T H E S I S

L. P. Anderson, M. S.

Graduate School
Michigan State College of Agriculture
and Applied Science
1939

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PENETRATIVE POWERS
of
DISINFECTANTS

iy

. .-'{By
L. PJ, Anderson

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfillment of the
requirements for the degree of
DOCTOR OP PHILOSOPHY

Bacteriology Department
East Lansing, Michigan
1939

^

ACKNOWLEDGEMENTS

The writer desires to express his appreciation to
Dr. W, L, Mallmann who suggested the subject for investigation
and who made many helpful suggestions as the work progressed.
The writer is also deeply indebted to Dr, W. L, Chandler for
guidance and timely suggestions concerning certain procedures.
The writer also wishes to express his appreciation to the other
members of the Bacteriology Department of Michigan State College
for their many suggestions and hearty cooperation whenever
assistance was required.

±27466

INTRODUCTION

Many authorities in the field of antiseptics and
disinfectants have freely and justifiably criticized and readily
altered the only accepted standard method of testing these compounds
for their actual value.

Since this method does not, obviously, give

the user of commercial preparations more than a vague idea of their
practical effectiveness in actual use, the attempt has been made to
devise a method that will attain this end.

It was decided that if

any one common characteristic which all compounds must possess to be
of practical value could be chosen and a feasible but relatively
simple test devised to evaluate this characteristic a step forward
would be made.
In view of the foregoing it seemed that the one
characteristic which must be common to all compounds irrespective of
their chemical constitution, is that of penetrative power, since in
order to kill quickly they must be able to pass through the wall, or
ectoplasm, of the organism and reach the life center.

On first

consideration it might appear a relatively simple matter to select
or devise a test which would evaluate this characteristic, giving
the proper evaluation to all compounds to which it was applied.

The

method so chosen is one to be described later as a speed test, and
which in order to withstand critical analyses must have a substantial
foundation that will reach beyond mere theoretical considerations.
It was deemed advisable to first ascertain, as far as practically

-2-

possihle, just how a few of the outstanding representatives of the
various groups of compounds operated in "bringing ah out destruction of
organisms.

Therefore, such methods were devised and employed to

determine this particular phase hut at the same time keeping in mind
the specific item, penetrative power, which theoretical consideration
demands must he present.

-3-

HISTORICAL REVIEW

The literature abounds in studies designed to systematize
the investigation of the effect of killing agents upon organisms*
Only the more important works will he reviewed in order to show the
general trend.

Robert Koch (32), 1881, started mercury on the road

to questionable fame as an excellent bactericidal agent by the use of
bacterial impregnated threads.

In I8S9-I89I* Geppert (20) proved that

the unneutralized sublimate carried by the thread in Koch's method was
responsible for the exceptionally high values obtained with mercuric
chloride.

To eliminate this factor Kronig and Paul (33)*1^97»

employed bacterial coated garnets as test objects and in their
thorough study formulated tenets which have served as a foundation for
subsequently devised methods.

Rideal and Walker (39)» L903* started

the trend toward the present day phenol coefficient method by devising
a test tube method of examining chemicals for their killing action.
Chick (10), 1908, did much to ferret out the probable mode of action
of killing agents, and also introduced the use of added organic matter
(Chick and Martin (ll),1908) in the testing methods.

In 1911* Anderson

and McClintic (2) published a method designed to eliminate some of the
objections! features of the Rideal-Walker method.
known as the Hygienic Laboratory Method.

This new method was

Shippen (HU) combined the

best features of the Rideal-Walker and Hygienic Laboratory Method in a
test which he put into practice about 1916 and which was published in
1927 by Reddish (37) s-s the !,R-W Modified Method”. Very little change

-H-

has been made in this test which is known today as the "Food and
Drug Administration Phenol Coefficient Method".
In I92U Conover and Laird (l*0 published a method utilizing
petri-plates instead of seeding tubes.

This was designated as a

Direct Unit Phenol Coefficient but has received little recognition.
Allen (1), 1929, proposed a method for examining antiseptics and
disinfectants consisting of direct application of the test solution
to the agar slant of a specially prepared culture.

In

1933 Jensen

and Jensen (23) proposed a modification of the method of Kronig and
Paul (33) *>7 using cover glasses instead of garnets.
Since that time more attention has been directed towards
the toxicity factor of antiseptics and disinfectants in order to
give a more practical aspect to the phenol coefficient.

This is

demonstrated by the work of Salle, McOmie and Shechmeister

1937»

employing embryonic chick heart, Iffye (36), 1937» using leucocytes, and
Samuels (^-2), 193^, using frog pharyngeal epithelium.

All demonstrated

that the majority of chemicals ordinarily employed as bacteriostatic
and bactericidal agents in the presence of tissue were more toxic for
tissue cells than for bacteria.
As illustrated by this brief review the bulk of the
evolutionary work since the time of Koch has been directed toward
introduction of new methods and refinement of the ones existing.
Scant attention has been given to the underlying factor of penetration.
To avoid a lengthy review only those works unquestionably dealing with
penetration will be reported.

-5In 1S97 Vincent (^7) examined the ability of various
chemicals to sterilize fecal matter pointing out that it was quite
difficult to obtain penetration of the organic particles to kill
the enclosed bacteria.

Claudius (12), 1902, while searching for a

method of sterilizing and preserving catgut, obtained penetration
with iodine as demonstrated by the death of the organisms enclosed
in the minute folds of the tissue.

Kendall and Edwards (25), 1911*

employed short uniform cylinders of seeded agar from which a small
center core was removed at the end of the exposure period to
demonstrate the penetrative ability of various substances. Seelig and
Gould (U3), 1911* used celloidin capsules and living animal skin to
demonstrate the penetration of alcohol and iodine.

In 191# the

committee on Standard Methods of Examining Disinfectants (13) ln
making their recommendations state that HA complete study of a
disinfectant must also include its physical and chemical behavior in
the medium in which it is employed, as for example, its diffusibility
or powers of penetration, and especially its chemical permanence11.
Carnot and Dumont (7), 1913, employed seeded agar plates
containing a perforated glass cup centrally embedded to determine the
penetrative powers of the compounds used in dressing war wounds.
Chambers (8), 1922, worked with starfish eggs demonstrating the
penetration of their walls by the acid and alkali groups of NaHCO^
and EH^Cl, respectively.

Hirschfelder Malmgren and Creavy (2l),1925,

produced artificial edema in the dog and observed penetration of an
intravenously injected dye from the blood vessels into the edema

-6-

fluid.

Hirschfelder and Wright (22), 1930# ln studying the action of

dyes upon proteins, state that an antiseptic acts to "prevent growth
of microorganisms hy merely adhering to and altering the “bacterial
surface so as to prevent cell division; and it kills them either by
altering the surface layer more intensely or by penetrating into the
cell body and injuring its internal metabolism or by both processes
simultaneously or successively".
In 1930 Knaysi (27-31) published a series of papers on
disinfection including an extensive review dealing with the various
conflicting theories as to the mode of action of killing agents*
Knaysi (30) points out that penetration of the cell wall of bacteria
is the one all important factor which has been consistently neglected*
Knaysi and Gordon (29), 1930, demonstrated the penetration of yeast
cells by iodine and mercuric chloride.
Karns (2*0, Karns, Cretcher, and Beal (25), 1932, studied
various solutions of iodine using silk and guinea pig skin to demon­
strate the mechanism of penetrative superiority of aqueous-potassium
iodide solutions of iodine over alcoholic solution of this element.
Biskind (5 , 6), 1932, worked in conjunction with this group but
employed frog skin to demonstrate that the aqueous forms of iodine
were superior to the alcoholic forms from the standpoint of speed of
penetration*

Uyiri and Jannitti (35)# 1932, studied the fate of

iodine placed upon the unbroken skin of dogs and rabbits.

They found

that iodine penetrated the live skin in a period of 162 hours but
only in the form of its compounds such as KI, and never as free iodine.

Mallmann and Chandler (3*0* 1933* found that of a group of
recognized disinfectants only colloidal iodine was able to penetrate
and render sterile the finely divided particles of avian fecal
material*

-s Stateraent of the Problem

The tendency of the general consumer to employ one
disinfectant or antiseptic for a variety of purposes rather than the
specific purpose for which it is adaptable necessitated choosing the
most severe of all the conditions which compounds must undergo in
practical use.

These conditions are:

(1) Brief contact with the

surface to be disinfected, usually under five minutes; (2) organic
matter such as blood, serum, pus, dirt, in or beneath which the
organisms are located; and (3 ) possible subsequent dilution.
Since disinfectants, unlike antiseptics and allied compounds
of the bacteriostatic type, must kill rapidly, they are not allowed
the privilege of rendering organisms innocuous by interfering with
their enzymatic processes thereby leading to gradual extinction, but
must reach and destroy the life center of the organism immediately.
Therefore, assuming the conditions chosen to be sufficiently severe
it was decided that the most important single feature which these
compounds must possess (disinfectants primarily, antiseptics to a
lesser degree, or not at all) is the ability to penetrate organisms.
It is difficult to prove just how a substance kills micro­
organisms and because of their minute size it would be equally as
hard to prove that the substance had penetrated the bacterial cell;
hence, it was decided to employ methods of a grosser nature and by
analogy apply the information obtained to bacteria thereby deriving
the single test desired.

-9It would be well, "before proceeding further, for the sake
of clarity, to draw a line of distinction, even though arbitrary,
between the commonly interchanged words antiseptic and disinfectant
in order that a consistent meaning may be conveyed in this paper.
The term antiseptic will be applied to compounds which render
organisms innocuous, or incapable of exhibiting evidence of life,
from the moment of contact, but only as long as the two are in
contact.

The term disinfectant will be used in the sense that the

compound employed will kill the organisms during the time of contact
implying that its subsequent removal will not result in the organisms
exhibiting life.

The terms disinfectant, germicide, bactericide, and

parasiticide will be used synonymously; and the terms antiseptic,
bacteriostat, and parasitistat will be used in like fashion.

Further,

to avoid confusing terminology, the term compound will be used
throughout the work in a general sense to represent the substances
used whether they are elements, or compounds.
Phenol Coefficients
To have information from an accepted standard method with
which to compare, several phenol coefficients were run on each
compound and the average taken as the basis for comparison with the
results of other workers as well as for the experimental methods*
to be described below.

The method employed was that described in

* All media (nutrient broth and nutrient agar) employed throughout
this work was prepared according to the specifications given in
TJ.S.D.A. Circular Ho. 198.

-10-

the U.S.D.A. Circular Ho. 198, as "The F.D.A. Method, Staphylococcus
aureus, 20°C 11.

To avoid inhibitory effects of the compound conveyed

to the subculture a second subculture was made into plain broth
(Shippen).

At the same time that subcultures were made from the

test solutions a subculture was also made into a broth containing a
substance which stopped the action of the compound that was introduced.
The Staphylococcus aureus culture (originally obtained from
Food and Drug Administration) used in the phenol coefficient tests as
well as throughout the remainder of the work was taken from the stock
collection of the Bacteriology Department of Michigan State College
and answered all the requirements demanded by the F.D.A. Method.

The

Bberthella typhosa strain used in one part of the work was likewise
obtained from the same source and met standard requirements for this
species.
Colloidal Iodine* was selected as the chief representative
of the halogen group while lugol's solution and Tincture of Iodine
(both prepared according to TJ.S.P.X) were included to demonstrate
the important role different solvents play in disinfection.
Merthiolate was chosen as representative of the newer type of
mercurial compounds which are supposed to occupy a more or less
intermediate position between the theoretically ideal disinfectant
and the theoretically ideal antiseptic.

Brilliant green was selected

as representative of the dye compounds and as probably the nearest to

* Throughout this paper the term colloidal iodine will be used to
refer specifically to Iodine Suspensoid Merck according to
Dr. W. L. Chandler.

-li­

the theoretically ideal antiseptic or bacteriostatic type of
compound*

Phenol was employed as a reliable standard representative

of the coal tar series.

Chlorine, though another representative of

the halogen group would seem superfluous, was, nevertheless, includ­
ed because of the large variety of agents on the market which have
this element as the active ingredient.

Two types of such compounds

were included, both commercial products, HTH as representative of
the inorganic chlorine compounds, and azochloramid as a representa­
tive of the slower acting organic compounds.
The data derived (Table 1) show without doubt that
elemental iodine is, according to the phenol coefficient test,
obviously superior to all the other compounds used.

In addition

there is no difference between the three forms, colloidal, Lugol's,
or tincture, when used in the high dilution necessary for testing,
the reason for which will be emphasized later.

Chlorine in the

hypochlorite form, HTH, is apparently effective when there are
150 p.p.m. available chlorine, yielding a phenol coefficient of 1^.5.
However, the stable type compound, azochloramid, in a saturated
solution of one (l) part in a thousand could not be tested because
it did not yield sufficient chlorine to kill in 15 minutes.

It was

necessary to extend the time of the test to 50 minutes before
sterilization of the seeding was effected, therefore a coefficient
is not given.
The necessity of the Shippen (*+U) modification is demon­
strated in the results obtained with the two compounds, brilliant

-12-

TABLE I. - Determination of Phenol Coefficients, Pood and Drag
Administration Phenol Coefficient Method, S.aureus 20°C.

Compounds

Dilutions killing in 10 min,
hut not in 5 min.
Subcultures in
Sodium
Plain Broth
thiosulphate
1st
Broth
2nd

Phenol Coefficient
Sodium
Plain
Broth
thio2nd sub­ sulphate
Broth
culture

Iodine - Tincture

151^,000

1 :1^,000

1 :1^,000

200

189

Iodine - Lugol's

isiM

oo

1 :1^,000

1 :1^,000

200

189

Iodine - Colloidal

IslH.COO

lslH,000

isii+,000

200

189

Merthiolate - Tincture*

l: 8,000

is 1,500

is 1,500

21.7

20.2

Merthiolate - Aqueous*

1 : 8,000

IS 1,000

l: 1,000

ii».5

13.5

Azochloramid

11 1,000

is 1,000

IS 1,000

Pailed

Pailed

HTH 15 - Aqueous

is 1,000

IS 1,000

1 : 1,000

1H .5

13.5

Brilliant Green*

1 :30,000

IS 1,900

Is 8,000

30.0

108.0

Potassium Iodide
Solution

Ho action

Ho action

Ho action

Alcohol 50$-Acetone 10$

Undiluted

Undiluted

Undiluted

Phenol

1:69

1:69

u fb

-

0.0285
-

0.027
-

♦ These are the nearest to killing dilutions; stronger dilutions could not
he tested owing to the amount of compound carried over from seeding pots.

-13-

green and Merthiolate.

Brilliant green is a well known bacteriostatic

compound and like practically all compounds of this class shows
specificity, especially for Gram-positive organisms.

It is not

surprising to find that it apparently killed in a dilution as high
as 1 to 30»000 for the particular stock dye that was employed in this
work.

However, with the double subculture procedure in use it was

found that 1 to 1900 failed to kill in 15 minutes.

Che reason that

lower dilutions gave negative subcultures, even when double
subcultures were made, was due to the amount of compound carried
over.

This was indicated by the fact that the broth had a strong

greenish tinge at a dilution of 1 to 1000 and the color was also
perceptible at 1 to 1600. failure of subsequent plantings into
these tubes to produce growth provided conclusive evidence that
there was an inhibitory amount of compound present even in the
second subculture tubes.

It is doubtful if the 1 to 1000 concentra­

tion killed within 15 minutes.
Merthiolate, in both aqueous and tincture (solvent;
alcohol 50 percent, acetone 10 percent, water Ho percent) solutions,
showed much the same picture as brilliant green.

The 1 to 1000

aqueous solution failed to kill in 15 minutes whereas the 1 to 1000
stock solution of tincture did kill in 5 minutes.

This was evidently

due to the alcohol concentration since dilution up to 1 to 1500,
lowering the alcohol concentration to 30 percent, failed to kill in
15 minutes.

This information, of course, was obtained by using the

Shippen (U4) double subculture technique.

Phenol coefficients of the

-1U-

nearest estimated or strongest dilutions used were calculated and
placed in the table.
The few items given in the above paragraphs illustrate
some of the weaknesses of the test even when it is uncomplicated by
additional factors such as added organic matter of various types or
definite particle size as suggested by Garrod (19)» 1935*

These

items illustrate why Reddish (38), 1937* ks-3 30 strongly emphasized
the fact that the test has been badly misused in expecting it to
yield any, let alone equally as much, information for antiseptics.
For, no matter what other arguments may be offered the paramount one
is that the test has been designed for disinfectants only.

In

addition there is the potential possibility of misinterpretation
depending upon the viewpoint of the investigator.

Reddish (38)

illustrates by giving the phenol coefficient of a certain percentage
of carbolic acid which is straining the interpretation slightly since
comparison is based on this compound and any strength compared to it
should always be unity.

In other words, comparing a yardstick with

itself accomplishes very little.

Another type of interpretation aptly

illustrated by Dunn (15)* 1937* is to give the phenol coefficient on
the basis of solid substance or dry powder resulting in a very large
figure which is definitely misleading if the compound is insoluble at
high concentrations or so toxic that to be usable it must be diluted
to such a weak concentration that the phenol coefficient of the usable
dilution may be even less than unity.

-15-

Salle and Lazarus* et. al., (1935-37)

utilizing a

tissue culture technique in conjunction with the regular phenol
coefficient method devised a toxicity test for evaluating
disinfectants which Salle, McGmie, Shechmeister and Foord (Hi), 193&»
found needed revision in the form of added organic matter.

The

incorporation of horse serum, however, did not seriously alter the
results hut merely lowered the figures for all compounds.

They

showed by their first technique that Merthiolate, with a rather high
toxicity index, had a phenol coefficient of 70.

let when their test

was revised adding the horse serum factor and taking into account
the marked inhibitory properties of Merthiolate, they found that a
H percent solution, the strongest that could be prepared, failed to
kill S. aureus in 10 minutes.

The well known dye, mer euro chrome,

which had by their first test a phenol coefficient of 0.6 (killing
in a dilution of 1 to Ho) was found by the modified (bacteriostatic
determination) method to fail to kill the same organism in an
85 percent solution, the strongest solution they could prepare.

On

the other hand they found iodine (Lugol*s solution) by their first
technique to have a phenol coefficient of 308 or killing in a
dilution of 1 to 20,000.

By the revised technique the killing

dilution was 1 to 3520, Just a little less than a saturated water
solution of iodine.

* Series of articles published in Proc. Soc. Exper. Biol, and Med.
were condensed and published with complete description of test by
Salle, McOmie and Shechmeister (Ho), 1937-

-l6-

These facts are presented to illustrate how closely the
findings in this work parallel those of these investigators*
Agar Cup Plate

The next method employed was the first step in demonstrat­
ing that penetrative power (assuming all other factors such as
medicinal-organism contact, constant) was the chief factor
responsible in the ultimate rapid destruction of organisms.
method is presented in U.S.D.A. circular 198.

This

It has been credited

to Dr. L. C. Himebaugih by Reddish but apparently was originated in
1918 by Carnot and Dumont (7) and known today as the agar cup plate
method.
Preliminary trial runs indicated that the chief objections
to this method are:

(l) The phenomenon observed is evidently more

that of diffusibility than penetration and (2) volatile compounds
will give a false picture, even of diffusibility.

The latter is

accomplished by saturating the atmosphere immediately above the agar,
redissolving in the moisture of the surface diffusing downward over
the entire area at the same time the compound is diffusing outward
from the center.
This last objection was easily eliminated by covering the
agar with a layer of paraffin prior to cutting out the center portion
with a cork borer.

Repair of the center cup so produced was

accomplished by filling with agar level with the paraffin and again

-17-

cutting out the center portion with a smaller cork borer leaving a
wall or collar of agar.

This formed a seal over the opening

existing between the agar and the paraffin layer thereby preventing
any of the compound subsequently placed in the cup from seeping out
between the layers of capillary action.

The bottom of the cup was

repaired by a drop of agar to prevent capillary seepage between the
agar and glass.
Employing 0.1 percent soluble starch and 0.1 percent
potassium iodide as an indicator in the agar, data were obtained
for iodine and chlorine (Table Z) which show that these volatile
compounds gave fairly consistent readings, i.e., diffusing outward
from the center of the plate a definite distance.

On the other

hand the data on uncovered agar plates (Table 2) appear to show
that the iodine containing compounds (according to the starchpotassium iodide indicator) had, within the limits of reading time,
2b hours, diffused out to the periphery of the plate.
After noting some of these peculiarities of agar cup
procedure a series of agar cup plates was set up using the test
organisms, E. typhosa and S. aureus, for indicators rather than
starch and potassium iodide.

This change was necessary as the

majority of the compounds listed were employed, and two, aqueous
merthiolate and tincture of merthiolate, cannot be tested by color
indicators.

The plates were observed at IS and 2U hours and

readings made at US hours*

-1S-

TJiBLE 2. - Determination of Penetration,

Agar Cup Plate Method **

Temperature 2^C
Inhibition Zone width* in cm.
Nutrienlb Agar
Plain Agar
Paraffin Paraffin
Uncovered
Uncovered Top
Top

Compound

Concentration

Iodine - Tincture

1:20

*+.0

l.ZS

2.07

i*.e

Iodine - Lugol's

1:20

Iko

1.5

2 .1*5

!*.0

Iodine - Colloidal

1:20

U.O

1.0

2.0

**.0

HTH 15

150 p.p.m.

-

0.1

0 .1*

-

* The average distance of all the plates used from the edge of the cup
to the periphery of the plate was 1*.0 cm. Hence, this figure denotes
penetration of entire plate.
** Starch and potassium iodide used as indicators.

-19-

As was expected from the preliminary observations the
5 percent solutions of tincture of iodine and Lugol's iodine gave
sterile plates in all seventeen trials (Table 3)*

®he 5 percent

suspension of colloidal iodine gave only two sterile plates out of
the series, the remaining fifteen plates having well marked zones.
The apparently larger zones given by colloidal iodine against
E. typhosa was due to the two sterile plates occurring in this
series, thereby raising the average.

The 0.1 percent concentrations

of Merthiolate, aqueous and tincture, gave identical results against
E. typhosa and also S. aureus. These two solutions were apparently
slightly more specific for the Oram-positive organism than the Oramnegative organism.

HTH 15 in a concentration of 150 p.p.m. available

chlorine gave the smallest inhibition zones obtained but was equally
effective against both organisms.

The more stable chlorine compound,

azochloramid, in a saturated aqueous solution of 1 part in 1000 parts
of water gave inhibition zones comparable with the Merthiolate
solutions but showing slightly more activity against E . typhosa than
S. aureus.
Since extent of penetration varies with the time, it was
thought more definite information might be obtained about the
relative penetrative power of the three iodine preparations, alcohol
solution, potassium iodide solution, and aqueous iodine (colloidal
suspension).
clearly shown.

Conversely the faults of the method would be more
To observe relative penetrative power a third series

-20-

TABLE 3 . - Determination of Penetration.

Agar Cup Plate Method

__________________________________ Temperature 37°C
Compound

Concentration

Inhibition Zone, width* in cm.
Indicators**
E . typho sa
S. aureus

Iodine - Tincture

1:20

**.0

k.O

Iodine - Lugol's

1:20

k.O

k.O

Iodine - Colloidal

1:20

2.9

2.3

Merthiolate - Tincture

1:1000

1.7

2.0

Merthiolate - Aqueous

1:1000

1.7

2.0

Azochloramid

1:1000

2.0

1.6

HTH 15

150 p.p.m.

0.7

O.S

* Prom the edge of the cup to the periphery of the plate the average
distance for all the plates used was ^.0 cm., hence, complete
penetration is denoted.
** Average of ten trials using E.typhosa, and seven trials using S.aureus.

-21-

of agar cup plates with paraffin tops were prepared and readings
made at frequent intervals up to
progress of diffusion.

hours to detect variation in

From Table ^ and the companion graph (Fig.l)

it appears evident that by this method Lugol's and tincture of
iodine penetrate or diffuse much more rapidly than does colloidal
iodine.

This is explained by the fact that the particles which

represent the reservoir in colloidal iodine settle down to the
bottom of the cup so they do not exert the full vapor pressure
force evenly throughout the suspension as does occur when the
suspension is kept agitated during short time contact.
This last item, as will be further clarified by following
tests, rather clearly demonstrates one of the definite objections
to this method.

The necessary lgpse of time before reading the

plates is so great that the worker has very little chance to place
a correct interpretation upon his findings, which can be just as
readily called diffusibility as penetrative power, since it is
frequently necessary for hg hours to lapse before reading the
results.

Reiterating, the most superficial reasoning would demand

that, to be effective, compounds should have the ability to penetrate
within a very short space of time.

In a test of this sort where

there is no way of knowing what might be taking place up to 2h hours
(when the very first evidence of growth is detectable) about the
most valuable information that can be obtained from it is whether or
not a compound will yield any zone of inhibition.

The size of the

-22-

TABLB

- Determination of Penetration.
Method Using Paraffin Tops. *
________________

Tim© from
filling, in
hours.

Agar Cup Plate

Temperature 25°0

Inhibition Zone, width in cm.
Tincture
Lugol1s
Colloidal
of Iodine
Solution
Iodine

00:30

0.25

0 .*+

0 .1

00:60

0.37

0 .6

0 .2

1:30

0 .H5

0.7

0.25

2:00

0 .6

0.85

0.33

2 iko

0.7

0.95

0.3 8

U:U0

0 .9 2

1.11

0A 6

10:00

1.3

1 .6

0.55

13:00

1 .U5

1.76

0.5 8

2^:00

1.95

2.15

0.6 5

38:00

2 .2

2.3

0 .7

* Efutrient agar containing 0.1 percent soluble starch.

Pig. 1. - Comparative Rates of Penetration of Tincture (A),
Lugol*s (B) and Colloidal Iodine (C) in Paraffin
Covered Agar,

Distance penetrated in

* 5

3

z
/

0

4-

Q

/Z

/£>

ZO

Z4-

Time of Action in Hours

ZQ

3Z

3i>

4G

-23-

zone produced is definitely misleading as will "be shown in the next
section.

Tor example, Merthiolate frequently gave plates that were

entirely sterile as did some of the iodine preparations acting on
uncovered plates.

Tet when these two compounds were confined within

an object that they had to penetrate in order to give any zone in
the agar, the areas of the zones were surprisingly similar.

One

other minor objection to this method is that special porous plate
tops must be employed to permit evaporation of moisture produced by
bacterial metabolism.

In addition considerable care must be

exercised to prevent slopping of the compounds over the edges of
the cup, particularly tinctures which have a tendency to "crawl".
Those objections are readily eliminated by the technique employed
in the next section.
Modified Agar Cup Plate.

As a result of the series cited, it was thought that an
alteration mi^it be made in the procedure which would show, without
question, that penetration had been accomplished and that
diffusibility had been entirely eliminated.

The technique was as

follows:
(1) A freshly collected length of adult chicken intestine
with a fairly constant diameter and thickness was washed in Locke's
solution,
(2) Cut in k cm. lengths, tied at one end with a loop of
No. UO thread,

-2U-

(3)

^b® section was filled with 1 ml. of compound and the

open end tied with a loop of thread,
(b) The tied section was washed by dipping into a neutral­
izing solution and thence into two changes of distilled water,
(5) It was now placed in the center of a petri-dish,
(6) Agar seeded with S. aureus was poured in a thin layer
into the petri-dish and allowed to set (this prevented floating),
(7) Additional seeded agar was poured to cover the tissue,
(S) The plates were incubated at 37°C. and,
(9 ) Readings were made at 2b and bS hours, noting the size
of the inhibitory zone obtained around the tubular section of tissue.
Employing this procedure the area of the zone need not
bear the brunt of interpretation as to the penetrative power of the
compound involved but rather serve as a crude yet definite indication
that the compound had penetrated the tissue*
Table 5 shows the relative penetrative power of the
compounds employed given in actual measurements obtained direct from
the plates.

It is to be borne in mind that while the elliptical

zone sizes given in cm. (length and breadth measurements) represent
the total area of inhibition minus the size of the tissue, the size
of these areas are not to be interpreted in the sense that one
compound is better than another simply because the zone is bigger.
In fact, the tables show that the majority of the zones are of very
nearly the same area.

Several observations were made which are

-25-

TABLE 5* - Determination of Penetration.

Compound

Concentration

Adult Chicken Intestine Test.*

Ho. of
trials

Average size
of zone in cm.

Average area
of zone in cm. 4

Iodine - Tincture

1:20

22

3.65 x 2.57

9.28

Iodine - Logoi's

1:20

23

3.25 x 2.1*

7 .so

Iodine - Colloidal

1:20

23

2.3 5 x l M

3.3S

Merthiolate - Tincture

1:1000

17

2.77 x 1.86

5.15

Merthiolate - Aqueous

1:1000

20

3.00 x 2.16

6 .1*8

Azochloramid

1:1000

12

0.85 * 0.15

0.13

32,500 p.p.m.

2

3.1* X 2.3

7.82

HTH 65
Brilliant Green

1:20

2

3 .9 X 2.8

. 10.92

Brilliant Green

1:100

1

3.8 x 1.9

7.22

Phenol

1:20

0.87 x 0.32

0.28

10

* 1 ml. of compound placed in 3 x 1 cm. length of intestine and embedded
in s« aureus seeded agar. Clear zone produced by compound diffusing
into the agar read at 1*8 hours.

-26-

cons idered worthy of inclusion here because of the bearing they
have on the subsequent data.

In addition these facts are more

valuable in this interpretation than the actual measurements
recorded.
The chlorine compound, HTH 15, employed in previous tests
in a dilution containing 150 p.p.m. available chlorine (l part
powder - 1000 parts of water) had given such consistent negative
results that a stronger solution of HTH 65 was prepared (5 percent
solution of the powder) containing approximately 32*500 p.p.m.
available chlorine which did give a zone of inhibition in this test but
only after it had MeatenM its way throu^t the tissue.

The three forms

of iodine employed while giving variable size zones, but within the
limits of technical error, never failed to render sterile the tissue
section in which they had been placed*

On the other hand the two

solutions of Merthiolate used while also giving a variable but
frequently good sized zone in the seeded agar consistently failed to
sterilize all parts of
This failure

the tissue section.
was noted at the ends

of the section where the

tie off was made indicating that the mechanically produced added
thickness of the tissue at

this point slowed the penetration of the

compounds sufficientlyto allow development of the organisms
present on the washed intestines.

normally

These organisms, as a group, are

predominately Gram-negative and less susceptible to these compounds
than the S. aureus used as the indicator.

The interpretation that

-27-

penetration was retarded is based upon the fact that subculture
from these hazy zones at the ends of the tissue section gave no
growth, nor would these subcultures support growth upon subsequent
seeding.

This was due to the amount of compound carried to the tube

of broth in the small loop of fished agar, indicating that while the
myriads of colonies had time to develop partially they were killed
within the ty-S hour time limit.

This is a notable contrast to the

sections containing iodine which invariably had a cleared zone all
the way around the tissue that upon subculture was always negative
and yet the subcultures would support growth upon subsequent seedings.
The slow acting chlorine compound, azochloramid, which In
comparison with the more commonly known brands of chlorine compounds,
is very stable in the presence of organic matter, gave a zone
indicative of just bare penetration of the tissue as the zone rarely
ever encompassed the whole tissue section.

In other words only

partial sterilization of the tissue section was obtained.

This was

also true of 5 percent phenol which was carried along in this series
of experiments as the representative of the coal tar group of
compounds.

The tissue section filled with phenol was usually, but

not always, surrounded by a visibly cleared zone of inhibition.
Brilliant green, when employed in a concentration of
1 to 1000 utterly failed to penetrate in a number of trials.
Stronger concentrations of 1 to 100 and 1 to 20 were prepared and
penetration was obtained*

The zone sizes given in the tables for

-28-

these latter concentrations of the dye are the measurements of the
area of dyed agar, and not a readable zone of inhibition, since the
dye did not penetrate soon enough with the 1 to 100 concentration
to prevent development of colonies within the zone covered by the dye
in the 4-8 hour reading time.

In both trials with the 5 percent

concentration of the dye the penetration of the tissue was fairly
rapid occurring shortly after pouring the agar, but not in sufficient
concentration to give rapid diffusion into the agar so as to prevent
the development of all colonies within the dyed area.
Penetration of Adult Chicken Ceca
The above findings led to the following experiment to
determine the relative time necessary for these compounds to penetrate
tissue of a slightly different type.

The technique was as follows:

(1) Adult chicken ceca were obtained, washed in Locke's
solution and filled with 10 ml. of compound,
(2 ) The open end tied with No. 40 thread,
(3 ) Washed by dipping in neutralizing agents and two
changes of distilled water,
(4 ) Placed in a flask containing 100 ml. of a 24 hour broth
culture of S . aureus,
(5) Shaken at intervals, and
(6) Subcultures made frequently to determine when the
compound had penetrated the tissue as indicated by death of the
bacteria in the surrounding medium.

-29-

To eliminate the possibility of variation in the structure
of the ceca from "bird to bird, pairs of ceca were obtained from two
birds> one of each pair being used for colloidal iodine and the
other of each pair being used for Lugol's iodine and tincture of
iodine respectively,
The findings recorded in Table 6 are in reasonable accord
with assumptions made in the preceding section*

Lugol's solution

proved to be the most rapid, in fact penetrating in just a few minutes
whereas tincture of iodine required 39 minutes and colloidal iodine
needed 5 hours to penetrate the tissue.
Both Merthiolate solutions penetrated this type of tissue in
6 1/2 hours while phenol required approximately 33 hours to sterilise
the culture.

All the other compounds, 1 to 1000 concentrations of

HTH 1 5 , azochloramid and brilliant green failed to sterilize the
S. aureus culture or tissue in Wk days.

At this time equal size

portions of the negative cecal tissue were snipped from the terminal
ends of the respective ceca, washed and embedded in S. aureus seeded
agar to ascertain if there were any residual compound in the tissue.
All compounds (but phenol), Lugol's, tincture, and colloidal iodine,
aqueous and tincture of Merthiolate, showed inhibitory zones.

The

latter two zones being somewhat larger than those produced by the
iodized tissue sections.

-30-

TABLE 6, — Penetration of Adult Chicken Ceca
. ______ ______________ ________________Temperature 20°C.
Compound

Concentration

Time when
1st subculture
became negative

Inhibition zone
of plated tissue
cm.

Iodine - Tincture

1:20

39 min.

Iodine - Lugol's

1:20

6 min.

0.1

Iodine - Colloidal

1:20

5 hrs.

0.1

Merthiolate - Tincture

1:1000

6i hrs.

2.2

Merthiolate - Aqueous

1:1000

6J hrs.

2 .4

HTH 15

150 p.p.m.

44 days positive

-

Azochloramid

1:1000

*44 days positive

-

Brilliant Green

1:1000

44 days positive

-

Phenol

1:20

33j hrs.

0

0 .0 5

-31Penetration of Microscopic Living Units

Having determined that dead tissue could "be readily
penetrated by the compounds in use and having in a measure determined
some of their limitations it was decided to conduct further tests
upon living objects of sufficient size to make the end point visible
with the aid of a microscope.

Coccidia from fresh acute cases of

fowl coccidiosis and freshly hatched long-tailed strongylid larvae of
horses were chosen for these tests.

These two organisms were

selected because in both instances there exists a very definite
membrane which must be penetrated before the life center is reached
with ensuing death, namely the wall of the oocyst or the protective
cuticle of the larvae.

Penetration of Coccidlal Oocysts.
The selection of coccidial oocysts to meet the investigative
purpose of this paper was based upon the evidence accumulated by
Chandler (9), Becker (4 ), Pish (IS) and others that the shell or wall
of the oocyst is a membrane extremely difficult for compounds to
penetrate.

Chandler and Schalm * have demonstrated (with another

objective in view, i.e., death of the organism) that a distinct differ­
ence exists between the three iodine preparations employed here,
finding that, under constant controlled conditions, they kill the
oocysts in the following order* (l) axjueous solution of iodine

* Unpublished data.

-32-

suspensoid, (2 ) Lugol's solution, and (3 ) tincture of iodine.

It is

well to toear in mind that thus fax the tests made on agar cup plates
and dead tissue have indicated that, if these tests are fair measures
of penetrative power, that the atoove order should toe (2), (3), (l).
The technique employed toy Chandler and Schalm was modified
slightly for this determination of penetrative power, which, as
previously stated, varies only as the time of contact.

In order to

ascertain this end-point the following method was used:
(1) 0.25 ml, of a suspension of freshly collected, washed,
coccidial oocysts was placed in U.75 nil* of compound,
(2) Thoroughly shaken at intervals,
(3 ) At definite periods 0 *5 ®1 * of the suspension was placed
in H.5 ml. of "brilliant green-sodium thiosulphate and allowed to
sporulate at room temperature.

This procedure sufficed for all iodine

and chlorine containing compounds,
(H)

For the merthiolate preparations the sample of oocysts

prior to placing for sporulation were first washed in saturated H^S
water then three washes of tap water toy centrifugation,
(5) For phenol, "brilliant green, and the acetone-alcohol
mixture three washes with tap water sufficed,
(6) At the end of Ug hours the greater portion of super­
natant liquid was drawn off and a drop of the sediment placed on a
slide under a cover glass,
(7) Handom fields were counted until a total of one hundred

-33-

oocysts had "been observed, noting those that were sporulated,
non-sporulated and fragmented,
(8 ) Control samples were sporulated at the same time,
(9) 2?he total sporalation for the control samples which
averaged 95 Per hundred oocysts was taken as the 100 percent base
upon which the percentages shown in the table were calculated for
the test samples, and
(1 0 ) The recognized sporulation medium, 2 percent potassium
dichrornate was also used to show that the brilliant green-sodium
thiosulphate medium produced maximum sporulation.
Of the rapid acting compounds (Table 7 ) It caja

seen that

colloidal iodine was the most rapid of all compounds in penetrating.
Observation showed that it actually entered and stained the cytoplasm
of the organism in less than 5 minutes.

The time for Lugol's solution

to enter and kill was over 15 minutes while 5 percent phenol needed
approximately h hours and the 5 percent HTH65 solution (32,500 p,p,m.
available ehlorine) required better than 7 hours to give complete
mortality.
From Table 8 it can be readily seen that the compounds which
under other penetrative conditions (cf, agar plate methods) appeared
possibly superior to colloidal iodine are in the light of these data
actually inferior.

Five percent tincture of iodine killed only

28 percent of the oocysts in 12 hours.

Aqueous Merthiolate in the

three concentrations 0 .1 , 1 *0 , and *K0 percent was sli^itly better.

-3'4-

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-35-

TABLB S. - Penetration of Coccidial Oocysts. *

Compound

Concentrati on

Percent of Coccidia Killed
4 hr. 6 hr. 8 hr. 10 hr. 12 hr.

Iodine - Tincture

1:20

k

12

16

26

28

Merthiolate - Tincture

1*1000

0

0

1

9

26

Merthiolate - Aqueous

1:1000

6

7

8

17

69

Merthiolate - Aqueous

1:100

8

Ik

23

^7

59

Merthiolate - Aqueous

1 :2 5

5

7

10

19

71

HTH 65

650 p.p.m.

0

1

9

15

25

AzocKtoramid

1:1000

1

8

2

3

5

8

3

5

11

13

Alcohol 50$ and.
Acetone 10 $ '
Brilliant Green

1:1000

1

2

0

1

0

Controls;
Potassium Dichromate

1 :5 0

5**

6

5

5

6

6

6

5

k

Brilliant Green and

* Oocysts suspended in compounds; Aliquot portions removed at intervals
and placed in 'brilliant green-sodium thiosulphate medium to sporulate.
Test and sporulation conducted at room temperature 22°C. Read at
tyg hours.
** Actual number dead per hundred oocysts counted.

-36-

Sufficient of the compound passed through the wall of the oocysts in
12 hours to prevent subsequent cyclic division of the cytoplasm in

69, 59 and- 71 percent of the oocysts, respectively.

Apparently the

1 .0 percent concentration of Merthiolate seems to he the optimum

concentration for uniform action under the conditions of the test.
The 0 .1 percent tincture of Merthiolate shows the inhibiting effect
of the

solvent upon the chemical as only 26 percent of the

oocystswere

killed

in the 12 hour period in contrast to the 69 percent

mortality

obtained with the same concentration of the aqueous solution.

Part of

the 26 percent mortality obtained by the tincture can be attributed to
the solvent itself since the alcohol-acetone mixture alone produced 13
percent mortality in 12 hours.

The saturated aqueous solution of

azochloramid 1 to 1000 concentration proved to be a very poor killing
agent, producing only a 5 percent mortality.

On the other hand the

inorganic compound HTH 65 in a concentration of 650 p.p.m. available
chlorine killed 25 percent of the oocysts.
of brilliant green failed to kill any of
In order to point out the role

The 1 to 1000 concentration
the oocysts.
solvents may play

in the

penetrative power of a disinfectant the three iodine preparations were
used with a much more resistant stock sample of oocysts so as to give
a clearer picture with no border line distinctions.
given in Table 9.

The results are

Here we have three decidedly different solutions of

the same strength element, 5 percent available iodine.

In colloidal

iodine the only solvent present (and a very poor solvent, 1 to 3000 at

-37-

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2 Q°C.) is water, the suspending menstruum for the particulate iodine

which forms the reservoir of instantaneously soluble iodine.

This

feature of instantaneous solubility constantly maintains the
surrounding solution at a strength of 1 to 3000, higher or lower
depending upon the temperature*
again all water.

In Lugol's compound the menstruum is

However, all the iodine present is in actual

solution, made possible by the strong solvent action of the potassium
iodide present.

The third compound, the well known tincture of iodine,

has but a small amount of water and two tenacious solvents acting,
namely potassium iodide and alcohol.
The results shown in Table 9 demonstrate that with all other
factors identical (concentration, substrate, temperature) there is
only one left which can be held responsible for the difference in
penetrative power of the three solutions of iodine and since the
solvents employed are the only variables present, it follows then that
they must be responsible for the fact that colloidal iodine kills
within ten minutes; Lugol's iodine requires three hours and tincture
of iodine is not effective in 2^ hours.

This simple experiment also

explains why identical results are obtained with these three solutions
of iodine when they are used in dilute form (1 to 1000 or higher
dilutions) as for example in the phenol coefficient method of testing
where the solvent is essentially diluted out of existence, yet in
practical use (concentrations of 1, 3 o r 5 percent) yield such marked­
ly different results.

To add more information to this particular

factor the following experiments were devised:

-39-

Penetration of Larval Cuticle

For this series freshly collected, hatched, long-tailed
strongylid larvae of horses were employed.

The technique consisted

simply in placing a drop of the larval suspension containing not less
than 100 larvae in an esmarch dish, adding 2 ml. of the test compound
and observing under a low-power (20x) binocular microscope the changes
that ensued.

In some instances neutralizing agents were employed to

stop the action of the compound after certain changes had occurred.
This will be explained as each compound is discussed.

One factor

which recommends the choice of larvae for these tests is that by
their constant, active movements in the solution the criticism of
lack of continual agitation is obviated.
Here again as in the previous test we have a definite set­
up which has an unquestionable end-point, namely irreversible demise
of the organism.

Further we have an analogous situation of a visible

barrier, the cuticle or protecting sheath of the larvae, which must
be penetrated by the compound before the life center of the organism
is reached resulting in death.
From Table 10 it is obvious that, of the group of compounds
studied, elemental iodine, irrespective of the original solvent
present, proved obviously superior to every other compound employed
in the rapidity with which the larvae were penetrated and killed.

In

addition it will be noted that no detectable difference existed
between the iodine preparations when employed as previously stressed,

-40-

TABLE 1 0 . - Penetration of Strongylid Larvae Cuticle.*

Compound

Concentration

Killing Time

Iodine - Tincture

1:1000

5 sec.

Iodine - Lugol's

1:1000

5 sec.

Iodine - Colloidal

1:1000

5 sec.

Merthiolate - Tincture

1:1000

Merthiolate - Aqueous

1:1000

62 hrs.

Merthiolate - Aqueous

1:100

62 hrs.

Merthiolate - Aqueous

1:25

62 hrs.

HTH 65

650 p.p.m.

75 min.

HTH 65

6,500 p.p.m.

15 min.

HTH 65

32,500 p.p.m.

3 min.

3i hr.

28 min.

Azochloramid

1:1000

312 hrs.

Brilliant Green

1:1000

2SS hrs.

Brilliant Green

1:100

l44 hrs.

Brilliant Green

1:20

107 hrs.

Phenol

1:100

U5 min.

Phenol

1:20

Alcohol 50$ + Acetone 10$
Control - in Tap HgO

undiluted

3i min.

2i hr.

15 min.

alive 30 days

* Two ml, of compound added to one drop of larval suspension
(approximately 100 larvae). Observed at intervals for visible
changes or death of the larvae (20x binocular)* Test conducted
at room temperature 20°C.

-41-

in dilutions of 1 to 1000 or higher.

All three compounds of iodine

of 0 .1 percent concentration killed within five seconds.

These

sharp end-points were obtained by neutralization with 0.1 ff sodium
thi ©sulphate, followed ty three washes of aspirations! sedimentation*
The next quickest killing compound was aqueous HTH containing
3 2 ,5 0 0 p.p.m. available chlorine.

While this compound required only

three minutes to bring about death of the organism it was noted that
in order to do so the organism was first dissolved.
process occurred in this way:

Briefly the

The thin double layer of cuticle which

formed the tail was the first part to disappear followed by a
splitting or shredding of the posterior end of the organism simultan­
eously with appearance of bubble-like rupture of the cutieule along
the whole length of the organism.

Inactivity and death were almost

immediately followed by dissolution.

If the digesting action of the

hypochlorite were stopped by neutralization with 0.1 N sodium
thiosulphate at the instant the last organism ceased moving, followed
by aspirational washing, none ever recovered and all had the appear­
ance of floating shreds in the dish.
This picture was obtained by using the weaker concentrations
of HTH 65 containing 6 ,5 0 0 p.p.m. and 650 p.p.m. available chlorine,
respectively.

The former concentration required only 15 minutes while

the latter concentration needed 75 minutes to kill all the larvae.
Maceration of the organisms followed in those samples not neutralized.
The action of this hypochlorite is in accord with that reported by

-b2~

Bashford C3 ) * 193-7 )* Baylor and Austin (^-6), 1912 , Piessinger and
Clogne (17 )» 1912 , who found that in attempted disinfection employ­
ing the classical Dakin*s solution that digestion of the tissue is
a paramount feature.

Piessinger and Clogne pointed out that in the

treatment of wax wounds by the Caxrel continuous irrigation method
that improvement was due more to the proteolytic action of the
compound upon mortified tissue than to any sterilizing action.
Chandler (9), 1933 » demonstrated that while hypochlorite
solutions were very inefficient, free chlorine was quite active in
killing coccidial oocysts.

In the light of these observations it

is well to recall that the HTH 65 solution containing 3 2 ,5 0 0 p.p.m.
available chlorine required 7 hours to kill all the coccidial
oocysts in the test sample (see Table 7 )*

I*1 connection with the

mode of destruction of larvae given above it can now be pointed out
that the prevention of cyclic division of the cytoplasm of the
oocysts was accomplished by the hypochlorite digesting and entering
the oocysts through the operculum followed by destruction of the
life center in the cytoplasm.

There is demonstrated then the

imperviousness of the oocyst shell to ordinary chemical agents.

At

the same time it is shown that the mechanism of action of the
hypochlorite type compound is not one of penetration and killing as
with elemental iodine but instead death results from digestion or
oxidation.

-*3-

Phenol in. a concentration of 1 to 100 "brought ah out death
in

minutes as an irreversible procedure without any external

changes of the organism while the 1 to 20 concentration killed in
three and one-half minutes.

Attempts to revive the organisms "by

washing with ammonium sulphide ((UHl^S) water followed by washing
with plain tap water were futile indicating that the phenol had
evidently penetrated sufficiently to bring about irreversible
changes in the protoplasm.
Por the two Merthiolate preparations, aqueous and tincture,
the killing time was established by washing various lots of organisms
at different intervals with water saturated with hydrogen sulphide,
for 3 minutes, followed by not less than four washes of tap water.
Control larvae kept in saturated hydrogen sulphide water for five
minutes followed by not less than ^ washes with plain tap water
showed no marked loss of activity or subsequent ill effects.

It was

found that whereas aqueous Merthiolate in 0 .1 , 1 .0 or U.Q percent
concentrations would cause the organism to begin less active move­
ments in 10 to 12 hours, irreversible death of all the organisms in
any given test lot could not be brought about even by the strongest
concentration used under 62 hours at 20°C.

Apparently the tincture

of this compound in 0.1 percent concentration was much better,
killing the organisms under 3 1/** hours, but when the menstruum,
alcohol-acetone, was examined for its killing power and found to
accomplish death in approximately the same time it was concluded

that the tincture was no better than the same concentration of
aqueous Merthiolate.
Brilliant green in three concentrations of 0.1, 1.0 and
5*0 percent proved to be a very poor killing agent being unable to
inactivate the more resistant organisms under 2SS, 1^4 and 107 hours,
respectively.

Likewise the stable chlorine compound, azochloramid,

in a 0.1 percent concentration proved to be too stable to give up
enough chlorine to inactivate the organisms in less than 312 hours.
At the end of this period there was still available chlorine present
as could be shown by titration.

As already emphasized, readily

available chlorine will bring about death of the larvae in a very
short period if the concentration is high enough for rapid oxidation,
but with azochloramid death is attained without microscopically
visible oxidation as the organisms were all intact after death.
After the above stated time period had elapsed washing once with 0 .1 IT
sodium thiosulphate and several times with plain water failed to bring
about recovery of any of the organisms.
To emphasize again the role solvents may play, several tests
were made using the three iodine preparations in 5 percent concentra­
tions and following the technique described above.

It was noted

(Table 11 ) that aqueous iodine (colloidal suspension) killed in less
than 1 second and visibly stained the interior of the organism in less
than 3 seconds, whereas the potassium iodide solution, Lugol's,
required 15 seconds to kill and 30 seconds to stain the interior; the
tincture needing U5 seconds and 5 minutes to accomplish the same

- t y j-

T&BLE 11, - Penetration of Strongylid Larvae Cuticle "by Selected
Compound with Limited Time Periods. *

Compound

Concentration

Iodine - Tincture

1:20

^5 seconds

5 minutes

Iodine - Lugol's

1:20

15 seconds

30 seconds

Iodine - Colloidal

1:20

1 second

3 seconds

Iodine - Tincture

Killing Time

Staining

Hecovery
none

** 1:1000

5 seconds

1:1000

5 seconds

none

1:1000

5 seconds

none

** 1:10,000

3 minutes

none

Iodine - Lugol's

1:10,000

3 minutes

none

Iodine - Colloidal

1:10,000

3 minutes

none

Iodine-

Lugol•s

Iodine - Colloidal
Iodine - Tincture

* Two ml, of compound added to one drop of larval suspension
(approximately 100 larvae). Neutralized at the intervals
given as killing time. Different trial lots were neutralized
at various intervals to determine interior staining. Obser­
vations done with 20x binocular. Test conducted at room
temperature 22°C.
* * The 1 to 1000 and 1 to 1 0 ,000 dilution results are included
to emphasize the role of solvent action.

-46-

results.

Again showing conclusively that, with iodine at least, the

weaker the solvent the more effective will he the iodine, or the
greater its penetrative power.
An observation made throughout the work reported in this
section on macroscopic living units which could he only hinted at hy
the data given in the tables is that the coccidial oocysts and
strongylid larvae when acted upon by the slower compounds (Merthiolate,
brilliant green,azochloramid, the higher dilutions of phenol and HTH)
behaved very much like bacteria.

Apparently each test sample contained

a small percentage of susceptible and highly resistant organisms.
Especially was this feature noticeable in working with the larvae.
Approximately 25 percent of the organisms were inactivated in the
first few hours or minutes of contact with the compounds and an
additional 50-60 percent were inactivated by the time the test period
had reached the half way mark for the particular compound in question.
The remaining 1 5 -25 percent of the organisms succumbed one by one
during the last half of the test period until the time reached when
they were recorded as dead or almost finished.

This behavior is

analogous to the death rates of bacteria under similar conditions of
slow acting compounds as frequently reported in the literature.

-*7-

Rabbit Skin Penetration

The next procedure employed to demonstrate the action of
the compounds studied was based upon a technique devised "by Etchells
and Fabian (l6), 1935 *

determining toxicity of compounds placed

upon the ahdomen of a rabbit.

Briefly the technique used was as

follows:
(1) 25 ml. graduate cylinders were cut at the 5 ml* divisions,
(2 ) The edges ground to right angle smoothness using alundum
and a smooth glass plate,
(3 ) The cup thus formed was attached to the shaved abdomen
of a rabbit (prepared 2U hours prior to the test) by the aid of rubber
bands terminating in hooks fashioned from paper clips,
(4 ) Collodion was then placed around the outside base of the
cup and allowed to dry.

The Hsettingn of the collodion and subsequent

drying gradually drew the cup against the skin until sufficiently
tight to prevent any leakage occurring,
(5) Several cups were placed in a row (usually two parallel
rows equidistant either side of the midline were employed) and as
each cup “set" sufficiently the rubber bands and hooks holding it
were moved to a new position for holding another cup,
(6) Following placement of the last cup and removal of all
bands and hooks the animal was ready for intravenous nembutal and
intermittent ether anesthesia,
(7) Blunt needles were inserted subcutaneously beneath each
cup for culture placement,

-kz-

(8) Two ml* of compound were then placed in each cup,
(9 ) Zero time was taken as each cup was filled, the
filling toeing done at intervals so as to allow for subculturing and
final cup removed, so that each compound was in contact with the
skin the same length of time,
(1 0 ) One-tenth ml. of a saline suspension of a 2U hour agar
slant culture of S . aureus was then injected into the pocket "beneath
each cup,
(11) At intervals as shown in the tatoles subcultures were
made toy withdrawal of 0.01 ml. of the inoculum and,
(12 ) Seeding into broth tubes, incubated ^-8 hours at 37 °C.
A study of Tatoles 12-16 show the culturing method to toe a
failure as apparently none of the compounds penetrated the live skin
in sufficient concentration to sterilize the pockets since subcultures
were all positive even after 11 hours.
rabbit No.

However, upon autopsy of

(Table lH) it was found that the iodines were

macroscopically present in the muscular layer beneath their respective
cups.

These circular areas of muscle tissue beneath each cup were

removed and plated in S. aureus seeded agar, incubated bZ hours and
the zone of inhibition noted.
Because of certain technical difficulties encountered,
including absorption of the inoculum, such that the time period could
not be practically extended, the culturing phase of the technique was
abandoned and attention directed toward determining in an unquestion-

-1+9-

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-52-

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TABLE 15. - Penetration of Rabbit Skin by Selected Compounds Acting 5 1 /2
Hours Before and an Additional 5 Hours After Death of the Rabbit.

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Zone Sub­
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1

TABLE l6. - Penetration of Rabbit Skin by Selected Compounds Acting 62 Hours Before
and an Additional Hi Hours After Death of the Rabbit.

V£>*
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Skin** stretched over
short tubes 1 ml.
culture inside.
Subcultures at

-53-

-5^-

abl© fashion the length of time required for these compounds to
penetrate both live and dead skin.

The term wlive skin8 will he

used in referring to the skin when it is on the intact, anesthesized
animal with circulation unimpaired.
employed in referring

The term Bdead skin'* will he

to the skin from the moment when the animal

has heen sacrificed and the general circulation stopped irrespective
of whether or not the skin is used on the animal or removed
immediately at death for test tuhe work.
In each test the animal was sacrificed as indicated hy the
termination of the experiments on live skin, Tables 1*4- 1 9 . The cups
were emptied and the interior washed with a neutralizing agent and
several changes of distilled water followed hy swabbing dry, then
cups were removed.

The circular area of epidermis covered by each

cup was removed as was the underlying areolar tissue and first layer
of muscle, except where inflammatory reactions had occurred to such
a degree that the fascia was more or less adherent to the skin.
These bits of tissue averaging 1 .3 cm. in diameter were placed upon
a previously poured thin layer of S. aureus seeded agar, and covered
with another layer of seeded agar.

Incubation was carried out the

usual *+8 hours to allow a dense growth to clearly outline the area
around the tissue where the compound had diffused into the agar,
killing or inhibiting the colonies.
Inspection of Tables lU-1 9 show that the compounds to which
definite killing action had been previously assigned were capable of

-55-

penetrating the skin, the areolar tissue, and into the muscle layers
if certain conditions obtained.
in more detail later*

(These conditions will "be described

Babbit Ho* ty, Table 1^, was sacrificed

immediately after the suspension of S . aureus was placed in the
pockets so that insight might be gained as to whether or not the
failure to sterilize the inoculum through, live skin was due to factors
beyond reasonable control*

This was evidently the case since positive

subcultures were obtained even though the iodine compounds had
macroscopically penetrated*

Inspection of Table l4 shows that

¥ percent aqueous Merthiolate and the three 5 percent iodine compounds
penetrated the epidermis of the dead animal in some time under 7 hours
since tissue plating demonstrated they were present in the first layer
of abdominal muscle*
Brom Table 15 it can be seen that these same compounds were
barely able to penetrate the dead skin in 5 hours, even though they
had acted on the live skin 5 V 2 Iwmrs before the animal was
sacrificed, a total of 10 1 /2 hours action upon the epidermis.
Incidentally, the stronger solution of aqueous HTH 65 used, (32,500
p.p.m. available chlorine) showed why it was virtually impossible of
demonstration in the subcutaneous tissue by this technique.

Since

HTH penetrated by oxidation, causing necrosis and digesting the
tissue as it went deeper, the compound itself was used up in the
procedure.

Therefore, insufficient residual compound was present in

the tissue to be demonstrable when the epidermal layer was plated.

-56-

At the time the animal was sacrificed (5 1 /2 hours) the skin was
dissected loose from the muscle and the under side examined for
macroscopic evidence of any of the iodine compounds*

Hone were

visible until 5 hours later, but edematous reactions in the areolar
tissue were evident under all cups except b- percent Merthlolate,
Brilliant green was omitted because of the obvious length of time
(by observation) necessary to penetrate the epidermis, while aqueous
HTH and phenol were dropped (Tables lH and 15 ) from further consider­
ation because the amounts penetrating were apparently used up or
bound by the epidermal cells so that no residual could be demonstra­
ted by plating the washed epidermis*
Considering Table l6 next, it is readily seen that aqueous
Merthiolate and the three iodine compounds penetrated the epidermis
and the areolar tissue in 11 hours.

In this test the compounds acted

on the skin of a live animal the first 6 1/2 hours and on a dead
animal the last b 1/2 hours.
To distinquish between the compounds from the standpoint of
speed, a section of the unused epidermal layer was removed at death,
cut into sections an inch square and stretched over one end of a
sterile two inch length of test tube.

A ml. of a saline suspension

of S» aureus was placed therein and the skin end of the tube then
immersed beneath the surface of a compound.

Subcultures from the

inside at intervals proved colloidal iodine to be the most rapid,
penetrating the skin in something less than two hours while over

-57-

three hours were required for tincture of iodine.

The

4

percent

aqueous solution of Merthiolate did not penetrate in sufficient
amounts to sterilize the seeding in 20 hours.

However, the compound

had penetrated in sufficient quantity "by this time such that the
Shippen procedure, i.e., a double subculture, gave no growth in the
first tube but excellent growth in the second broth tube.
The next two experiments (Tables

17

and IS) were designed

to explode the myth of easy penetration of live skin.

In these two

tests a run was performed with the cups set up on one side of the
mid-line of the abdomen with the animal alive.

Duplicate cups were

set-up on the other side of the mid-line and filled just as the
animal was sacrificed.
the tissues plated.
manner.

The first set of cups was then removed and

The second set of cups was treated in the same

The only variable introduced was time, the compounds being

allowed to act upon the live skin longer than upon the dead skin.
Acting upon dead skin 7 3/^ hours (Table 17) all of the
compounds employed (l and h percent aqueous Merthiolate; 5 percent
tincture, Lugol's and colloidal iodine) penetrated the areolar tissue
in sufficient concentration to give definite zones on plating.
Whereas, acting upon live skin almost an hour longer k percent aqueous
Merthiolate was the only member of the group apparently able to
penetrate the epidermis.

It is evident from the areolar tissue zones

and the relative size of the epidermal tissue zones produced by the
1 percent aqueous Merthiolate and the three 5 percent concentrations

-58-

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-6o-

of iodine that the dead epidermal layer was much easier to penetrate
than that of live skin*

The aqueous forms of iodine, Lugol's and

colloidal, show some superiority over the same strength of tincture
of iodine hy penetrating the first layer of abdominal muscle while
the latter compound reached only as far as the areolar tissue within
the allotted time period.

The 4 percent aqueous concentration of

Merthiolate showed similar superiority over the

1

percent concentra­

tion by penetrating the dead epidermis, areolar tissue and into the
abdominal muscle while the weaker concentration failed to penetrate
the muscle.
In the second experiment of this type (Table 18) the
allotted time for penetration of the live and dead skin was shortened
2 1/2 and 3 1 /b ^ours respectively.
distinction between the compounds.

This served to give a clearer
Acting upon live skin

6

hours none

of the compounds penetrated the epidermis whereas acting upon dead
skin b 1/2 hours three of the group, Lugol's iodine, colloidal iodine
and k percent Merthiolate penetrated the epidermis as demonstrated by
their presence in the areolar tissue.

It is noteworthy that with

these shorter time periods smaller amounts of the compounds were
present in the epidermal tissue as demonstrated by the relatively
smaller inhibitory zones when compared with those obtained on dead
skin in the previous experiment (Table 17).
Prom observations made upon the plated tissues given in
Tables 1^ to 18 it was noted that the zones given by the areolar

-61-

tie sue under the b percent Merthiolate cups were barely evident.

It

is felt that the evidence of penetration given by these zones is
false.

It is possible that these zones were due to contamination of

the areolar tissue by the Merthiolate when the epidermal layer was
being cut from the skin.

Even after most careful neutralization and

thorou^i washing of the epidermal layer under the b percent Merthiolate
cups there was considerable residual compound in this layer as demon­
strated by the majority of plates being sterile or almost so.

In

view of this fact it was felt that if true penetration had occurred
then the areolar tissue and muscle tissue layers would have enough
compound to produce much more definite, distinct zones than were
obtained.

In this connection it is well to remember that the

bacteriostatic properties of mercury are high and not easily inacti­
vated by nutrient media, hence even a minute amount of the compound
carried from epidermal layer to the subcutaneous layers by the
instruments at time of dissection could easily account for the results.
Before proceeding to the final table of this series, it
would be well again to call attention to the more obvious differences
among the three iodine compounds employed.

Whether acting on live or

dead skin, colloidal iodine penetrated faster and deeper than did
Lugol's iodine.

The platings of surface skin, areolar and muscle

tissue from colloidal iodine cups gave much larger inhibition zones
than the Lugol's, indicating that more iodine was taken up by the
tissue from the colloidal iodine.

The same concentration of tincture

-62-

of iodine was an outstanding failure, in several instances not even
penetrating the epidermal layer of tissue.
The data given in the final Table of this series (19) were
obtained by the same procedure as used in Table l6 , i.e., tying the
skin over the open end of a short sterile test tube and placing a
ml. of a saline suspension of S. aureus on the inside, replacing the
cotton plug and inserting the skin end of the tube in the compound
allowing it to float undisturbed, except to subculture at intervals.
Using the Sfcippen subculture procedure with Merthiolate
and brilliant green it was possible in this lengthy test to demon­
strate that the dye could penetrate dead skin but required
even to tint the saline suspension inside the tube and

99

66

hours

hours to

reach sufficient concentration to produce a negative subculture.

The

b percent concentration of aqueous Merthiolate required less than 37
hours to give a negative subculture but the

1

percent solution of

Merthiolate needed something over 60 1/2 hours to give negative sub­
cultures with the

0 .1

between

hours.

^7

und

60

percent tincture yielding negative subcultures

The iodine group was again obviously superior.
iodine penetrated in
15

2

Colloidal

3 /k hours while Lugol*s solution required

minutes longer to render the seeding sterile.

Tincture of iodine

was 3jnost as slow as the strongest solution of aqueous Merthiolate,
requiring 1^- to 17 hours to penetrate.

Here, too, is strikingly

shown why, in practical use, tincture of iodine is of little value

-63-

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- Relative Penetrative Time of Selected Compounds Acting
Tissue Stretched over One End of a Class Tube*

on Epidermal

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-64-

unless allowed to dry upon the skin releasing the iodine held in
solution.

The alcohol instead of acting as a penetrative assistant

is in reality a retarding agent*

Because of its great solvent

power it allows very little of the iodine to penetrate the skin*
As shown by the two experiments (Tables 17 and IS) where the
compounds acted upon live skin

6

and

8

hours, barely enough iodine

from the tincture penetrated to give a residual sufficient to
record (Table

17)

even a small zone when the live epidermal layer

was plated.
The double subculture method employed with the organic
mercurial and the dye (Table
emphasis.

19)

illustrate a point worthy of

This point is used as a basis for interpretation of an

observation of a constant phenomenon which was noted throughout the
entire study.

Wherever double subculturing technique (Shippen) was

employed it was found that just at the breaking point, i.e., where
sterilization occurred, the first subculture would show no growth
in 24 hours yet slight but definite growth in 48 hours.

The seoond

subculture tube would show no growth even after several days
incubation.

The purpose of the second subculture is to dilute

beyond inhibition any of the compound which might produce
bacteriostasis in the first subculture, thereby render it negative
and give a false presumptive of sterilization.
Apparently a condition obtains in the test suspension,
wherein, although there is insufficient compound to cause inhibition
when transfer is made to the primary subculture, nevertheless there

has been enough action to result in a slow reduction in the number
of viable organisms*

A degree of scarcity is finally reached

resulting in a primary subculture which is negative at 24 hours but
becomes positive in 48 hours*

At the same time there are initially

so few viable organisms present in the primary subculture the
secondary subculture seeded therefrom failed to receive any viable
organisms at all yielding a bacteriostatic paradox of obtaining a
negative secondary subculture before obtaining a negative primary
subculture.
The reason is that the compound had penetrated the skin in
sufficient concentration to kill as was macros cop ically evident by
the presence of the dye.

Due to the slow penetration of the organisms

many hours longer were necessary to obtain positive evidence (negative
subcultures) of killing.

This will be demonstrated conclusively in

the next section.
The entire phenomenon is illustrated by the subcultures of
brilliant green in the last two columns of Table 19, items 81 hours
and 99 hours respectively.

The primary subculture at 81 hours being

negative at 24 hours and becoming positive at 48 hours indicates two
coincidental effects, (l) probable reduction in the number of vigorous
growing organisms in the test suspension and (2 ) temporary inhibition
in the primary subculture which was overcome within 4o hours.

The

secondary subculture in this instance showed excellent growth in 24
hours.

The fact that there was a faint trace of dye in the primary

-66-

sub culture tube and no visible evidence of dye in the secondary sub­
culture tube indicates that the further dilution hy double subculturing performed its office of eliminating the bacteriostatic action of
the dye.

The subcultures taken at the

9 9 "^

hour of the test demon­

strate the validity of the premise of slow reduction in the number
of organisms*

In this case a

^-8

hour positive primary subculture

and a negative secondary subculture were obtained*

However* at this

time sufficient dye had not penetrated the skin to result in any
color being transferred to the second subculture tube.

This tube

supported good growth upon subsequent seeding with S* aureus* proving
that the negative result could not be due to bacteriostasis.
Through the various tests previously described data have
accumulated which when correlated point to what has been rather
definitely demonstrated in these skin tests, i.e., that in order for
a compound having any antiseptic or germicidal characteristics to
penetrate any living cell it must bring about definite physico­
chemical changes.

These changes, whatever their nature —

coagula­

tion, denaturation, chemical combination with parts of the protein
molecule —

are, one and all, synonymous with death.

This point can­

not be emphasized too heavily when speaking of penetration of
individual cells, especially bacteria.

In the more complex organisms

of the multicellular variety such as the larvae used emphasis on this
point is less important since the functions (protection, food getting
and assimilation, reproduction) of the organism as a whole are

-67-

delegated to various groups of cells rather than all heing contained
in a single cell as in the case of the bacterium*
3Troiii the standpoint of practical surface disinfection,
particularly skin disinfection, it is well to hear in mind the above
premises when speaking of penetration of any epidermal surface.
Penetration of the live skin as such, by any compound that is
definitely germicidal or bacteriostatic is most improbable since as
it goes deeper it must kill each stratum of cells in its path*

Hence

it is difficult for a compound to penetrate deeply into the skin and
underlying tissue because as one layer of cells after another are
penetrated and killed the adjacent tissue juices, lymph and capillary
flow will carry away the compound.

This condition makes it increas­

ingly difficult for the compound to reach a concentration high enough
to conquer the next barrier of cells*

If comparison is made between

the extremely rapid bactericidal agents, iodine and chlorine, on the
one hand, and the slow acting almost strictly bacteriostatic compounds
represented by the bland dye on the other hand, it becomes obvious
that the penetrative powers of these compounds shown by the skin tests,
particularly those shown in table

19

(three hours for the aqueous

iodine; 3/4 to 2 1/2 days for Merthiolate and almost 3 days for
brilliant green) parallel the known relative rapidity of these compounds
when used in practice*

Thus it becomes necessary to admit that any

compound when placed upon any epidermal surface for the purpose of
killing organisms must be deleterious to the surrounding tissue cells

-68-

in order to be of any value against the organisms present.

The only

saving grace is that when effective concentrations of some compounds
are considered there are usable dilutions which will, in a specified
time of contact, be much more destructive to organisms than to
tissue cells.

Admitting that all compounds having any right to bear

the name antiseptic or disinfectant are toxic to tissue cells, our
attention must then be turned to the matter of relative toxicity*
That is, the compound which in a usable concentration combines the
least toxicity to tissue cells with the greatest toxicity to
bacteria (relative penetration) becomes the article of choice.

That

the short-acting aqueous solutions of iodine possess this feature in
a manner superior to all other compounds tested here, is obvious.
This view has been definitely substantiated by the work of Salle and
Lazarus (4o), Nye (3 6 ), Samuels (42) and others.
It is noticeable that little effort has been made to
demonstrate borderline differences in this tissue work.

While

realizing that some of the niceties of technical precision and more
precise analyses of results such as interface phenomena, surface
charge, etc., have been omitted, it was felt that more concrete worth
could be drawn from the work if the cruder approach yielded more
definite, inescapable facts.

Penetration of Bacteria
Speed Test

Upon the basis of the information derived from the foregoing
tests the following simple procedure was devised for determining the
ability of compounds to penetrate bacteria.
The technique employed was as follows;
Materials:

(l) 32 tubes of broth (each containing 9

of

broth) were arranged in pairs, numbered, and l6 sterile petri plates
were labelled to correspond,
(2 )

200

ml. of sterile nutrient agar was melted and cooled

to 45°C,
(3 ) Ample, not less than 30* one ml. sterile tip delivery
pipettes were procured,
(4) 4-99

o f the dilution of compound to be tested was

placed in a sterile one liter Erlenmeyer flask,
(5 ) The flask was warmed to 37°C. and
(6 ) A saline suspension of a 24 hour agar slant culture of
S . aureus was prepared making sufficiently turbid to contain not less
than

1 0 ,0 0 0 , 0 0 0

organisms per ml.

Method:

(l) culture control - 1 ml. of the seeding

suspension was transferred serially through
1

6

broth tubes plating

ml. from the last tube,
(2 )

flask, mixed,

1

ml. of the seeding suspension was placed in the test

-70-

(3) At intervals of 15, 30, 4-5 and 60 seconds,
5* 7* 9* 11* 13* 15*

2 0 , 25

and

30

minutes

1

2

, 3* 4,

ml. was transferred

from the flask to each of the tubes in the first line of broth
tubes,
(4) After the 4th transfer (at

60

seconds) there was

sufficient time, while waiting the next interval, to return to the
15

second subculture and transfer one ml. to the second broth tube

and thence

1

ml. of this mixture to the corresponding petri plate,

(5 ) The succeeding time interval subcultures were
treated likewise,
(6 ) At the first opportunity usually between the fourth
and fifth minute subcultures the previously seeded plates were
poured with

10

ml. of sterile nutrient agar,

(7) Second culture control - after the 30 minute subculture
was completed, Step (l) was repeated to detect any increase in
organisms.
(8 ) At the close of the test all broth tubes and petri-plates
were placed for 48 hours incubation at

3 7 °C.

and,

(9 ) The findings were recorded, positive or negative tubes
at 24 hours and 48 hours and plate counts at 48 hours.
Table 20 is presented as illustrative of the typical record
obtained from the above procedure.

For the sake of brevity this table

is condensed with ten other tables to form Table 21 containing the
pertinent facts.

With the facts gleaned from the work described in

-71-

table

20. - Penetration of Bacteria "by Elemental Iodine
Within Limited Time Intervals

Tincture of Iodine

Number

1 :1 ,0 0 0 , 0 0 0

Subcultures *
Exposure
Time

Culture Control **

Temperature 30°C
Plate Count
at *48 Hours

1 st

2 nd

o

Total Number
of Surviving
Bacteria

70

7 0 .0 0 0 . 0 0 0

1

15

sec*

+

+

12*40

6 2 .0 0 0 . 0 0 0

2

30

sec.

+

+

22*4

11 200,000

3

*45 sec.

+

4*

123

6 ,1 5 0 , 0 0 0

*4

60

sec.

5

2

min.

6

3

min.

7
8

,

1 ,100,000

+

22

+

+

1

+

+

0

0

*4 min.

0

0

5

min.

0

0

9

7

min.

0

0

10

9

min.

0

0

5 0 ,0 0 0

* One ml. subcultures from flask of compound seeded with bacteria.
Subculture made into 9 ml. broth, mixed, and subsequently sub­
cultured. One ml. from each of the second subculture tubes plated
to note decrease in number of organisms. Tubes and plates
incubated at 3 7 °C.» read at *48 hours.
** One ml* of saline seeding suspension of S. aureus transferred
serially through 6 broth tubes plating 1 ml. to obtain number of
bacteria employed in seeding the flask.

- 7&

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-73-

the preceding sections it was decided to employ only iodine,
Merthiolate and "brilliant green for this test.

It was felt that in

the selection of these three compounds the entire range of rapid —
moderate —

extremely slow penetration would be best demonstrated

by iodine, Merthiolate and brilliant green, respectively.
i

The data obtained (Table 20) are in harmony with the
accumulated data of the preceding tests*
tion of

1

to

1 ,0 0 0 , 0 0 0

while a

1

to

1 0 0 ,0 0 0

Elemental iodine in a dilu­

penetrated and killed the bacteria in

dilution sterilized the seeding within

2

30

minutes
seconds.

These results occurred consistently irrespective of whether tincture,
Lugol's or colloidal iodine was the stock solution used.
Merthiolate in a dilution of 1 to 10,000 effected a 75 Per­
cent reduction of the initial seeding within

minutes with little

20

additional effectiveness noted up to 30 minutes.
dilution of

1

to

5 ,0 0 0

was obtained within
in

30

15

a

95

When used in a

percent reduction in the initial seeding

minutes but complete sterility did not result

minutes.
Brilliant green employed in a dilution of 1 to 10,000 was

as efficient as Merthiolate at the same concentration reducing the
initial seeding by 77 percent within 20 minutes.
concentration was cut in half (l to

2 0 ,0 0 0 )

However, when this

this compound failed to

give any reduction in the initial seeding within 30 minutes.

That

the compound was definitely bacteriostatic was proved by the observa­
tion that of the two series of broth tubes used as dilution blanks
in which the concentration of the compound was
2 ,0 0 0 , 0 0 0

1

to

2 0 0 ,0 0 0

and

respectively none of the tubes showed any evidence of

1

to

-71*-

growth even after extending the incubation period from Ug hours to
96

hours.
It was found necessary to use four broth tubes instead of

two

asdilution blanks

for each time interval when the stronger

concentrations of Merthiolate (l to
(l to 10,000) were tested.

5 ,0 0 0 )

and brilliant green

When only two dilution blanks were

employed sterile plates resulted.
These results explain some of the points emphasized in the
preceding sections and

support the contention held that in order for

a given compound to be an effective germicide it must possess the
power to penetrate bacteria rapidly.
It is to be noted that no organic matter was employed in
this test.

Organic matter was omitted because true penetration of

the bacteria was desired without confusing side reactions to consider.
For organic matter to be of any value in this particular test the
organisms would have to be enclosed by the particles i.e., grown
within the particles so that the compound would have to penetrate the
organic coating before reaching the organism.

To employ any

particular organic matter even of uniform size as suggested by
Garrod (19), 1935* would merely serve to introduce another variable
into the test.

Those compounds that penetrate rapidly would enter

organic matter and bacteria alike, being removed from the solution
in the procedure.

Consequently, stronger solutions of such compounds

are required to effect sterilization.

Compounds which penetrate

-75-

bacteria slowly would also penetrate organic matter slowly*

This

virtue of slowness enable weak dilutions of slow penetrating compounds
to do the same total work that requires, comparatively, much stronger
dilutions of the rapid acting compounds to accomplish*

This

comparison of the slow penetrating compound with the rapid penetrating
compound under conditions of organic matter testing would make the
former stand out as the superior bactericidal agent which is the
reverse of the true state of affairs as demonstrated by this work*

-76-

SUMMAHT

The compounds employed in this work are summarized in
Table 22 under each test used according to their relative effective­
ness.

In arranging this table* the factors of temperature, substrate,

concentration, quantity, and time of action were all considered.

The

information embodied in this table may be condensed by arranging
these compounds in the order of their penetrative ability:
1

.

Iodine - Colloidal

2

.

Iodine - Lugol's

3.

Iodine - Tincture

h.

Merthiolate - Aqueous

5*

Merthiolate - Tincture

6

. Phenol

7.

H T H

g.

Brilliant Green

9*

Azochloramid

The phenol coefficients of a group of selected disinfectants
and antiseptics were found.
The mechanism of the agar cup plate method was shown to
demonstrate diffusibility rather than penetrative ability.
Modification of the agar cup plate method was accomplished
and the penetrative powers of the iodines, Merthiolate, brilliant
green, azochloramid and phenol unequivocally established.

1—1

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Merthiolate

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Merthiolate
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Penetration
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i

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** Hot demonstrable by technique employed
*** Tincture, Lugol's, and Colloidal Iodine gave identical

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Iodine
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Iodine
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Iodine
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Iodine
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Penetration
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Merthiolate
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Iodine
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Iodine
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Iodine
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Penetration
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cuticle

*
*
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Iodine***

1

______

Modified agar iPenetration Penetration
of coccicup plate of adult
penetration of chicken
dial
Oocysts
adult chicken ! ceca
i
i
intestine
i
Iodine
Iodine
L -Lugol1s
-colloidal 1

to Their Relative

•
*
*
<D
d
3
O
h-t

!
j Iodine
-colloidal

Iodine
-tincture

Phenol
co-ef­
ficient
Agar cup
plate

GO
ID r-i
•d
rH P
W.
)
rd d
O
i
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Order
of
effec­
tive­
ness.

TABLE 22. - Summary of Compounds Employed According
Effectiveness in Each Test Used.

-77-

©

-78-

The time was determined which is required for the compounds
to penetrate adult chicken coca*
Unquestionable penetration was accomplished in determining
the time required to kill coccidial oocysts by certain concentrations
of the iodines, phenol, H T H, Merthiolate, and azochloramid*
To show actual penetration strongylid larvae were exposed
to various concentrations of the group of compounds, the death of the
larvae indicating penetration of the cuticle and the time required*
To determine the penetrative powers of the various compounds
acting upon skin a series of trials was made on the live and dead skin
of rabbits*

The results show that some of the compounds can penetrate

dead skin; and can penetrate live skin only by destroying the cells as
they go deeper*
In the experiments to determine the actual time required by
selected compounds to penetrate the minute cells of bacteria and
destroy them, it was found that the usual high dilutions of iodine
killed within 30 seconds to 2 minutes*
15

Merthiolate required 20 and

minutes to reduce the total number of viable cells by

percent respectively*
bacteria within

30

and

75

95

Brilliant green would not destroy any of the

minutes when used in a concentration of

1

to

2 0 ,0 0 0

Practical methods have been shown to yield more usable
information concerning the penetrative powers of disinfectants and
antiseptics than the present accepted routine bacteriological tests*

.

-79-

CONCLUSIONS

Penetration of "bacteria "by an antiseptic or disinfectant
is absolutely essential to their ultimate destruction*
Activity, or power to penetrate rapidly, is a prime requisite
of all compounds designed to kill bacteria within a short time interval*
Penetrative power is indispensable to all compounds even
when allowed an extended time interval of action*
Of the selected group of compounds studied, iodine is the
only substance consistently penetrating under all the varied tests
employed*

These results are in harmony with its rapid, irreversible

destruction of bacteria as compared with the slower reversible action
of the organic mercurial and the dye*
Iodine in the true colloidal state possesses inherent
penetrative powers definitely superior to the same element in true
solution.

This greater activity is probably due to an increased

distribution coefficient in favor of the organic test object*
Chlorine penetrates only by oxidation, i.e., first
destroying the cell surface*
All compounds which have the power to penetrate tissue
cells or bacteria in the original unaltered state of the compound
do so only by killing the cell penetrated.
The speed test employed upon bacteria is a true means of
obtaining the penetrative power of disinfectants and antiseptics.

-8 0 -

literatuee cited

1#

Allen, A. W.: A Comparative Study of the Bactericidal Values
of Twenty-one Commonly Used Antiseptics. Arch. Surg.,
1 9 :5 1 2 - 5 1 7 . 1 9 2 9 .

2.

Anderson, L. T., and McClintic, T. B.: A Method for-the
Bacteriological Standardization of Disinfectants. Jour.
Inf. Bis., 8:1-26. 1 9 1 1 .

3*

Bashford, E, P.: Sterilization by Dakin Solution and the
Occurence of Secondary Hemorrhage. Lancet., 2:595-597*
1917.

4.

Becker, E. R.: Coccidia and Coccidiosis of Domesticated,
Game and Laboratory Animals and of Man. Collegiate Press,
Ames, Iowa. p. 15,17* 193^*

5*

Biskind, M. S.: Penetration Through Tissue of Iodine in
Different Solvents. Proc. Soc. Exper. Biol, and Med.,
30:35-37. 1932.

6

.

Ibid.: Effect of Iodine in Different Solvents on Permeability
of Prog Skin to Ringer Solution. Ibid., 30:37-3®* 1932.

7

.

Carnot, P., and Dumont, J.: Technique D'Etude de la Penetration
des Antiseptiques en Milieux Solides. Compt. Rend. Soc.
de Biol., 81:1199-1200. 1918.

8

.

Chambers, R*: Permeability of the Cell: The Surface as
Contrasted with the Interior. Proc. Soc. Exper. Biol,
and Med., 20:72-7**. 1 9 2 2 .

9

.

Chandler, W. L.:
Some Observations on Chlorine as a Disinfectant.
Jour. Am. Vet. Med. Assoc., 35!95“99* 1933*

10.

Chick, H.: An Investigation of the Laws of Disinfection.
Jour. Hyg., 8:92-15®* 190®.

11.

Chick, H., and Martin, C. J.: The Principles Involved in the
Standardization of Disinfectants and the Influence of Organic
Matter upon Germicidal Value. Ibid., 8 :6 5 ^— 6 9 7 * 190S

12.

Claudius, M.: A Method of Sterilization and Sterile Preserva­
tion of Catgut. Hospitalstidende,
Raekke 10:305-310.
1902.

-81-

13*

Committee on Standard Methods of Examining Disinfectants.:
Heport Am. Jour. Pub. Health, 8 :5 0 6 -5 2 3 . 1918.

l4.

Conover, J. E., and Laird, J. L.: Direct Unit Phenol
Coefficient. Am. Jour. Pub. Health, l4:1043-1047. 1924.

15*

Dunn, C. G.: Pungicidal Properties of Sec-amyltricresol,
Orthohydroxyphenylmercuric Chloride and a Mixture of the
Two. Jour. Inf. Dix., 6 1 :3 1 -3 6 . 1937.

l 6 . Etchells, J. L., and Fabian, P. W.: Technic for Skin Irritation
Tests: Technic for Determining Irritating Effect of Chemical
Compounds. Jour. Indus. Hyg., 17:298-300. 1935,
17*

Piessinger, H., and Clogne, R.: Action of Hypochlorites. Revue
de Chirurgie, 36:264. I9I8 . Original not seen. Reviewed in
J.A.M.A. 71:231. 19I 8 .

18.

Pish, P.: The Effect of Physical and Chemical Agents on the
Oocysts of Eimeria tenella. Science, 735 292-293. 1931*

19*

Garrod, L. P.: Testing of Disinfectants in the Presence of
Organic Matter. Jour. Hyg,, 35:219-237. 1935*

20.

Geppert, J.: Zur Lehre von dem Antisepticis. Berl. Klin.
Wchnschr., 2 6 : 7 8 9 and 8 I9 . I8 S9 . Ibid: Zur Desinfectionsfrage. Deutsche. Med. Wchnschr. 17:797»825 and 855* 1891.
Ibid: Die Wirkung des Sublimats anf Milzbrandsporen,
Ibid: 17:1065. 1891. Originals not seen. Cited by
G. Knaysi. I.

21.

Hirschfelder, A. D., Malmgren, G., and Creavy, D.: The
Penetration of Mercurochrome, Acriflavin, and Gentian
Violet into Edematous Tissues. Jour. Pharm. Expt. Ther.,
24:459-464. 1 9 2 5 .

22.

Hirschf elder, A. D., and Wright, H. H.; Studies on the
Colloid Chemistry of Antiseptsis and Chemotherapy. I. The
Mode of Combination of Antiseptic Dyes with Proteins.
Jour. Pharm. Expt. Ther., 38:411-431. 1930*

23 . Jensen, V., and Jensen, E.: Determination of the Phenol
Coefficient of Disinfectants by the Cover-slip Method.
Jour. Hyg., 33:485-484. 1933*
24.

Karns, G. M.: The Behavior of Iodine Solutions at LiquidSolid Interfaces. I. The Wetting Power of Iodine from
Various Antiseptic Solutions. Jour* Am. Pharm. Assn.,
21 :779- 782. 1932.

-82-

25.

Earns, 0. M., Cretcher, L. H., and Beal, a. D. J Ibid.
II* The Importance thereof in the Preparation of New
Iodine Antiseptics. Ihid., 21:783-787. 1 9 3 2 .

26.

Kendall, A. I*, and Edwards, M. R, : A Method for Determin­
ing the Germicidal Value and Penetrating Power of Liquid
Disinfectants. Jour. Inf. Dis., 8:250-257. 1 9 1 1 .

27*

Knaysi, G.: Disinfection. I. The Development of Knowledge
of Disinfection. Jour. Inf. Dis., **7 :2 9 3 -3 0 2 . 1 9 3 0 .

28.

Knaysi, G. , and Gordon, M.: II. The Manner of Death of
Certain Bacteria and Yeasts when Subjected to Milk
Chemical and Physical Agents. Ibid., **7 :3 0 3 -3 1 7 . 1930.

29.

Ibid.: III. The Taking Dp of Iodine by Yeast Cells.
Ibid., **75318-321. 1930.

30.

Knaysi, G.J IV. Do Bacteria Die Logarithmically?
1*7:322-327. 1930.

31.

Ibid.: V. Some Properties of Frequency Curves and Their
Dse in Studies of Disinfection. Ibid., **7:328-333* 1930.

32.

Koch, E.: Deber Desinf ection. Mittheil. a.d. Kaiserl.
Gesundheitsamte., 1:23**. 1881. Original not seen.
Cited by G. Knaysi.I.

33.

Kronig, B., and Paul, T.: Die chemischen Grundlagen der
Lehre von der Giftwirkung und Disinfection. Zeit. f .
Hyg. u. Infektionskrankh., 25:1. 1S97* Original not
seen. Cited by G. Knaysi. I.

3**.

Mallmann, W. L*, and Chandler, W. L.: On the Disinfection
of Avian Fecal Material. Jour. Am. Vet. Med. Assoc.,
3 6 :1 9 0 -1 9 6 . 1933.

e

Ibid.,

35

. Nyiri, W., and Jannitti, M.: About the Fate of Free Iodine
Upon Application to the Unbroken Animal Skin: An Exper­
imental Study. Jour. Pharm. Expt. Ther., **5:85-107. 1932.

36

. Nye, H. N.: The Belative in Vitro Activity of Certain
Antiseptics in Aqueous Solution* Jour. Am. Med. Assoc.,
108:280-287. 1937*

37

. Heddlsh, G*: Examination of Disinfectants.
Health, 1 7 :3 2 0 -3 2 9 . 1927

Am* Jour. Pub.

-83-

38.

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Table of Contents

Introduction
Historical Review

^

Methodology

^

Penetration

^

Statement of the Problem

g

Methods

9

Phenol Coefficient

g

Organisms Employed

10

Compounds Employed

10

Table

1

.

Phenol Coefficients

Agar Cup Plate

l6

Procedure
Table

12

16

Halogens - paraffin covered agar

18

Table 3«

Organisms as indicators

20

Table

Iodine compounds, speed

22

Graph showing relative speed ofiodines

22

Pig.

2

.

1.

Modified Agar Cup Plate - Chicken Intestine
Procedure

23

Table 5»

Zones of inhibition

Penetration of Adult Chicken Ceca
Procedure
Table

23

25
28
28

6

.

Relative effectiveness

30

Table of Contents (cont*d)

Penetration of Microscopic Living Units
Penetration of Coccidial Oocysts
Procedure
Table

7

.

Speed of Iodine, phenol and HTH

Table

8

* Group of compounds employed

Table

9

* Solvent effect upon iodine

Penetration of Strongylid Larval Cuticle
Procedure
Table

10#

Group of compounds employed

Table

11.

Solvent effect upon iodine

Penetration of Rabbit Skin
Procedure
Table 12.

Subcutaneous pocket subcultures

Table

Subcutaneous pocket subcultures

13.

Table l4

Penetration of Dead Skin

Table 15*

Penetration of Dead Skin

Table l6 .

Speed of Penetration of Dead Skin

Table

Comparison of live and Dead Skin

17.

Table 18.

Comparison of Live and Dead Skin

Table

Speed of Penetration of Dead Skin

19.

Penetration of Bacteria - Speed Test
Procedure
Table 20.

Sample Table

Table 21.

Grouped results

Table of Contents (coat'd)

Page
Summary
Table 22,
Conclusions
Literature Cited

Relative effectiveness of compounds
in all tests

77
IS