.4'1 ''''''

H
’
\

NM

1

M

H

l

l

l

—
—__,._
—

'

it

\

 

A COMPARATIVE STUDY OF
MERTMOLATE, PHENOL. AND SEVERAL
QUATERNARY AMMONIUNK
COML‘T'GUHDS AS PRéSERVATIVES
FOR SERUM

Thu}: Em- ?he Dag-vast cf M. S.
MECHEGAN STATE COLLEGE
'Wéfiiam C. Aiegnani
1950

 

 

This is to certify that the

thesis entitled

A Comparative Study of Herthiolate, Phenol, and
Several Quaternary Ammonium Compounds as

Preservatives for Serum

presented by

William C. Alegnani

has been accepted towards fulfillment
of the requirements for

Masters degree in Wogy

Date May 26; 1950

Maj r professor

i1; ., ‘
‘4 (I, i
-- f‘iz‘

. v I l
. O I
. I a
l u _ I I
I \-
p
4
u
' I
I
\
A
I
l I '
5
Q
I
1‘ " -
'v‘ . -n.‘3 I ‘q‘.’s' It.

 

    
 

...\
’35."

 

 

.-
-

 

O
. -" ”-‘_"-

~.
v
-
I
.-——-———-——-—
I-u-|§ -

 

.
v v --—'

 

A COMPARATIVE STUDY OF MERTHIOLATE, PHENOL,
AND SEVERAL QUATERNARY AMMONIUM COMPOUNDS AS
PRESERVATIVES FOR SERUM

by
William C. Alegnani
.3,“

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Bacteriology

1950

THESIS

 

ACKNOWLEDGMENT
The author wishes to express his
appreciation to Dr. Walter L. Mallmann
for his guidance and helpful criticism

given this work.

r367,s s>~4cp
‘ ,,‘-.“.-‘, ,‘v I .

TABLE OF CONTENTS

Page

IntrOduCtion.0.00..OOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOO
ROV16' or literaturOOOOOO0.00.0...OOOOOOOOCOOOOOOOOOOOOO

Description Of compound‘oeoeeeeeee00000000000000.0000...
HerthiOI‘toeeeeeeee0000000one00000000000000.0000...
PhenOIOeeoeeeoeoeeeeeeoeeeooeeeeeeeeooeoeeeeeeeeeee
Quaternary ammonium compounds......................

(a) BTCeoeoeeeeeesoeeeeeeeoooeeeeeeeeeeeeeeoeeoee
(b) Tetrosan eooeeeeoeeeeeeeeeeeeeoeeeeeeoeeeeeee
(C) Hyaminfi #23890.eoeeeeeeeeeeeeeeeeeeeeeeeeeeoe
(d) Cetyl-pyridinium chloride....................
(9) ISOthEH Q'lseeeeoseeeeeeeeeeeeeeeeeeeeeeeeeee

Experimental and discussion.............................
Effect of temperature on serum.....................
Determination of germicidal activity

(a) Activity ever a ninety minute period.........
(b) ACtiVity at 370 Ceeeeeeeeeeeeeeeeeeeeeeeeeeeee
(c) Activity at refrigerator temperature.........

sumryOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCCOOOOOOOOOO...

ReferODOOSCOOOOOOO.OOOOOOOOOOOOOOO.OOOOOOOOOOOOOOOOOOOO.

(D0ldsfi<l®€fl¢ub P' F4

pa
CH”

13
16
23
28

3O

INTRODUCTION

The preservation of serum in the liquid state is of
paramount importance in the field of therapeutic medicine.
Preservation in this form is simple, economical, and provides
the phlebotomist with an instantly available form of the
blood derivative. The use of serum.and plasma played a
significant part in the treatment of casualties in World War II
and in civilian defense. Many attempts have been made to
find a preservative which could be added to the serum.in a
concentration that would be bacteriostatic and yet remain
non-toxic. The establishment of the quaternary ammonium
compounds as excellent bactericides and bacteriostats, and the
fact that they are relatively non-toxic in use-dilutions,
stimulated the undertaking of this work., The two major
difficulties encountered in preserving serum in the liquid
state are: (a) rapidity of bacterial growth when contaminated,
and (b) precipitation and loss of essential elements.

At present, there are four common methods for the
preservation of serum; (a) simple desiccation; (b) storage
in the liquid state with or without chemical preservatives;
(c) vacuum desiccation from the frozen state; and (d)
preservation in the frozen state. Each method has its own
advantages and disadvantages.

REVIEW OF THE LITERATURE
Emerson (4), in 1925, introduced a method of preserving

plasma and serum by subjecting it to high pressures. A
chamber was built to withstand considerable pressure. The
material was introduced into the chamber and subjected to
concussive impact by the action of a plunger. Since that time,
it has become evident that microorganisms are capable of with-
standing pressure far greater than is practicable to apply,
and that under those conditions denaturation of the original
serum proteins would occur.

Harper (13) offered a method of preserving serum by ;
simple desiccation. The material is dried in a vacuum distilling
apparatus at reduced pressure (about 15 mm mercury) between
4o°-45°c. It was found that the serum remained stable in this
form over long periods of time.

Strumia (27) described a method for preserving serum and
plasma in the frozen state. The technique involved three
essential features; (a) fairly rapid freezing, (b) maintenance
at a temperature well below the melting point (-5°to -78°C),
and rapid thawing in a water bath at 37°C and warming at
room temperature.

Flosdorf and Mudd introduced the "LyOphile” (7) and
"Cryochem" (8) processes for the preservation of biological
materials (serum, plasma, etc.). The procedures are
essentially the same with the exception of the method of
elimination of water vapor. The material is first frozen and
placed in a vacuum apparatus. In the Lyophile process the

water vapor is condensed as ice in a container kept cold by

dry-ice. In place of dry-ice, the Cryochem equipment utilizes
a regenerative chemical desiccant - Calcium sulfate.

Noble (19) used varying dilutions of glycerol, chinsol,
phenol, chloroform, mercuric iodide, dibromin, ortho-chloro-
phenol, trikresol, and chloretone for preserving agglutinating
serum. He showed that the sterility of the individual samples
was retained over a period of four years at 60-800.

Other chemical agents which have been used as preservatives
for serum are quinsol, formalin, carvasept, eucupin, hexyl-
resorcinol, and yatrene (an iodine derivative of pyridine).

The organic mercury compounds are among the more common chemical
agents now used to preserve sera. These will be discussed more
thoroughly later. Gershenfeld (10) stated that "mercurials
appear to be unsuited in the preservation of sera to be
concentrated and refined. The composition and amount of the
.preservative are important. Liquid serum must not contain more
than 0.5 percent of preservative, nor more than 20 percent

total solids. Products intended for intravenous use must be
clear, free from excessive coloration and viscosity."

Novak (20) made a comparative study of the sulfone
derivatives (sodium sulfathiazole, sodium sulfapyridine, and
sulfanilamide) for preserving plasma. He concluded that a
final concentration of 0.2 percent sodium sulfathiazole
solved the problem of gross bacterial contamination in plasma,

and that actual sterilization of the material occured when there

was minimum.contamination. He also demonstrated that it was
desirable to add the sulfonamide compounds to plasma and
maintain it at room temperature for several days previous to
storage in the refrigerator. If the plasma was placed directly
into the refrigerator, a longer period of time was required and
sterilization occured only in the higher concentrations. This
was, no doubt, due to the reduced activity of the sulfone
derivatives at lower temperatures.

DESCRIPTION OF COMPOUNDS

A description of the compounds tested and a review of the
literature regarding their use as applicable to this work is
as follows:

Merthiolate

Herthiolate - sodium ethyl mercuri thiosalicylate - is
produced by the Eli Lilly and Company, and is marked "Released
for use as a biological preservative".

Jamieson and Powell (16) noted that merthiolate was free
of precipitating prOperties when mixed in strong concentrations
with protein solutions, and that under these conditions
maintained its germicidal properties. They also noted that it
had a low degree of toxicity and was harmless toward labile
antigen fractions and antibodies. MacKay (17) showed that
a 1-10.000 dilution of merthiolate in human serun.prevented
the growth of Staphylococcus aureus and Bacillus subtilis.
Studies with the Agar-cup Plate Method by Rose and Miller (24)
indicated that the effectiveness of merthiolate diminished as

the blood concentration increased. Smith, Czarnetzky, and
Mudd (26) found that mercurial antiseptics exert their action
almost wholly by.virtue of their mercury content, and that the
killing power per unit weight of mercury falls off rapidly
with dilution when used in the presence of serum. Fildes (6)
demonstrated that mercury combines with -SH groups in the cell,
thus depriving the cell of R-SH groups which are essential for
its metabolism. Boogerheide (14) demonstrated that the
critical bacteriostatic dilution of merthiolate against

Staph. aureus was not changed in the presence of 10 percent

 

serum.
2.1.1.222;

In testing merthiolate (l-l0,000)and phenol (1-200),
Rosenstein and Levin (25) noted that the failure of the preser-
vative to destroy the organism tested could not be attributed
to the lessening of the potency of the preservative due to
storage in the presence of serum. It was also shown that the

efficiency of phenol against staphylococci, B. pyocyaneous, and

 

diphtheroids decrease during 10 month storage at 5°C, and that
maximum.effect of the preservative on the organism is usually
reached in 48 hours. Falk and Aplington (5) showed that the
effect of using phenol and merthiolate in mixtures is additive.
HOOgerheide (14) noted that the critical bacteriostatic

dilution of phenol against Staph. aureus was reduced from

 

1-450 to 1-280 in the presence of 10 percent serum.

Quaternary Ammonium.Compounds

There is an extensive literature presentingsevidence te-~
show that the high molecular quaternary ammonium.compounds
(cationic) are effective antiseptics and disinfectants. An
excellent review of the literature concerning their chemical
structure, physical properties, and interactions between
proteins is presented by Glassman (11). The author was not
able to find any literature concerning their use as
preservatives for serum. The specifications adapted by the
Food and Drug Administration utilizing 10 percent normal horse
serum as organic matter in testing disinfectants, have supplied
much information regarding the effect of serum.on the germicidal
potency of quaternary ammonium compounds and the effect of the
quaternary ammonium compounds on the serum.

Anson (1) showed that proteins may be denatured by surface
active agents of either anionic or cationic type. He attributed
the denaturation to the type of hydrOphobic-hydrophilic
structure. Putnam.and Neurath (22) noted that precipitation
of serum proteins appeared to be caused by electrostatic forces.
The maximum concentration of the quaternary ammonium.compound
required for complete precipitation corresponded closely to the
total acid binding capacity of the proteins.

Hotchkiss (15) demonstrated that surface active cations
cause an irreversible damage to the cellular membrane of bacteria,»
which results in the release of soluble nitrogen and phosphorus

compounds from the cell and the death of the cell.

gig

BTC - a mixture of alkyl—dimethyl-benzyl-ammonium
chlorides.

Using Salmonella typhosa, Harley (12) noted that alkyl-
dimethyl-benzyl-ammonium chloride had its killing dilution
reduced from l-S0,000 to l-6,000 in the presence of 10 percent
serum. Boogerheide (14) demonstrated that the critical
bacteriostatic dilution of this compound against Staph. aureus
was reduced from 1-400,000 to l-l40,000 in the presence of
10 percent serum.

Tetrosan

Tetrosan - alkyl-dimethyl-3-4-dichlorobenzyl-ammonium
chloride.

DuBois and Dibble (S) noted that the killing dilution of
Tetrosan against Staph. aureus, when determined by the F.D.A.
method was approximately l-50,000.

gyaminegfiZSBQ

Hyamine #2389 is a derivative of Phemerol (p-tertiary-
octyl-phenoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium.chloride),
having essentially the same bactericidal and bacteriostatic
effect on microorganisms. No information was awailable on
Hyamine #2589, however, there was on Phemerol. Mallmann (18)
noted that the killing dilution of Phemerol against Sal. typhosa
was reduced from l-22,000 to l-5,2OO in the presence of 10
' percent serum. Hoogerheide (14) noted that the critical

bacteriostatic dilution of Phemerol against Staph. aureus was

reduced from l-400,000 to 1-140,000 in the presence of 10
percent serum.

Cetyl-pyridinium chloride

 

For brevity, cetyl-pyridinium chloride will be referred
to as CPCl for the remaining part of this work.
Quisno and Foter (23) showed that 10 percent serum
reduced the germicidal activity of CPCl from 12 to 25 times
when tested against gram negative organisms. Hoogerheide (14)
noted that the critical bacteriostatic dilution of this compound

when tested against Staph. aureus, was reduced form l-l,OO0,000

 

to 1-420,000 in the presence of 10 percent serum. Pharmaco-

logical and toxicological studies carried out by Warren 33 31 (28)

on rabbits, revealed that the MLD of CPCl was 20 mgm per kgm

of body weight, and the LD50 was 35 mgm per kgm of body weight.
Isothan_Q-15

 

Isothan Q-15 - lauryl isoquinolinium bromide.

Information on this compound was obtained directly from the
Onyx laboratories (21). The killing dilutions of Isothan Q915
in the absence of serum were found to be l-7,000 for Escherichia
221$ and l-20,000 for Staph. aureus. Toxicological studies on
guinea pigs showed that the MLD was 80 mgm per kgm of body
weight, and the LD50 was 200 mgm per kgm of body weight.

EXPERIMENTAL AND DISCUSSION

Normal horse serum was obtained from the Michigan Department

of Health, Lansing, Michigan. Three Lots* were required for this

* Lot #652 - 2,800 m1
Lot #0-654 - 1,400 m1
Lot #A-644 - 1,400 ml

work. Prior to delivery the serum was passed through a bact-
eriological filter and collected in a sterile one gallon glass
Jug. The serum.was then dispensed under aseptic conditions into
sterile screw-cap test tubes (25 m1). These tubes were kept
in the refrigerator (2°-10°C) until ready for use. Representative
samples were added to Brewer-Modified Thioglycollate medium (2)
to check for sterility. The tubes were incubated for 48 hours
at 37°C. No contamination was found.

The organisms used in this work were Micrococcusgpyogenes

var. aureus #209 and Escherichia coli #9637. All cultures used

 

in this study were 24 hour slant cultures which had been trans-
ferred at least three successive days on plain nutrient agar
slants. The 24 hour slant cultures were washed with saline and
diluted to compare with a set of standard nephelometer tubes.

The desired number of organisms was obtained by diluting with
sterile saline. A check on the final number of organisms was made
by plating 1 ml portions of the dilution on four Tryptose Glucose
Extract (T.G.E.) agar plates. An average count was obtained from
the four plates.

An attempt was made, not to determine the exact amount of
compound necessary to kill the organisms in the presence of
serum, but to determine if the organisms were killed or'
inhibited by the preservatives at concentrations compatible
with the serum and within the limits of safety used.in bio-
logical products for regular distribution.

All dilutions of the compounds and serum are reported in

terms of final concentration.

For convenience, stock solutions were prepared by diluting
with sterile distilled water. These dilutions were kept in
screw-cap test tubes at room temperature. The dilutions
prepared were as follows: (a) Merthiolate 1-250, 1-500, and
l-l,250. A l-l0,000 dilution of merthiolate had a pH of 6.75.
(b) Phenol 1-20 and 1-50. A 1-200 dilution of phenol had a
I pH of 6.8. (c) BTC 1-250, 1-500, and 1-1.250. A 1-10.000
dilution of BTC had a pH of 7.0. (d) Tetrosan 1-250, 1-500, and
l-l,250. A l-l0,000 dilution of tetrosan had a pH of 6.75.

(e) Hyamine #2389 1-250, 1-500, and l-l,250. A l-10,000 dilution
of Hyamine #2389 had a pH of 6.95. (f) CPCl 1-500 and l-l,250.
(g) Isothan 0’15 1-250, 1-500, and l-l,250. A l-10,000 dilution
had a pH of 5.55.

The first phase of this study consisted of testing the
compatibility of the various compounds with serum at different
temperatures. Two sets of tubes containing 10 ml of serum
served as controls. To sterile cotton plugged test tubes were
added 9 ml of serum and 1 ml of the preper dilution of compound.
Individual sets were incubated at refrigerator (2°-10°C),
room (220-2400), and 37°C temperatures. The results are
presented in Tables I and II.

From these data it is apparent that refrigerator temperatures
are more compatible with the serumpcompound mixtures than either
room or 37°C temperatures. The concentration of the various
compounds seemed to have little or no visible effect on the

serum proteins at these temperatures, with the exception of the

TABLE I

The Precipitation of Normal Horse
Serum at Various Temperatures.

 

 

 

 

 

 

 

Temperature
Control 2°-lO°C H 220-2400 H
Tubes Time in Days '
l 5 15 l 5 15 1 l5
#1 - - — — SP P SP P
#2 - - - - SP P SP P

 

 

 

 

 

 

 

 

 

 

(-) No visible precipitate
(SP) Slight precipitate
(P) Precipitate

-11-

e322»: sea 85
33338." r:
3333.3 953» 23
3328.3 35:» on TV

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.eeuleuee-eu 33.3» as
13.0 atom defies 33.3." 0m 5:. 5509.8 me banana—8

HH H43

 

mmhmmfimmhmfi a3-m&u-nsauuuun segues
000.7” 00.?” 084 80;..." 00min 08.." 80.1" 00m..." 0004 e338
.. .. 3.0

m a .a a m B m m m a h m a t a m .a 558”
m m B a A .5 0a m B a at. .. an .. u. .. .. .. .. an H06
a m B a m B a m an m a m B m .a ii .532
a .— mm m m an a a .3 a mu m .a .. m B .. .. .. .. .. u, .. .. .. .. 05
m «mum m as «as an m mm. was .. n- .. .. .. .. .. .. 3335.3.
H m a ma m S m m m are
m a new. mamamanamamamamauma Sosa
000.84 000.07g 0006A 000.8.A 000.01” 000.m.A 000.84 000.07." 0006...” 8333
0eR 0395mm 307% tflfieeeea

-12-

l-5,000 dilution of Isothan Q-15, which seemed to enhance
precipitation.

A preliminary experiment was carried out to determine the
effect of sodium thiosulfate on merthiolate at bacteriostatic
dilutions. A medication tube containing 18 m1 of serum, 1 ml
of merthiolate (1-250), and 1 ml of diluted M. pyogenes var.

 

aureus culture containing 58,000 organisms was mixed and
allowed to stand for five minutes. Four 1 ml portions were
pipetted into four test tubes containing 9 ml of F.D.A. broth
and mixed. One ml was transferred to another tube containing
9 ml of F.D.A. broth and another 1 m1 portion was transferred
to a tube containing 4.5 ml of sodium thiosulfate (1-50) and
4.5 ml of F.D.A. broth. All tubes were incubated at 37°C for
48 hours. The results are presented in Table III.

The results of this preliminary test showed that sodium
thiosulfate was capable of exerting a neutralizing effect on
merthiolate at the bacteriostatic dilution (1-500,000). For
the remainder of this work sodium thiosulfate was used to
neutralize the effect of merthiolate in the second dilution tube.

The second phase of this work involved a method similar to
that used by Smith, Czarnetzky, and Mudd (26) for determining
the germicidal activity of mercurials in the presence of serum.
Only those modifications were made which were necessary to meet
the demands of this experiment. The purpose of this experiment
was to determine the germicidal activity of the compounds in a

the presence of serum over a 90 minute period. In most cases

TABLE III
Neutralizing Effect of Sodium Thiosulfate on Merthiolate

(1-500,000) against H. o enes var. aureus* in
the Presence of I gercent NormaI
Horse Serum.

 

 

 

 

 

 

 

l Dilution 2 Dilution
Tube # #
**
number FoDeAe F.D.A. H‘29" 03
Broth Broth F.D.A. Broth

1 C - /

3 - - x

4 . - - /

 

 

 

 

 

s Number of organisms used in test - 58,000
rs 4.5 ml of sodium thiosulfate (1—50) was added to 4.5 ml of
F.D.A. broth.
(-) No visible growth
(K) Visible growth

-14-

a final concentration of 90 percent normal horse serum was
used. This facilitated the amount of preservative and diluted
organisms to be added, and also provided a total volume from
which representative samples could be taken without introducing
a large error. Thus, to 18 m1 of serum were added 1 ml of
preservative and 1 m1 of diluted culture. Owing to the limited
solubility of phenol, it was necessary, in order to obtain a
final volume of 20 ml, to use 17 ml of serum, 2 ml of phenol
(1-20), and 1 ml of the diluted culture, resulting in a final
85 percent serum mixture and a 1-200 dilution of the compound.
Individual medication tubes were prepared containing 18 ml of
serum. To these were added respectively, 1 m1 of merthiolate
(1-250), 1 ml of BTC (1-250), 1 ml of Tetrosan (1-250), 1 m1 of
Hyamine #2389 (1-250), 1 ml of CPCl (1-500), and 1 m1 of Isothan
0915 (1-250). All tubes were placed in a water bath maintained
at room temperature (220-2400) during the time of exposure. At

the initial time, 1 ml of the diluted culture of M. pyogenes var.

 

aureus was added to the medication tube and thoroughly mixed
with the serum and preservative. At intervals of l, 5, 10, 15,
30, 60, and 90 minutes, a 1 m1 portion of the mixture was
removed, pipetted into a sub-culture tube containing 9 ml of
F.D.A. broth and mixed. One ml of this mixture was then
transferred into another tube containing 9 m1 of F.D.A. broth
and mixed. From this second tube four Petri dishes received

1 m1 portion each. Ten ml of melted T.G.E. agar (48°C) was

poured into the plates, mixed thoroughly and allowed to solidify.

-15-

All plates were incubated at 37°C for 48 hours. Sodium thio-
sulfate was used in dilution tube #2 to neutralize the action
of merthiolate. For the other compounds, the #2 dilution
tubes contained only F.D.A. broth. The F.D.Aa broth tubes
were used to dilute the compounds beyond their bacteriostatic
ranges. Thus the absence of bacterial colonies on the plates
would indicate germicidal activity. Results of this experiment
are presented in Tables IV - X.

The results of this experiment showed that the compounds
were not able to kill the organisms in the presence of serum
over a period of 90 minutes. Bacteriostasis was observed in
many of the dilution tubes. Garrod (9) noted that the bact-
eriostatic concentration varied according to the number of
organisms present. This explains the difference in observable
growth obtained in the dilution tubes. The number of organisms
used in the test was kept at a minimum.to facilitate plate
counts, saving both time and materials.

The third phase of this work was to determine if the
germicidal activity of these compounds in the presence of serum
could be detected on incubation of the mixtures at 37°C for
periods of 3, 7, and 25 days against M. pyogenes var. aureus
and E. coli.

Two sets of screw-cap test tubes were prepared containing
serum, compound, and organisms. One set was inoculated with

M. pyogeneg var. aureus and the other with E. coli. A separate

 

set was prepared containing only the serum and organisms; these

served as controls. Duplicate sets of tubes were prepared contain-

- 15 -

TABLE IV

Effect of l-5,000 Merthiolate on M. pyogenes var.
aureus* in the Presence of 90*Percent
Normal Horse Serum at 220-2400.

 

 

 

 

Tim” °f E‘p°’u” 1 5 10 15 so so 90
in Minutes
Dilutions
1n #1 1-50,000 . O C - - - -
F.D.A. **
Broth
#2 1-500.000 ; / / / / g ;

 

 

Average Count of

Four T.G.E. Plates 25 51 23 25 22 23 25

 

 

 

 

 

 

 

 

fl Number of organisms used in test - 132,000

es A 1-100 dilution of sodium thiosulfate in F.D.A. broth.
(-) No visible growth

(li Growth

TABLE V

Effect of 1-200 Phenol on M. pyogenes var aureus* in
the Presence of 85 Percent Normal Horse
Serum.at 22°-24°C.

 

 

Time of Exposure

 

 

 

 

 

 

 

 

 

in Minutes 1 5 10 15 30 60 90
Dilutions
in #1 1-2.ooo / K I l / / /
FODOA.
Broth
#2 1-20.ooo / / l I K l /
F§X§r3§3.§?u3le§§s 2° 18 23 22 20 21 19

 

 

 

* Number of organisms used in test - 44,000

TABLE VI

Effect of l-5,000 BTC on M. pyogenes var. aureuss in
the Presence of 90 Percent Normal Horse
Serum at 220-2400.

 

 

 

 

Timgnogifigggzur° l 5 10 15 so so 90
D11§31°n’ #1 1-50.000 - - - - - - '
F.D.A.

 

 

Average Count of
Four T.G.E. Plates 24 21 19 2° 25 21 21

 

 

 

 

 

 

 

 

e Number of organisms used in test - 52,000

TABLE VII

Effect of l-5,000 Tetrosan on M. pyogenes var. aureug“

in the Presence of 90 Per entoNormal Horse
Serum.at 22 -24 C.

 

 

 

 

Time of EXPosuro 1 5 10 15 so so 90
in Minutes
Dilutions - - - -
in #1 1 50,000
F.D.A.
Br°th #2 l-soo,ooo - - - - ' / l /

 

 

Avera e Count of
Four TIG.E. Plates 19 20 21 18 19 22 22

 

 

 

 

 

 

 

 

* Number of organisms used in test - 52,000

-18-

TABLE VIII

Effect of l-5,000 Hyamine #2389 on M. pyogenes var.
aureus* in the Presence ofo90 gercent
Normal Horse Serum at 22 ~24 C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time of Exposure
in l 5 10 15 so 60 90
Dilutions - - - -
in #1 1 50,000
F.D.A.
Br°th #2 1-500.000 - - - - - - -
Average Count of
Four T.G.E. Plates 18 18 19 2° 2° 17 19

 

* Number of organisms used in test - 43,000

TABLE IX

Effect of 1-10,000 Cetyl-pyridinium chloride on M; pyogenes
var. aureus* in the Presence of go Percent Normal
Horse Serum at 220-24 C.

 

 

 

Time of Expo sure

 

 

in Minute, 1 5 lo 15 so 60 90
D11§31°“' #1 1-100.000 / I l l l x /
FODCA.

Br°th #21-1,000,000 / / / / / / /

 

 

Average Count of
Four T.G.E. Plates 35 38 40 41 46 52 31

 

 

 

 

 

 

 

 

* Number of organisms used in test - 65,000

-19-

TABLE X

Effect of l-5,000 Isothan Q—15 on M. pyogenes var

BLIPGUI*

in the Presence of 90'Percent

Normal Horse Serum at 220-2400.

 

Time of Exposure

 

 

 

 

D1123l°n3 #1 l-50,000 - - - - - - _

F.D.A.

Broth #2 1-500.000 - - - - , - _
Average Count of 26 18 -19 16 17 18 18

Four T.G.Ea Plates

 

 

 

 

 

 

 

 

s number of organisms used in

test - 43,000

ing respectively, 1 ml of merthiolate (1-250) and 18 ml of
serum; 2 m1 of phenol (1-20) and 17 ml of serum; 1 ml of
BTC (1-250) and 18 ml of serum; 1 m1 of Tetrosan (1-250) and
18 ml of serum; 1 ml of Hyamine #2389 (1-250) and 18 ml of
serum; 1 ml of CPCl (1-500) and 18 ml of serum; 1 ml of Isothan
Q-15 (1-250) and 18 ml of serum; and one set containing 19 ml
of serum to serve as a control.

To one set was added 1 ml of a diluted culture of M. pyogenes
var. aureus containing 90,000 organisms per ml. To the other
set was added 1 ml of a diluted culture of E. coli containing
275,000 organisms per ml. All tubes were incubated at 37°C. At
intervals of 3, 7, and 25 days, 1 ml was removed, transferred
to 9 m1 of F.D.A. broth and mixed. One'ml was removed from
this dilution, transferred to a second tube containing 9 ml of
F.D.A. broth and mixed. Sodium thiosulfate was used to neutral-
ize the action of merthiolate. Four ml from the second dilutions
was removed, 1 ml portions transferred to Petri dishes, mixed
with melted T.G.E. agar (48°C) and allowed to solidify. Plates
were incubated at 37°C for 48 hours. Results are presented in
Table XI.

It was noted from the results in Table XI, that all of the
compounds exhibited germicidal activity against M. pyogenes
var. aureus in the presence of serum after contact for three
days at 37°C. E. coli proved to be resistant to the action of
the compounds over the entire period of 25 days with the exception
of merthiolate. Merthiolate was the only compound to show any

germicidal activity against E. coli.

-21-

TABLE XI

Effect of Compounds on the 0rganisms* in the Presence
of 90 Percent Normal Horse Serum** at 37°C
over a Period of 25 Days.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time M. pyogenes var.
aureus £2.22};
Compounds in 2.0.1. F.D.A9T.G.EJ .D.AAE.D.A.T.G.E.
Days Brgih Brpgh Plate Brgih Brggh Plates
m 3 _ _ _ _ _ _
Merthiolate
1-5.000 7 ' r ' - - -
25 - '- D I- a -
Ph 1 3 - - - l / TNTC
en 0
1-200 7 C / TNTC
25 - - - K / TNTC
ETC 5 - - - / / TNTC
1-5,000 7 - - - / / TNTC
25 - - - f / TNTC
6 - - - l / TNTC
Tetrosan 7 - - - g / TNTC
1‘5'000 25 - - - / / TNTC
Hyamine 3 - - - [ / TNTC
#2389 7 - - - - f ,{ TNTC
l-5,000 25 - - - / { TNTC
s - - - / { TN
CPCl 7 - - - / / TNTC
1'10’000 25 - - - / / TNTC
Isothan 3 - - - / / TNTC
Q’15 7 - - - / / TNTC
_ 1-5 p 000
. 25 - - - / / TNTC
:5 l / / 1 / TNTC
Control 7 w/ / / / f TNTC
25 x x / x ,1 mo

 

 

 

 

 

 

 

 

 

* Number of organisms used in test:

M. pyogenes var. aureus - 90,000
0 CO - 76,000

** 85% Normal horse serum was used for phenol.

use Sodium thiosulfate used in dilution #2 for merthiolate.
(-) No visible growth (K) Visible growth

TNTC - Too numerous to count.

The fourth phase of this work was to determine if the
germicidal activity of these compounds against M. pyogenes
var. aureus and E. coli in the presence of serum at refrigerator
temperatures (2°-10°C) could be detected.

All tubes were prepared in the same manner. To duplicate
sets of screw-cap test tubes were added serum, compound, and
organisms.

Two controls were run on each organism. To 19 m1 of serum
was added 1 m1 of culture. The M. Pyogenes var. aureus culture
contained 48,000 organisms per ml. The E. coli culture contained
143,000 organisms per ml.

Phenol dilutions were prepared by adding to duplicate tubes,
containing 17 m1 of serum, 2 ml respectively of 1-20 and 1-50
phenol. One set was inoculated with 1 ml of M. pyogenes var.
aureus culture containing 48,000-organisms per ml; the other
set was inoculated with 1 m1 of E. coli culture containing
143,000 organisms perml.

Three tubes each were set-up for most of the remaining
compounds. To each set containing 18 m1 of serum per tube
were added respectively, 1 ml of merthiolate (1-250), (1-500),
and (1-1.250); 1 ml of arc (1-250), (1-50m, and (1-1.250); 1 ml
of Tetrosan (1-250), (1—500), and (1-l,250); 1 ml of Hyamine
#2389 (1-250), (1-500), and (l-l,250); 1 ml of CPCl (1-500) and
(1-1,250); and 1 m1 of Isothan Q-15 (1-250), (1-500), and (l-l,250).

To one set was added 1 m1 of M. Eyegenes var. aureus
culture containing 29,000 organisms per ml; to the other set,

1 ml of E. coli culture containing 223,000 organisms per ml.

Tubes were placed in the refrigerator and at intervals of 3,

- 25 -

5, 12, 25, and 50 days poured plates were made to determine
bacterial survival.

One ml samples were removed from the tubes, transferred to
9 m1 of F.D.A. broth and mixed. From these tubes 1 ml was
transferred to a second tube containing 9 ml of F.D.Ac broth and
mixed. Sodium thiosulfate was added to the merthiolate tube.

From the tubes containing M. pyogenes var. aureus 1 ml portions

 

were pipetted from the second dilution tube into each of four
Petri dishes. From the tubes containing E. coli one-half m1
portions were pipetted from the second dilution tube into

each of four Petri dishes. Ten ml of melted T.G.E. agar (48°C)
was poured-into the Petri dishes, mixed and allowed to solidify.
All plates were incubated at 37°C for 48 hours.

Results of the control plates are presented in Table XII;
those for phenol in Table XIII; and the results for the remain-
ing compounds in Table XIV.

The results from this experiment indicated that the
germicidal activity of all of the compounds, as compared to
germicidal activity at 37°C, is greatly reduced at refrigerator
temperatures. The control plates showed that M. pyogenes var.
aureus was gradually being destroyed at this temperature.

E. coli proved to be more resistant at this temperature, however,
on the fiftieth day some reduction in number was noted.
Merthiolate (1-5,000) proved to be the most active ormpound of
the group, preventing growth on the M.gpyogenes var. aureus

plates on the twelfth day, and preventing growth on the E. coli

-24-

TABLE XII

Effect of Temperature (2°-10°C) on the Viability of
0rganisms* Grown in Normal Horse Serum.

 

M. pyogenes var.

 

 

 

 

 

 

 

 

 

 

 

 

aureus ELL—9.23:3;
Time
Control Tube Control Tube
in
#1 #2 #1 #2
Days
Average Count of Average Count of
Four T.G.E. Plates 1 Four T.G.E. Plates
5 29 25 71 ‘ 72
5 l9 . 21 71 81
12 8 16 65 73
25 7 9 67 7O
50 3 5 50 50

 

 

 

 

 

a Number of organisms used in test:

M. o enes var. aureus - 48,000
E. colI - I43,000

- 25 -

TABLE XIII

Effect of Phenol (1-200) and (1-500) on the Viability of
Organisms* in the Presence of 85 Percent Normal
Horse Serum.at Temperatures of (20-10 C).

 

 

 

 

 

 

 

 

 

A 2:53:33: ..._.....E-
in Average Count of Average Count of
Four T.G.E. Plates Four T.G.E. Plates
Days
1-200 1-500 1-200 1-500
3 21 27 69 68
5 19 32 78 66
12 9 ll 71 75'
25 1 5 54 61
50 0 0 44 56
A

 

 

 

 

 

 

* Number of organisms used in test:
M.gpyogenes var. aureus - 48,000

E. coli - 143,000

 

.eeaean .néta upon me oases ens-mobs on» assuage." gee-Pa seven
dosages-e0 002 8:3
. 08....E
oooemm cinema is» segue».- .2
See» 5 deep awake we nee—en e

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mm an on 9 mm on an 3 ma am mm mm -. H: on
.3 on mm mm mm. an R an .3 R an em as a mm 1
.2 R R 3 3 8- 9 on m: an "R S as n ma m
- mm 3 .R an Hmm. an no me an m: 9 mm m: m.- m
- a r. s a a ash—Wm a s e. a s a a n
,. 0 0 0 TN 0 0n 0 0 0 0 0 0 0 0 1 on
a n 0 m u an m H .e. a a 0 0 0 mm w
m s m 3 m an m .n m .0. a o o o N... m
- ..
Us a. m 3 s an 0 m n a. u m n a m m
a: mu -. m 0 an a m 2 or. 8 m s n n a
9W4 _..§A_ 34151434 an..." 10m...” enl— 0m 0.1,- EA STA—woman RA and m
9.0 sods: F 88 F leases __ 8n 3335.3. he .

 

-27..

4003003 no send-81min 0e
lumen eeuem Hesse: aseouem am no eeaueean
es. s.- eeseussuwo we acumen-8 we ensued-.3 unease» we aeeuun

Baa

plates on the fiftieth day. A marked reduction in the number
of E. coli was noted on the twelfth day in the 1-5,000 dilution
of merthiolate. A similar reduction of E. coli was noted on the
fiftieth day in the 1-10,000 dilution of merthiolate. The
remaining compounds had little or no germicidal effect on either
of the organisms at this temperature.

SUMMARY

A comparative study of merthiolate, phenol, and five
quaternary ammonium compounds as preservatives for serum was
made.

Dilutions of the compounds studied had little or no visible
effect on the serum.proteins.' Temperature, in this case,
appeared to be the influencing factor in the precipitation of
‘the serum proteins.

Germicidal activity of the compounds studied could not be
demonstrated when left for a contact period of ninety minutes
with the test organism.in the presence of serum.

On incubation of the serum, compound, and organism.mixture
at 37°C for three days, the germicidal activity of the
compounds could be demonstrated.

At refrigerator temperatures (20-1000) the germicidal
activity of the compounds is greatly reduced. Merthiolate
was the only compound to exhibit germicidal action against both
of the organisms tested. Merthiolate in concentrations of
1-5,000, 1-10,000, and 1-50,000 exhibited germicidal action

against M. pyogenes var. aureus on the twelfth day of incubation

 

at refrigerator temperature. Merthiolate 1-5,000 exhibited

germicidal activity against E. coli on the twelfth day, and

in a concentration of 1-10,000 on the fiftieth day.
Merthiolate, as compared to phenol and the quaternary

ammonium compounds tested, appeared to be the most satisfactory

compound to use as a preservative for serum.

- 29 '

(l)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)
(11)

(12)

(13)

REFERENCES

Anson, M.S., 1939 The denaturation of proteins by
synthetic detergents and bile salts. Jour. Gen. Physiol.
23, 239-246.

Brewer-Modified Thioglycollate, Products for the bact-
eriological laboratory. Balt. Biol. Lab., Balt. Md..

DuBois, A.S., and Dibblee, D., 1946 Death-rate study on
a high molecular quaternary ammonium compound with
Bacillus motions. Science 103, 734.

Emerson, V.L., 1923 Preservation of serums. U.S. Patent
1,440,868. '

Falk, C.R., and Aplington, S.P., 1936 Studies on the
bactericidal action of phenol and merthiolate used alone
and in mixtures. Amer. Jour. Hyg. 24, 285-307.

Fildes, Paul, 1940 The mechanism of the antibacterial
action of mercury. Brit. Jour. Exptl. Path. 21, 67-73.

Flosdorf, E.W., and Mudd, S., 1935 Procedure and apparatus
for preservation in "Lyophile " form of serum and other
biological substances. Jour. Immunol. 29, 389-425.

Flosdorf, E.W., and Mudd, S., 1938 An improved procedure
and apparatus for preservation of sera. micro-organisms,
and other substances - The "Cryochem" process. Jour.
Immunol. 34, 469-490.

Garrod, L.P., 1935 The effect of bacterial numbers on
minimum bacteriostatic concentration. Jour. Infect.

Gershenfeld, L., 1939 Biological products. Romaine Pierson
Publishers, Inc., New_York.

Glassman, H.N., 1948 Surface active agents and their
application in bacteriology. Bact. Reviews 12, 105-148.

Harley, R.J., 1949 Some factors influencing detergency
and germicidal activity of detergent-sanitizers.
Unpublished Master's thesis, Department of Bacteriology,
Michigan State College, East Lansing, Michigan.

Harper, S.B., Essex, H.E., and Osterberg, A.E., 1940 The

preparation and experimental use of dried blood plasma.
Staff Meetings of the Mayo Clinic 15, 689-694.

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

Hoogerheide, J.C., 1945 The germicidal properties of
certain quaternary ammonium salts with special-reference
to cetyl trimethyl ammonium bromide. Jour. Bact.

49, 277-289.

Hotchkiss, R.D., 1946 The nature of the bactericidal action
of surface active agents. Ann. N.Y. Acad. Sci. 46,

Jamieson, W.A., and Powell, H.M., 1931 Merthiolate as a
preservative for biological products. Amer. Jour. Hyg.
14, 218-224.

MacKay, M.E., 1941 The bacteriostatic effect of disinfectants
in human serum and citrated plasma. Brit. Med. Jour.
1, 747-750.

Mallmann, W.L., 1944 Evaluation of disinfectants. Soap
and San. Chem. 20, August.

Noble, A., 1930 Effect of preservation on agglutination
serum. Jour. Bact. 19, 31-32.

Novak, M.V., 1942 Use of sulfone derivatives in plasma to
control bacterial contamination. Blood Substitutes and
Blood Transfusion. Edited by Stuart Mudd and William
Thalhimer Chpt. 28, 267-273. Charles C. Thomas: Balt. Md..

Onyx laboratories, Technical data sheet - Isothan Q-15,
Jersey City, N.J.

Putnam, F.W., and Neurath, H., 1943 Complex formation
between synthetic detergents and proteins. Jour. Biol.
Chem. 150, 263-264.

Quisno, H., and Foter, M.J., 1946 Cetyl pyridinium chloride.
1. Germicidal properties. Jour. Bact. 52, 111-117.

Rose, 8.3., and Miller, R.E., 1940 Studies with the agar
cup-plate method. II. Effect of blood on mercury antiseptics.
more Jouro ”eds 301. 199’ 338-342.

Rosenstein, D., and Levin, 1., 1935 The bactericidal and
antiseptic action of preservatives frequently used in
biological products, and the effect of these preservatives
on the potencies of the products. Amer. Jour. Hyg. 21,
260-279.

Smith, D.E., Czarnetzky, E.J., and Mudd, S. 1936 The
mechanism of inactivation of mercurial antiseptics by
serum, and its implication regarding the possibility of
intravenous antisepsis. Amer. Jour. Med. Sci. 192, 790-808.

- 31 -

(27) Strumia, M.M., 1941 Preparation and preservation of human
plasma. III. Freezing of plasma and preservation in the
frozen state. Amer. Jour. Clin. Path. 11, 388-401.

(28) Warren, M.R., Becker, T.J., Marsh, D.G., and Shelton, R.S.,
Pharmacological and toxicological studies on cetyl
pyridinium chloride, a new germicide. Jour. Phar. Exptl.
Therapeutics 74, 401-408.

- 32 -

‘.

 

f- 7"?—

 

 

, ‘. .
I J
‘. , 4 a -
l
.
I
\ ‘ ‘
.
.
\
.
W
.
I
4 , i/
.
' |
l
. ' .
I 1 r
I e
O
D
‘ D
\
' \
I . ‘

s O A
’ 4
'.

\ ‘ t
1 l A I
I
1

| . .

. L

.

. 1 .

I

J ;
s'
o I

l I
‘ ‘ I
A I
I » . . ,
\
I
. l
I . “
a \
. .
/
. v
1
. ‘ \
. ‘ J
J x
' I
W ‘
. I
1
l
v
\
v ‘ ‘ | I
\ ~ ' I
‘ v
. i
a
A I 5
‘ I
l
l A
z I

. . I
I I "
' \
n! .
‘ 0
z
I

TATE UNIV RSITY LIBRARIES

293 03056 0795

AN

MICHIG S
1

3