
pH of Buffer
5.80
6.20
6.42
6.52
6.61
6.82
6.93
7.00
7.05
7.21
7.45
7.68
8.04
pH of Experlmental Mixture
6.18
6.40
6.62
6.75
6.78
6.98
7.05
7.18
7.20
7.35
7.55
7.73
8.04
Mg.#
Test
0.62
0.79
0.84
0.86
0.87
0.86
0.83
0.70
0.69
0.63
0.58
0.49
0.39
o6-Naphthol Blue Formed
Blank
Correction**
0.41
0.21
0.55
0.24
0.60
0.24
0.62
0.24
0.63
0.24
0.6£
0.24
0.59
0.24
0,51
0.19
0.49
0.20
0.43
0.20
0.37
0.21
0.23
0.26
0.23
0.16
# Buffers of various pH values were M/20 Clark and Lubs phosphate
mixture; test consisted of 5 ml. substrate mixture, 5 ml. M/20
phosphate buffers of various pH values and 1 ml. standard sus­
pension of normal gonococci. The blank had 1 ml. 0.15 M acetate
solution, pH 6.6 instead of organisms. Substrate mixture con­
sisted of M/100 alpha-naphthol in 50# alcohol, M/161 dlmetkyl-pphenylenediamine HC1 in distilled water and M/244 Na2C03 in dis­
tilled water in equal quantities.
#* This figure is obtained by subtracting the value of the blank
from that of the test.
From Table 64, pH 6.6 buffer was selected for the routine oxidase
test.
It should be noted that the final pH of the experimental
mixture in the test was 6.78.
In order to ascertain whether the quantity of substrate used
in the experimental fluid was more than ample for the maximum
oxidation by gonococcus under the conditions used, the following
test was made: Duplicate sets of tubes containing oxidase sub­
strate with gonococci and oxidase substrate alone were incubated
for 24 hours at 37° C.
imeter.
The dye formed was estimated in the color­
The organisms formed 6.49 mg, per cent alpha-naphthol blue
while the blank formed 5.57 mg. per cent of the dyestuff.
These
results indicate that the oxidlzable substrates available In the
indophenol oxidase test mixtures were more than sufficient for
oxidation by standardized suspensions of gonococci during the 15
minute reaction time adopted for the indophenol (cytochrome)
oxidase Inhibition tests.
The enzyme activity In this test was arrested by the addition
of KCN.
That KCN inhibits indophenol oxidase is recorded by
Yamagutchi (1935), Stotz, Sldwell and Hogness (1938), Keilln (1929)
and others.
The final concentration of potassium cyanide used in
this Investigation to arrest the enzyme activity was 0.046 M.
A number of solvents was used to extract the dyestuff formed.
Ethyl ether, xylol, toluol and petroleum ether gave a purple color
while carbon disulphide, chloroform, acetone and ethyl alcohol
gave a blue color when the dye was extracted.
It was noted that
although the colors of the alpha-naphthol blue standard or the
dyestuff extracted from the indophenol oxidaee test mixture dis­
solved in the same or different solvents may match when examined
by Inspection, a different shade and tint was elicited In the
colorimeter.
This difficulty was obviated by the use of a 1:1
chloroform-alcohol mixture; a very good colorimetric match was ob­
tained both in shade and tint between the extracted dyestuff from
the test reagent and the alpha-naphthol blue standard dissolved in
this solvent.
Considerable attention was paid to the preparation of a satis­
factory standard for this work.
were tried.
Over forty separate preparations
These included the method of Guthrie; the adjustment
of the substrate to various pH values; the addition of chromates,
ferrlcyanide, sodium hypochlorate to accelerate oxidation; pro­
longed exposure of the substrate in atmospheric conditions; the
bubbling of oxygen through the mixture; addition of suspensions
of gonococci; and the use of minced beef heart as a rich source
of cytoolirome oxidase.
The best reproducible results were ob­
tained by following the original method of Koechlin and Witt
(1881), and accelerating the reaction by the addition of ferric
chloride as suggested by Guthrie (1931).
Alcohol was found to be the
best solvent for purifying the dyestuff.
The characteristics of the alpha-naphthol blue prepared checked
with those given in Beilstein (1930).
A bright yellow color was
produced when the crystals were dissolved in concentrated sulphuric
and dilute acetic acids.
In the presence of stannous chloride in
an acid menstruum and with the application of heat, the dye was able
to be reduced to form a grey sediment (the stannic salt of leucoalpha-naphthol blue).
The dyestuff was more soluble in alcohol
than ethyl ether but was insoluble in distilled water.
Since the
dye prepared had the same general characteristics as for alpha-
naphthol blue recorded in the literature, it was considered a
satisfactory colorimetric standard for this investigation.
The
absolute purity of the indophenol standard is however not attested,
A probable error due to the separate oxidation of the dlmethylp-phenylenediamine in the "nadi" reagent by the cells, in addition
to the formation of indophenol as pointed out by Battelll and
Stern (1912), should not be overlooked when expressing results in
terms of alpha-naphthol blue.
This error must necessarily be small
since the oxidation product of dlmethyl-p-phenylenediamine catalyzed
by gonococcus produced a red color when extracted with chloroform
and a dark-brown color when alcohol was added direotly to the test
mixture,
A final consideration should be devoted to the nomenclature
of the dye standard.
The unsatisfactory state in the nomenclature
of Indophenols and indamines has been recognized by Gibbs, Hall,
and Clark (1928).
The correct name for the oxidation product of
dimethyl-p-phenylenediamine and alpha-naphthol is alpha-naphthol
blue as mentioned by Koechlin and Witt (1881), MiShlau (1883),
Heller (1912), Beilsteln (1930), and Schultz (1934).
Beilsteln
has also named this substance naphthoquinone-(1,4)-mono-(4-dlmethylamino-aniline).
4) Effect of Potassium Permanganate on the Indophenol Oxidase Test.
As shown In Table 55, tests for the Inhibition of indophenol
oxidase by potassium permanganate are difficult to access because
of the irregularity of the results.
Gonococcus suspensions sub­
jected to the effect of high concentrations of KMn04 gave alphanaphthol blue readings higher than the controls, indicating that
the effect was not entirely enzymatic in nature.
Organisms sub­
jected to 1- 100, 1-1 ,000, and 1-10,000 dilutions of KMn04 gave
correspondingly decreasing values for alpha-naphthol blue formed
while with the next three tenfold dilutions increasing values were
obtained.
This observation pointed to the probability that an
excess of KMn04 remained in the gonococcus suspensions and that
alpha-naphthol blue was being formed by direct oxidation of the
substrate by the permanganate.
Qualitative manganese tests were carried out with both the
suspending fluid and the centrifuged gonococci after being subjected
to the various concentrations of potassium permanganate.
The teste
were made after each stage of centrifugation and washing.
In the
routine process of washing the gonococcus suspensions free of drug,
three washings were carried out.
In the present experiments, the
suspensions were washed four times.
Volhard's reaction for the
presenoe of manganese was used as described In Treadwell and Hall
(1932) and Baskervllle and Curtman (1916).
The reagents consist
of lead peroxide in concentrated nitric acid to which is added the
material to be tested for the presence of manganese.
The mixture
is then boiled and when the residue settles a purple color in the
supernatant fluid signifies the presence of manganese.
The depth
of color is proportional to the concentration of the manganese
present.
The organisms were treated with tenfold dilutions of potassium
permanganate ranging from 1-100 to 1-10,000,000 as recorded in
Table 55.
The supernatant fluid and the sediment were tested
separately for the presence of manganese after each centrifugation
of the drug-treated gonococcus suspension.
It was found that the concentration of manganese decreased
with progressive washing, so that after the third centrifugation
a negative test for manganese was obtained In the supernatant fluid.
This observation applied to all of the dilutions of potassium
permanganate used.
With the sedimented cells, markedly different results were
obtained.
After the third and fourth centrifugation, the results
were as follows:
Concentration of KMnOa
Added to Organisms.
Moles
Volhard Test for Mn.
1-100
6.32 x 10-2 Strongly positive
1-1,000
6,32 x 10-3 Moderately positive
1-10,000
6.32 x 10-4
Doubtful
1-100,000
6.32 x 10-5
Negative
1-1,000,000
6.32 x 10-5
Negative
These results of the manganese tests on sedimented organisms par­
allel generally the physical appearance of the organisms, as shown
in the following table:
Concentration of KMnOa
Physical Appearance
Added to Organisms.
Moles
of Organisms.
1-100
6.32 x 10-2 Brown colored sediment;
very amorphous
1-1,000
6.32 x 10-3 Brown colored sediment;
very amorphous
1-10,000
6.32 x 10-4 Light brown colored sed­
iment ; moderately
amorphous
1-100,000
6.32 x 10-5 Brown tinted sediment;
not amorphous
1-1,000,000
6.32 x 10-6 Grey-white sediment;
not amorphous
The controls in all cases did not show the presence of manganese
and had the usual grey-white appearance.
It may he concluded that
the irregular results obtained In the tests on the effect of
potassium permanganate on Indophenol oxidase were Influenced by the
presence of varying concentrations of -oermanganate In the cells.
This conclusion was tested further by observing the results
obtained when various dilutions of potassium permanganate in acetate
solution, were added to the oxidase test mixture.
In other words,
the various dilutions of potassium permanganate were substituted
for the permanganate-treated organisms in the regular oxidase test.
The following constituted the test mixture:
5.0 ml. naphthol-diamine substrate, 5.0 ml. pH 6.6 M/20
phosphate buffer, 1.0 ml. various dilutions of KMn04
dissolved in acetate solution (0.15 M).
The tubes con­
taining these ingredients were Incubated for 15 minutes
at 370 c., after which two ml. of two per cent potassium
cyanide were added.
The dye formed was extracted as
usual with chloroform-alcohol.
The results were as
follows:
KMnOA Dilutions
in 0.15 M Acetate
Solution.
1- 1,000
1- 10,000
1- 100,000
1-1,000,000
1- 10 ,000,000
Blank.
Mg # Aloha-Naphthoi
Blue Formed
Found
Corrected
Moles
5.75
5.75
5.75
5.75
5.75
x
x
x
x
x
10-4
10-5
10-6
lO"?
lO"8
3.40
1.13
0.39
0.28
0.21
0.12
3.28
i.ol
0.27
0.16
0.09
The above results show that when the concentration of perman­
ganate was increased, more alpha-naphthol blue was formed.
It should
be noted that a concentration of potassium permanganage as high
as 1-1,000,000 (5.75 x 10-7 M) was still effective In oxidizing
the test mixture to a small extent.
5) The In Vitro Effect of Sulfanilamide on the Gonococcus (#1111).
Since sulfanilamide did not inhibit the growth of the
gonococcus in the routine germicidal tests as shown by subculture,
it was of interest to determine whether this strain waB sulfan­
ilamide-fast.
Torrey broth enriched with 20 per cent hydrocoele
fluid was used as culture medium.
To tubes containing 10 ml. of
the broth and one ml. of the gonococcus suspension were added a
standardized acetate suspension of a 48 hour culture of gonococcus.
1-10 and 1-100 dilutions from this standard suspension were also
prepared.
Sulfanilamide in a final concentration of 1:133
(4.36 x 10-2 M) was added and the tubes incubated at 35-36° C.
0.2 ml. was subcultured on Douglas chocolate agar plates at var­
ious intervals, as shown in the following table (Table 65).
TABLE 65.
of Gonococcus.#
Time of
Subculture
Standard Suspension
of Organisms
Control
Drue##
1/10 Number
of Organisms
Control
Drue##
1/100 Number
of Organisms
Control
Druf
20 minutes
+++■*•
24 hours
4+++
0
++ +4
0
+4+4
0
48 hours
4+4+
0
+4+4
0
+444
0
7 days
+444
0
+4+4
0
+4+4
0
++++
4 + 44
+ 4+4
+4
4+
* Control broth had a pH of 7.20 while that of the sulfanilamide
broth was 7.32. 0.2 ml. of inoculated broths plated on Douglas
agar at intervals indicated; plates incubated in 10 per cent COg.
## Broth contained 1-133 sulfanilamide.
0 Indicates no growth.
++++ Indicates profuse growth.
____
As shown in Table 65 a 1-133 dilution of sulfanilamide in
broth has no apparent effect on the gonocoocus when incubated at
36° C. for twenty minutes.
Complete inhibition of growth occurs
following exposure to the drug for 24 hours or longer.
6 ) Studies on the Effect of Age of the Culture on Glucose and
Pyruvic Dehydrogenases.
Barron and Miller (1932) demonstrated the oxidation of
glucose by gonococcus, and Barron (1936) using an eight hour cul­
ture, demonstrated the presence of a pyruvic dehydrogenase.
In
the following supplementary study cultures of the gonococcus which
had been incubated for Q, 24 and 48 hours respectively, were
tested for the presence of glucose and pyruvic dehydrogenases by
the methylene blue technique.
The results are shown In Table 66.
TABLE 66.
Effect of Age of Gonococcus Culture (#1111) on Dehydrogenases.*
Ace of Culture
Reduction Time to 90#
Leucometh.vlene Blue (minutes)
£Lucose_
Pyruvic
Dehydrogenase
Dehydrogenase
8 hour
9.75
30.00
24 hour
11.00
30.00
48 hour
0
0
* The test mixture consisted of 0.5 ml. 1-5000 methylene blue,
0.1 ml. peptone-NaCl solution, 0.5 ml. substrate (0.01 M
glucose or 0.55 M pyruvate) and 2.9 ml. M/20 pH 7,4 phosphate
buffer. The observation time was 120 minutes.
The results recorded in Table 66 represent the averages of
several tests.
Although eight and twenty-four hour cultures of
gonococcus dehydrogenated glucose and pyruvate, a forty-eight
hour culture failed to show the presence of these two enzyme
systems.
IV.
General Discussion.
In the interpretation of the experimental data presented in
this investigation, it seems necessary to discuss briefly the
problems and limitations involved in a study of the relationship
between respiratory enzymes and viability of bacteria.
Enzyme activities in common with viability tests, were de­
termined with apparently Intact gonococci.
It should, however, be
pointed out that the use of cell-free enzyme preparations may re­
sult in a different picture.
several investigators.
This view has been expressed by
Green (1940), for example, states that
while a complete study of enzyme activity should be carried out
with cell-free enzymes as well as with intact cells, Hlt is not
always possible to reproduce in enzyme systems reactions observed
in the intact cell."
The work of Penrose and Quastel (1930) is of
interest in this connection.
The activities of oertain enzymes
were compared when the intact cells of Mlorococous lysodeiktlcus
were used and when the cells were disintegrated by lysozyme.
In
general, with the cell juice they found a diminution or complete
inhibition of the dehydrogenases, and an increased activity of
"paraphenylenedlamine oxidase," catalase, urease and fumarase;
peroxidase wa6 unaffected.
Gonococci undergo some autolysis when
suspended in salt solutions and many manipulations of the suspen­
sions are made before the enzyme activities are finally determined.
These faotors should be taken into consideration when evaluating
the enzyme inhibition endpoints of such relatively fragile cells.
From the results recorded in Table 57, it can be seen that
with the exception of sulfanilamide viability ceased in all in­
stances before total enzyme inhibition occurred.
Sulfanilamide
did not cause complete inhibition of any enzyme tested.
case of silver nitrate and potassium permanganate,
In the
the concentra­
tions causing marked Inhibition of glyceric dehydrogenase approx­
imated that causing death of the organisms.
A marked discrepancy
between viability and enzyme inhibition endpoints is present In
the case of merthiolate and the silver protein preparations.
The general results obtained in this Investigation conform
somewhat to those of Yudkin (1937) on the effect of silver sul­
fate on viability and inhibition of certain enzymes of B. coll.
Yudkin found that viability was lost long before the enzymes were
completely inhibited.
These results raise again the question of
the relationship between the activity of respiratory enzymes and
the viability of the cell.
The investigations of Rettger and
his collaborators (Gasman and Rettger, 1933; Edwards and Rettger,
1937; Wedberg and Rettger, 1941) have been discussed in the review
of the literature.
It is well to point out, however, that in gen­
eral they found a distinct inhibition of the dehydrogenase systems
at the maximum temperature of growth, while catalase and "paraphenylenedlamine oxidase" varied considerably in this respect.
Sykes
(1939) demonstrated inhibition of succinic dehydrogenase by
germicides at a "slight excess over the concentrations which are
lethal to 3act. coll.11
Manometric studies have in general shown a
closer relationship between respiration and viability.
(1939)
Hershey
states that although the respiratory mechanisms are not the
sole determinants of growth, he found a constant quantitative rela­
tionship between respiratory activity and the rate of growth.
Gkrelg
and Hoogerheide (1941) have stated that the inhibition in the rate
of oxygen uptake caused by germicides corresponds closely with the
inhibition of growth.
Ely (1939) and Grelg and Hoogerheide (1941)
found that when respiration was completely Inhibited by a germicide,
the organisms were dead when tested by subculturlng.
Bronfenbrenner,
Hershey and Doubly (1939) have shown that bacteriostatic concentra­
tions of germicides usually depress the oxygen uptake by 10 per cent
while germicidal dilutions decrease the O2 uptake by more than 80
per cent of the total.
Ely states further that before a lethal
effect takes place the germicides suppress the respiration of bac­
terial suspensions to a considerable extent.
Various reasons may be advanced to explain the lack of coinci­
dence between the lethal and enzyme inhibition endpoints observed
in the present Investigation.
It is quite possible that the var­
ious germicides cause the death of the cells by inhibiting an
enzyme system for which no tests were made In the present study.
Other fundamental processes, such as the phosphorylating and
glycolytic systems, may be limiting factors whose inhibition af­
fects more directly the viability of an organism.
Secondly, the process of growth ceases with the disorganiza­
tion of the cell and this may occur even if the organism is only
slightly injured.
Some weight is lent to this suggestion by the
observation that certain enzymes may function independently of the
intact cell, or when the cell has been treated physically or chem­
ically so that the ability to grow is lost.
It has been mentioned
previously that Penrose and Quastel observed greater enzyme activity
with lysed cells than with intact cells.
Rahn (1932) believes that
poisons are catalysts for certain destructive reactions in the cell.
According to his theory poisons in general accelerate both the
constructive and destruotlve processes, the latter more strongly.
In the third place, other stable mechanisms, whether accessory
to the respiratory enzymes or not, may be operating and. hence com­
plicate the correlation of enzyme activities with death of the
organisms.
This was pointed out by Casman and Rettger (1933) in
explaining the thermostability of certain enzymes.
A contribution
by Fildes (1940a) Is important in this connection.
He came to the
conclusion that the anti-bacterial action of mercury is specifi­
cally neutralized by -SH compounds.
Mercury combines with the -SH
groups of the cell, as it does with glutathione, forming a compound
devoid of these groups and depriving the cell of sulphydryl com­
pounds essential for metabolism.
Since sulphydryl compounds are
necessary for cell metabolism, Fildes considers them "essential
metabolites."
Fildes (1940b) has advanced a theory which would
explain the mode of action of a drug by its interference with essen­
tial metabolites.
Another point to be considered is the well-known
observation that bacteria which have lost the power to multiply due
to chemical poisoning may regain viability if an "antidote" is
used within a certain time.
Thus, organisms poisoned with small
concentrations of mercuric chloride may not grow upon subculture,
but on the addition of ammonium sulphide or hydrogen sulphide the
poisonous effect of the mercury is neutralized and the organisms
will now grow out.
This situation complicates the definition of
death of bacteria.
Finally, it should be borne in mind that the methods for
testing enzyme activities may not have been adequate for correlat­
ing enzyme inhibition with the viability of the organisms.
The results obtained with sulfanilamide are not conclusive.
The time of action of this drug on the gonococcus suspension
was twenty minutes, the same as routinely used with other germ­
icides.
After twenty-four hours Incubation in 1-133 sulfanilamide
broth, the growth of the organisms was totally inhibited.
This
work confirms that of Cohen (1938) who correlated the dilution of
sulfanilamide and the time factor using various strains of gon­
ococci.
The effect of this drug on the enzymes studied was negli­
gible with the exception of indophenol (oytochrome) oxidase where
there was a 55 per cent average inhibition of activity and lactic
dehydrogenase, where the inhibition was 26 per cent.
Chu and
Hastings (1938) found a 34 per cent Inhibition of oxidation of
glucose by gonococcus, using 0.66 per cent sulfanilamide and a
reaction time of one hour.
Mellon, Locke and Shinn (1939) have
evolved a theory of the mode of action of sulfanilamide by postu­
lating an inhibitory action on catalase.
However, MacLeod (1939a)
has indicated that sulfapyridlne restrains the growth of pneu­
mococcus, an organism known to be devoid of catalase and peroxidase,
in a medium free of demonstrable catalase.
Likewise, Burton, McLeod,
M cLeod and Mayr-Hasting (1940) state that it is doubtful if catalase
Inhibition is important since the bacteriostatic action of sul­
fonamide drugs is demonstrable even with bacteria devoid of catalase
such as streptococci, pneumococci and various anaerobes and growing in media free of catalase.
Woods (1940) has suggested that sulfanilamide acts by compet­
itive inhibition of the enzyme system concerned in the utilization of
p-amlnobenzoio acid, which he considers an essential metabolite.
Para-aminobenzolc acid reverses the bacteriostatic action of
sulfanilamide.
In the present investigation, Indophenol oxidase
was found to be inhibited most by the aotion of sulfanilamide.
However, Collier (1940) has found that this drug has no effect
on mammalian catalase or cytochrome oxidase.
Nevertheless, there
is a possibility that the inhibition of Indophenol oxidase can
account for the bacteriostatic action of sulfanilamide since this
enzyme is the last link in the chain of hydrogen transport where
hydrogen finally combines with molecular oxygen.
Green (1940)
states that "it is significant that the respiration of yeast and
other microorganisms is independent of the oxygen tension.
This
and many other lines of evidence point to the cytochrome oxidase
as the essential catalyst in aerobic events."
The age of the culture is of Importance in determining the
activity of a germicide.
Forty-eight hour cultures of the gonococcus
were used in these experiments and reproducible results could be
obtained in the tests for activity of the various enzymes.
How­
ever, cells harvested after 48 hours incubation at 370 C. fall to
show dehydrogenase activity for either glucose or pyruvate, al­
though in cultures inoubated for 8 or 24 hours these dehydrogenase
systems are active.
For this reason it would be important to
carry out the enzyme inhibition and viability tests with cells
collected after shorter periods of incubation.
It should be
pointed out that the resistance of older cells to various inimical
agents is greater than in the case of young cells (Winslow and
Walker, 1939).
It is possible that the use of younger cells might
Indicate a closer correlation between germicidal and inhibition
tests.
Variation in results might also be brought about by alter­
ing the medium used in cultivating the organisms, and by
carrying out the tests with various strains of gonooocci.
The nature of the heat-stable bacterial peroxidases has been
a subject of discussion for some years.
vincingly shown by Callow (1926).
Their presence was con­
Casman and Rettger (1933)
found that catalase and succinic dehydrogenase may inhibit the
peroxidase reaction with benzidine,
Edwards and Rettger (1937)
and Wedberg and Rettger (1941) found that all the organisms
studied had a heat-stable peroxidase.
Guaiac, benzidine,
o-tolidine and 2,7 dlamino-fluorene were used with hydrogen per­
oxide as separate reagents for testing peroxidative activity.
In
the present investigation the gonococcus was shown to contain a
heat-stable benzidine peroxidase and a heat-labile pyrogallol
peroxidase.
Stapp (1924) has shown that the bacterial peroxidases
were somewhat stable to heat if tested with benzidine and hydrogen
peroxide as substrates, and resisted the action of salts, acids,
alkali, iodine and various solvents and narcotics.
Because of
this peculiar stability, Oppenhelmer (1926) concluded that Stapp
was working with a purely chemical oxidation system and not with
an enzyme.
Oppenhelmer states that pure peroxidases are complete­
ly heat-labile (70-80° C.), and although crude preparations lose
their activity upon heating It is soon recovered on standing.
A
considerable number of substances, both organic and inorganlo^
have been shown to give a positive oxidase or peroxidase­
like reaction (Xastle, 1909).
These substances Include blood,
inorganic salts of certain heavy metals, body secretions, sub­
stances of vegetable origin, all oxidizing agents, etc.
In this
connection, Green (1940) states (pg. 22): "Practically all Fe
porphyrins can act as pseudo-peroxidases, I. e. they catalyze the
same type of reactions as peroxidases but never approach its
catalytic efficiency.
Peroxidase is thermolablle, whereas the
pseudo-peroxidases found wherever Fe porphyrin occurs are all
thermostable."
A detailed study of the true nature of bacterial
peroxidase is therefore Indicated.
V. SUMMARY AND CONCLUSIONS.
The effect of eight chemotherapeutic agents has been studied
using Neisseria gonorrhoeae as test organism, in an attempt to
correlate loss of viability with the inhibition of one or other
of five respiratory enzymes.
The drugs tested were, silver nitrate
protargol, neo-silvol, silver nuclelnate, argyrol, merthiolate,
potassium permanganate and sulfanilamide.
The enzymes chosen for
Inhibition studies were, lactic and glyceric dehydrogenases,
catalase, peroxidase and Indophenol (cytochrome) oxidase.
Within
the limits of the experiments the following conclusions seem war­
ranted.
There is no obvious correlation between the concentration of
a drug which is lethal for gonococcus and either partial or com­
plete Inhibition of any one enzyme system at the same concentration
In general, death of the cells occurred before significant enzyme
inhibition appeared.
A possible exception to this general rule
is found in the case of the inhibition of glyceric dehydrogenase
by silver nitrate or potassium permanganate.
With both of these
compounds the lethal concentration causes approximately 65 per cent
inhibition of glyceric dehydrogenase, although inhibition of the
other enzyme systems tested is relatively small at the same con­
centration of drug.
It is worthwhile pointing out that lesser
amounts of the various germicides were required to inhibit glyceric
and lactic dehydrogenases than any of the other enzymes studied.
The effect of sulfanilamide on gonococci places it in a
different category from the other 'germicides' studied.
In the
case of sulfanilamide little effect on viability was observed within
the limits of the experiments, though exposure to the drug for 24
nours caused the death of the cells.
Furthermore, the inhibition
of respiratory enzymes by sulfanilamide was nil with the exception
of Indophenol (cytochrome) oxidase and lactic dehydrogenase, which
were Inhibited by 55 per cent and 26 per cent respectively at the
highest concentration of the drug (4.36 x 10~2 M ) .
Although the
significance of these partial Inhibitions is not known, it is
possible that the inhibition of these two enzyme systems may be
concerned in the bacteriostatic effect of the sulfonamide drugs.
The concentrations of the various compounds which caused the
death of the cells, when expressed on a molar basis, were found
to vary less from one compound to another than the concentrations
necessary to produce Inhibition of the various respiratory enzymes.
Of the eight drugs tested, silver nitrate, potassium permanga­
nate and protargol are the most efficient enzyme inhibitors.
The presence of catalase has been shown not to interfere with
the results obtained in tests for peroxidase activity.
Hydroxylamlne
hydrochloride (9.97 x 10-4 M) completely Inhibits catalase activity
and about 60 per cent of peroxidase activity of gonococci.
How­
ever, peroxidase inhibition tests with silver nitrate and potassium
permanganate gave the same endpoints whether hydroxylamlne in the
above concentration was present in the reaction mixture or not.
If pyrogallol and hydrogen peroxide are used as substrates,
only a heat-labile peroxidase can be demonstrated In gonococcus,
whereas if benzidine and hydrogen peroxide are used, peroxidase
activity can be demonstrated which is heat-stable.
Pyrogallol
is not oxidised by gonococcus in the absence of hydrogen peroxide.
A quantitative, colorimetric, indophenol (cytochrome) oxidase
test has been developed for use in studies with the gonococcus.
VI.
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