Examination of Germicides and Antibacterial Agents*

Vol. 35

Examination of Germicides and
Antibacterial Agents*

Laboratory Section

THE Report of the Standard Meth- reports from the several Referees and

ods Committee for the Examina- Associate Referees.
tion of Germicides and Antibacterial STUART MUDD, M.D., Chairman, Uni-
Agents comprises separate progress versity of Pennsylvania, Philadelphia

Chemical Antiseptics

In the preliminary work on the de- peptones produced by the digestion of

velopment of methods for testing anti- casein. Preliminary studies appear to

septics during the past year, two of the indicate that such peptones may help

Associate Referees, G. S3. Slocum and in a solution of the problem concerning

W. A. Randall, have studied a group the development of a medium which is

of media to determine their efficacy in uniform and reproducible.

the in vitro testing of antiseptics. A preliminary method has been de-

Through the courtesy of one of the veloped to determine the efficacy of

peptone manufacturers we were sup- germicides in the destruction of spores

plied with nine batches of peptone, all related to those preparations offered for

of which, from the standpoint of the use for cold sterilization of surgical in-

manufacturer, were as nearly identical struments. One of the Associate

as it was possible to produce. These Referees, Dr. J. D. Brewer, has devised

peptones have been carefully studied to a hinged joint which is similar to the

determine whether, when used in a test joint in scissors. It has a closure of a

medium, they were capable of main- few thousandths of an inch and prob-

taining the resistance of Staphylococcus ably represents the most difficult of all

aureus, and also whether there was any surgical instruments to sterilize. The

variation from one peptone to another. technique of test would be, in general,

Similar studies have been made by the to contaminate this hinged joint with

Referee and Associate Referees on spores in whole blood by allowing the

chemical disinfectants. The results of blood suspension of spores to flow into

the studies indicate definitely that there the joint by capillary action and coagu-

was a marked difference in the ability late. After subjection of the joint to

of these peptones to maintain the re- the chemical agent under test it would

sistance of S. aureus, and that there then be transferred to a tube of testing

was definite variation from one pep- medium such as thioglycollate medium.

tone to another. Work is now under The scissor joint has attached to it a

way in which tests are being made of circular loop to facilitate handling.

* Committee authorized 1941. Published Reports, These surgical joints can be made to
A.J.P.H., May, 1943, and Aug., 1944.
hold to a tolerance of a few thousandths

[839]

840 AMERICAN JOURNAL OF PUBLIC HEALTH Aug., 1945

of an inch and seem to represent a The referees involved in the investi-
reasonably accurate reproduction of the
joints of instruments which are not com- gations dealing with antiseptics and
pletely accessible to scrubbing with a disinfectants have worked closely to-
brush before immersion in a " steri- gether during the past year and be-.
lizing " fluid. cause of their common interests will
continue to do so.

HENRY WELCH, PH.D., Referee

Chemical Disinfectants

Unprecedented advance in the de- tive effort emphasized the need- for a
velopment of germicides has taken place consistent and uniform culture medium
in recent years. The standardized for use in testing germicides. However,
production lots of peptone are likely to
methods which were dependable for
evaluating coal-tar, cresylic and pine vary in their nutritive properties; and
oil types of disinfectants are not ade- in fact, collaborative work in the Anti-
septic and Disinfectant Subcommittees
quate as measures of proficiency for the together with the kind co6peration of
variety of chemical germicides in one of the leading culture media pro-
ducers demonstrated the difficulty of
current use. reproducing this material exactly.

It was pointed out in last year's re- Solution of this problem consequently
port on this subject that one of the lies in the use of a synthetic or semi-
principal difficulties interfering with the synthetic medium or in an available
adaptation of standardized testing meth- stock pile of composite batches of pep-
ods lies in the exaggerated effect that tone sufficiently large to supply germi-
slight differences in the test culture may cidal testing laboratories over a
have in the testing of certain types of substantial period. One of the col-
the newer compounds. One of the laborators of this committee, Dr. D. A.
Wolf, has succeeded in producing a
primary causes of variations in test cul- semi-synthetic medium of considerable
promise for the production of test
tures is the culture medium. During cultures. Also experimental evidence
the year the subcommittee compared the has been accumulated within the com-
phenol coefficient results on five widely mittee that a tryptic digest of casein,
different chemical compounds obtained supplied by Sheffield Farms, a sub-
by using the regulatory F.D.A. medium sidiary of National Dairies, under the
and a promising brand of dehydrated name of N-Z Case, is a satisfactory and
medium developed specifically for dis- consistent medium for the production
infectant testing. The study was sig- of test cultures of both Eberthella
nificant for the wide variation of re- typhosa and Staphylococcus aureus.
sults obtained by the qualified and This constituent produces a satisfac-
experienced operators. As might be tory culture medium without the use
expected, the most uniform figures of beef extract, thereby eliminating an
were obtained with a fortified coal-tar additional source of variation. It is
preparation and sodium ortho phenyl understood that this product can be
phenate while the most diverse resulted produced in sufficiently large quantity
from the tests on a halogenated syn- to allow a satisfactory and uniform
thetic phenol. One operator obtained composite stock supply.
widely different phenol coefficient
figures by culturing the test organisms
in media made from different batches
of regulation peptone. This collabora-

Vol. 35 GERMICIDES 841

Neither of these two media has been other hand, the need for reliable means
subjected to collaborative study by the of comparing potential germicidal
committee, although the casein digest quality is urgent. Present activities of
has been found by one of the commit- this and closely related committees are
tee members to equal the best available being devoted toward refining and
peptone as an ingredient for satisfactory modifying available methods with this
subculture medium. end in view. Sources of variation in a
biological test may be intricate, and
It has become increasingly apparent must be eliminated one by one, conse-
and is now well recognized that no quently spectacular progress in revising
single laboratory test of the coefficient- and developing a standardized reference
drop type can supply adequate informa- test for germicides should not be
tion concerning the proficiency of- a expected.
germicide in practical use. On the
CHARLES M. BREWER, Referee

Antibiotic Agents

The great potentialities of penicillin different substrates, to replace the less potent
strains now in use; several new strains were
for combating a number of infections thus obtained; the yields of penicillin ob-
caused by Gram-positive bacteria and tained from each strain were found to vary
a variety of other diseases, notably depending on the nature of the substrate and
gonorrhea and syphilis, led to tre- the conditions of culture, such as surface
mendous interest in the production and growth vs. submerged and agitated growth.
utilization of this antibiotic substance,
that by far overshadowed the interest 2. Studies of the nutrient requirements and
in all the others combined. The great conditions of culture favorable to the pro-
advantages of penicillin as a chemo- duction of penicillin; whereas at first the
therapeutic agent comprise its activity stationary method was most prevalent,
against aerobic and anaerobic Gram- gradually the growth of the penicillin-
positive bacteria; its relatively low producing strains in deep tanks in a sub-
toxicity to animals; the inability of merged and agitated state began to replace
blood, pus, and other body fluids to the stationary procedure; certain other
prevent its antibacterial action; its methods, such as the use of bran as a
activity upon sulfonamide-resistant medium, the trickling filter, the use of soft
strains of bacteria. However, penicillin agar, as well as the surface liquid culture, are
also has certain limitations, chief of still in use for the growth of the organism
which are, its relative instability against and the production of penicillin.
chemical and biological agents, the
adaptation or increasing resistance of 3. Methods of assaying penicillin, com-
sensitive bacterial strains to its potency, prising the dilution, cup and turbidimetric
and its limited activity against Gram- methods, and a variety of other less common
negative bacteria, fungi, and other procedures such as effect of penicillin upon
disease-producing agents. the production by suitable bacteria of
hemolysin, nitrite and other metabolic
The investigations carried out thus products.
far on penicillin can be divided into
several categories: 4. Production of penicillin by various other
fungi, in addition to above two groups.
1. Isolation and selection of more active
penicillin-producing strains of Penicillium 5. Studies of the chemical and physical
notatum and Penicillium chrysogenum from properties of penicillin.

6. Utilization of penicillin for disease
control.

7. Mode of action of penicillin upon bac-
teria in vitro and in vivo.

In addition to penicillin, other anti-

biotic agents received considerable at-

842 AMERICAN JOURNAL OF PUBLIC HEALTH Aug., 1945

tention. It is sufficient to mention gacin, when freed from a small amount
streptothricin and streptomycin, sub- of accompanying gliotoxin, was found
stances active upon both Gram-positive to be identical with helvolic acid.
and gram-negative bacteria, in contra- Clavacin, also designated as claviformin,
distinction to gramicidin and penicillin patulin and clavatin, depending on the
that are largely active against Gram- organism producing it, is a good illus-
positive bacteria. The production by tration; it has now been crystallized
bacteria of antibiotic agents, aside from and its chemical nature determined. It
tyrothricin, has also received attention. is active against both Gram-positive and
Various fungi were found to produce Gram-negative bacteria, and is fairly
more than one antibiotic substance. toxic to animals. Gliotoxin is produced
by species of Trichoderma, Gliocladium,
This is true of Aspergillus flavus (asper- and A. fumigatus; it is a highly potent
substance and is active both upon fungi
gillic acid and penicillin), A. fumigatus and bacteria.
(spinulosin, fumigatin, fumigacin, glio-
toxin), and others. Some of the anti- SELMAN A. WAKSMAN,
biotic agents are produced by a large .4ssociate Referce
number of different organisms. Fumi-

Disinfection of Air by Germicidal Vapors and Mists

The principal advances in this field controls the vapor output of the glycol
during the past year are the develop- disperser.3 The apparatus consists, in
ment of suitable apparatus for the dis- brief, of a constant beam of light which
persion of glycol vapors and the con- is reflected from the shiny rim of a
struction of a device for the automatic
regulation of the concentration of slowly rotating metal wheel, to a photo-
glycol vapor in the air. Two types of electric cell whose current controls the
glycol vaporizers have been made: one vaporizer. This wheel dips into water
operates on the principle of passing and is cooled by evaporation caused by
heated air over a number of slowly an air current directed against the rim.
rotating metal discs which dip into a Glycol vapor condenses on this cooled
pool of glycol.' The amount of glycol surface in proportion to its concentra-
vapor dispersed is controlled by varying tion in the air. With increasing deposi-
the temperature of the evaporating cur- tion of glycol on the wheel rim, the in-
rent of air. The other type of glycol tensity of the light reflected to the
vaporizer employs the method of boiling photocell is progressively diminished un-
water-glycol mixtures and provides a til it ceases to activate the relay inter-
certain amount of water vapor with the posed between the photocell and the
glycol vapor.2 The rate of glycol vaporizer, and vaporization of glycol
evaporation is controlled by varying the ceases. Conversely as the concentra-
ratio of water to glycol which deter- tion of glycol in the air falls, conden-
mines the boiling point of the mixture. sation on the rim, which is constantly
An automatic device for adding water cleaned by passage through water,
is employed to maintain the boiling diminishes, with the result that the in-
point at the desired temperature. tensity of the reflected light increases
to the point where the relay closes and
Automatic regulation of the concen- the vaporizer is set in operation again.
tration of glycol vapor in the air has Actual test of this glycostat under prac-
been achieved by means of an instru- tical conditions has shown that it is
ment designated as a glycostat which possible to maintain constantly a bac-

Vol. 35 GERMICIDES 843

tericidal concentration of triethylene the air of wards housing patients suf-
glycol vapor in the air below the fering from upper respiratory infections
saturation point, i.e., without the due to this agent showed that the
occurrence of a mist. glycol produced a reduction of ap-
proximately 65 per cent in the number
Since last year's summary of the sub- of air-borne streptococci. However,
ject in this section, there have been when the vapor was employed in con-
several reports on the effectiveness of junction with oil treatment of bed-
glycol vapors in controlling respiratory clothing and floors the reductions
disease and air-borne bacteria. Exten- exceeded 90 per cent.6 7
sion of the study carried on in the
wards of a children's hospital 4 revealed REFERENCES
a continued marked reduction in the
incidence of acute respiratory diseases 1. Coey, S. C., and Spiselman, J. W. To be
in the glycol treated wards. Observa- published.
tions on the bacterial population of the
air in glycol treated and untreated 2. Jennings, B. H., Bigg, E., and Olson, F. C. W.
Heat. Pip. & Air Cond., ASHVE Journal Section,
wards have shown a pronounced diminu- 16:538-545 (Sept.), 1944.

tion of air-borne bacteria in the 3. Puck, T. T., Wise, H., and Robertson, 0. H.
I. Exper. Med., 80:377, 1944.
former spaces.4' 5 A study of the effect
4. Harris, T. N., and Stokes, J. Am. J. M. Sc.,
of triethylene glycol vapor on a single 206:631, 1943.
respiratory pathogen, namely beta
5. Personnel of Navy Medical Research Unit No. 1,
hemolytic streptococcus group A which U. S. M. Bull., 42:1288, 1944.

was present in considerable numbers in 6. Hamburger. M., Jr., Puck, T. T., and Robert-
son, 0. H. J. Infect. Dis, 76:208, 1945.

7. Puck, T. T., Hamburger, M., Jr., Robertson,
0. H., and Hurst, V. J. Infect. Dis., 76:216, 1945.

0. H. ROBERTSON, M.D.
Associate Referee

Disinfection of Air by Ultra-violet Irradiation

In an important investigation, Lurie this virus is about twice as resistant
of the Phipps Institute' reported on as Escherichia coli to ultra-violet
" The Prevention of Natural Air-borne radiation.2
Contagion of Tuberculosis in Rabbits
by Ultraviolet Irradiation." This A number of studies on the control
paper establishes the " air-borne route " of air-borne contagion in day schools
for the transmission of tuberculosis in are in progress; however, no further
rabbits. Striking results in regard to publications on the results of these
control of tuberculosis by means of studies have been published.
ultra-violet were obtained. The prac-
tical application of these findings in The difficulty in obtaining reliable
the prevention of air-borne transmission epidemiology data on the control of
of tuberculosis in man will have to over- air-borne contagion has been empha-
come certain technical. difficulties. Not sized by a study of the National Insti-
the least is the fact that man can tute of Health on the use of ultra-violet
safely be exposed to only very little
radiation of 2,537A. upper air irradiation of sleeping
quarters in a boys' training school.3
In a comparative study of the This study which gave interesting in-
sensitivity of viruses and bacteria to formation on other points gave little
monochromatic ultra-violet radiation it information on the value of ultra-violet
was reported that influenza virus has in the control of upper respiratory in-
fection because the incidence of illness
a maximum of sensitivity at 2,650A during the period of study was very
low.
very similar to bacteria, and that
The use of ultra-violet radiation in

844 AMERICAN JOURNAL OF PUBLIC HEALTH Aug., 1945

the control of air-borne contagion in also been presented by Lurie' in his
crowded military sleeping quarters was study on tuberculosis..
tested by using upper air as well as
floor irradiation in an extensive study REFERENCES
at a naval training station.4 The re-
duction of upper respiratory infection 1. Lurie, Max B. .. Exper. Med., 79:559-573,
was about 25 per cent during a season 1944.
of relatively low incidence of illness.
It was brought out in this study that 2. Hollaender, A., and Oliphant, J. W. I. Bact.
the intensity of ultra-violet radiation (in press).
is of greatest importance for the control
of cross-infection. Such evidence has 3. Schneiter, R., Hollaender, A., Caminita, B. H.,
Kolb, R. W., Fraser, H. F., DuBuy, H. G.. Neal,
P. A., and Rosenblum, H. B. Am. J. Hyg. (in
press).

4. Wheeler, Lt. S. M., Ingraham, Lt. H. S.,
Hollaender, A., Lill, N. D., Gershon-Cohen, Jr., and
Brown, E. W. A.J.P.H., 35, 5:457 (May), 1945.

ALEXANDER HOLLAENDER, PH.D.

Associate Referee

Fungicidal and Fungistatic Agents

Proposed Method for Testing Fungicides against Trichophyton

The recently increased demand for The number of conidia in the
fungicides to be used in treatment and inoculum (2,500,000 per 5 ml. of fungi-'
in combating the spread of dermato- cide dilution) is not intended as a safety
factor to compensate for organic matter
phytosis has emphasized the immediate or other protective factors 'to be met
need for a standard method of testing in practical application, but was
fungicides. The following method is adopted to minimize false negatives re-
offered to meet this urgent need. sulting from the possibility of picking

The method is strictly an in vitro up loops of material without organisms
test and is not intended to provide in the course of conducting the test. It
criteria for judging proficiency in prac- is essential that enough conidia be in
tical use. However, it would seem that the inoculum to permit the making of
subtransfers. Otherwise failure to dif-
conicentrations of preparations unable ferentiate between fungicidal and fungi-
static action may completely invalidate
to destroy the organism under condi-
tions of the test would not be suitable conclusions. The method of detecting
as fungicidal agents for general use on fungistatic action by subtransfers is ad-
inanimate objects and surfaces such as
mittedly imperfect. Dilution of the
floors contaminated with pathogenic fungicide sufficient to prevent fungi-
fungi. Products intended for thera- static action, accomplished by trans-
peutic use and showing by this test a ferring a loopful of the fungicide-
suitable fungicidal activity must be ex- conidia mixture into a suitably large
amined separately for toxicity and volume of broth, becomes impractical
irritating properties, ability to pene- in cases where perhaps 1,000 ml. would
trate epidermis, and other characteristics be required. Therefore the operator
important in clinical use. must learn by trial within the procedure
as outlined, and to some extent for each
The method is applicable to water fungistatic agent tested, a compromise
soluble fungicides. Water immiscible ma- whereby a fair sample of spores is
terials may be tested, but unless stable carried to the subtransfer without
transferring at the same time enough
dispersions are obtained, reliable checks of the fungistatic agent to inhibit
growth in the subtransfer tube.
cannot be expected. It may be de-
sirable to adapt this method to testing
fungicides not soluble in water by using
some other diluent. Preliminary studies
indicate that this may be possible.

Vol. 35 VGERMICIDES 845

The use of a phenol control is speci- 2-5° C. At intervals not to exceed 3
fied solely for the purpose of providing months it shall be transferred to fresh
a standard check upon the resistance agar slants which shall be incubated at
of the conidial suspension used in the a temperature between 25° and 300 C.
test. It is generally recognized that it for 10 days and then placed in storage
is improper to compare the action of at 20-50 C. until the next transfer
phenol with unrelated fungicidal and period.' No culture which has been
bactericidal agents, and it is not in- kept at room temperature or higher for
tended that in this method the final more than 10 days should be used as
results be expressed in terms of phenol a source of inoculum for culture pur-
coefficients. poses. Cultures may be kept at room
temperature for preservation of the
TEST ORGANISM strain and for the inoculation of cul-
tures if transferred at intervals not
The test fungus shall be a typical exceeding 10 days.
strain of Trichophyton mentagrophytes
(T. interdigitale, T. gypseum) isolated PREPARATION OF THE CONIDIAL
from dermatophytosis of the foot. The
strain shall sporulate freely on artificial SUSPENSION
media. The presence of abundant
conidia is manifested by a powdery ap- Petri dish * agar cultures shall be
pearance on the surface of a 10 day prepared by planting the inoculum at
culture, particularly at the top of an the center of the agar plate and incu-
agar slant, and can be confirmed by bating the cultures at a temperature
microscopic examination. The conidia-
bearing mycelium should peel easily between 250 and 300 C. for 10 days
from the surface of dextrose agar.
Conidia of the required resistance shall and not exceeding 14 days. The
survive a 10 minute exposure at 200 C. mycelial mat shall then be removed
to a phenol dilution of 1:60, but not to from the agar surface to a flask con-
one of 1:45.* taining 20-25 ml. physiological salt
solution (0.85 per cent NaCl) per plate
CULTURE MEDIA by means of a sterile spatula or heavy
flattened wire. The conidia are freed
The fungus shall be carried on agar from the mycelium by shaking the
slants of the following composition: mixture gently with or without glass
C.P. dextrose 2 per cent, Neopeptone 1 beads, and the suspension is filtered so
per cent, agar 2 per cent, adjusted to that hyphal elements are removed while
pH 5.6-5.8. The same culture medium conidia pass through. Sterile ab-
shall be used in preparing cultures for sorbent cotton in a sterile funnel is
obtaining a conidial suspension, and a suitable. This should yield 10-25
fluid medium of the same nutrient com- million conidia per ml. The density
position (without agar) shall be used to of the conidial suspension is estimated
test the viability of conidia after by counting in a hemocytometer and
exposure to the fungicide. diluted with physiological salt solution
so that it contains 5 million conidia
per ml.

CARE OF THE FUNGUS STRAIN OPERATING TECHNIQUE

A stock culture of the fungus on Dilutions of the fungicide are pre-

dextrose agar slants shall be stored at pared. Methods of making dilutions

* Bottles 11 cm. high, 4.5 x 6 cm. in cross-section,

A* suitable strain (No. 9533) has been de- and with neck 3.5 cm. in diameter can be substituted
posited with the American Type Culture Collection. for Petri dishes.

846 AMERICAN JOURNAL OF PUBLIC HEALTH Aug., 1945

as outlined in U.S.D.A. Circular 198 for may be run at other time intervals.
determination of phenol coefficients are After 5, 10, and 15 minute periods of
suggested and the test procedures are exposure to the fungicide, a sample is
similar to the procedures outlined removed from each conidia-fungicide
therein. Five ml. portions of each mixture with a 4 mm. loop and placed
fungicide solution and the phenol con- in 10 ml. of the dextrose broth men-
trol solutions are placed in 25 x 150 tioned above. To eliminate the risk of
mm. culture tubes and arranged in faulty results due to the possibility of
order of ascending dilutions. These fungistatic action, subtransfers must be
are placed in a 200 C. water bath until made. This may be done by with-
the temperature of the water bath is drawing a loopful of the conidia-fungi-
reached. With a graduated pipette 0.5 cide mnixture, touching it to the surface
ml. of the spore suspension is placed in a tube of dextrose broth as described
in the first tube of the fungicidal solu- above,,and immediately immersing it in
tion, the tube shaken, and immediately a second tube of broth. The inocu-
replaced in the water bath. Thirty lated tubes are incubated at 25°-300 C.
seconds later 0.5 ml. of the conidial Final results are read after 10 days,
suspension is added to the second tube. although an indicative reading can be
This procedure is repeated at 30 second made in 4 days.

intervals for each of the fungicidal C. W. EMMONS, PH.D.,
dilutions. If more convenient the test
Associate Referee

Prepare for Winter Now!

The Solid Fuels Administration for or insist on getting more than your fair share.
War asks the JOURNAL to request pub- Take your dealer's advice on your coal
lic health workers, particularly health problems.
officers and health educators, to inform
the public of the tight fuel situation 2. " Check up on all heating equipment,
which the population will face this whether fired by oil, coal, or gas, to insure
winter. peak efficiency. Clean your furnace and
install controls or other heat-saving devices
All fuel will be scarce next winter, where available. Learn how to get the full
the Solid Fuels Administration declares. amount of heat from the fuel you use.
The need for conservation is urgent and
people should be asked to prepare for 3. " Protect your home against loss of heat
winter now by doing the following by installing insulation, storm windows, and
things: weather-stripping. The work can be financed
with convenient monthly payments suited to
1. "Store now whatever kind and quan- your income on the FHA plan. 'Heat-
tity of fuel your dealer can let you have. If sealing' pays big dividends by cutting your
you burn coal or coke, don't wait for some fuel bills."
preferred kind that may never be available,
Detailed information on saving fuel
by heat-sealing houses may be obtained
from the Solid Fuels Administration for
War, Washington 25, D. C.