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A Comparative Study of Various Digestive Agents Used
In the Isolation of mrcobacterimn Tuberculong From

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presented by

Joseph Wilson Whalen

has been accepted towards fulfillment
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Master of Meme—degree mhflefldogy

Ma] professor

Date July 16. 1951

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A COMPARATIVE STUDY OF VARIOUS DIGESTION AGENTS
USED IN THE ISOLATION OF MYCOBACTERIUM
TUBERCULOSIS FROM SPUTA

 

BY

Joseph Wilson Whalen

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

1951

33145.5. '3’

ACKNOWLEDGMENT

The author wishes to express his appreciation to
Dr. N. L. Mallmann whose guidance, suggestions, and
criticisms contributed extensively toward organization
of the experimental studies; and to Dr. A. S. Kimball,
Mr. D. A. Grover, and Dr. S. A. Yannitelli who pro-

vided laboratory facilities and materials.

7'. {‘27 M“
f3l~gO-‘- 7 I E}

TABLE OR CONTENTS

Introduction

I The Necessity of Cultural Examination for
a. tuberculosis in Diagnostic Bacteriology

P-'

 

The Evolution of Inhibitory Agents in Ciltured
fledia and Digestion Techniques for the Iso-
lation of iv tuberculosis--Their Inadequacies

 

I Advantages and Inadequacies of Contemporary
Digestants
Experimental
I Use and Comparative Study of Sodium Hydroxide
and Other Chemical Agents as Digestants of
Sputa
A. Materials and Methods
B. Results
II Determination of the Relative Efficiency of
Trisodium Phosphate Digestion as Contrasted
with Other Agents
A. Materials and hethods

B. Results

III Utilization of Trisodium Phosphate in Culture
of Routine Specimens

IV Study of Fungicidal Agents
A. Materials and Methods
B. Results

V Determination of the Relative Toxicity of
Dowicides For Er tuberculosis

 

A. haterials and hethods

B. Results

Page

25

26

29

52
52
51+

56
59
no

41

Ml
LLl
[+2

VI

VII

VIII

IX

Comparative Studies of Various Formulated
Digestion Rixtures

A. Materials and hethods
B. Results

Use of Formulated Digestion Mixtures in
Routine Culture of E.tuberculosis

 

Preparation and Study of Digestion kixtures
Containing "Dowicide A"

A. study of Fungicidal Activity--
Materials and kethods

B. Study of Fungicidal Activity
Results

C. Study of Digestion Mixtures Contain-'
ing "Dowicide A" in Cultivation of
E. tuberculosis from Sanatorbnn
Sputa-- Aaterials and hethods

 

D. Results
Formulation of Digestion Mixtures Contain-
ing Terramycin and Determination of their
Relative Efficiency

A. haterials and methods

B. Results
Comparison of Efficiency in Routine
Hospital Work of Terramycin Digestion
Mixtures with those Containing Penicillin

A. Materials and Methods

B. Results

Discussion

A?
ME
AS

A?
A9
A9

51

52
55

55

57
58

60
60
61

65

Swnma ry

Appendix

Literature Cited

75

lllllllllllllllll IIIV‘IIIIIIlII

Introduction

The clidcal manifestations of infection with

pgycobacterium tuberculosis have been described in vague

 

fashion since the advent of recorded history. Such
references to it may be noted in the Bible and other
literature of even greater antiquity. Mummies unearthed
in the ruins of ancient Egypt have presented patholog-
ical evidence of the ravages of the disease during the
period of that nation's zenith of power. The Middle
Ages saw many weird remedies advanced for the treatment
of "consumption", practically all of which were detri-

mental to the patient's chances of recovery.

However, despite the pronounced antiquity of tuber-
culosis, it was not until the relatively modern time of
1819 that Laennsc (49) described the clinical aspects of
the infection in humans. Villemin, in 1865 (87), pro-
duced tuberculosis in the rabbit by means of injection of
tissue from human tuberculous lungs and thus demonstrated
not only the probable presence of an undiscovered causa-
tive agent, but also its apparent communicability.

The actual discovery of the etiological agent was
made by Koch (46) in 1882, who also proved by a series
of experiments that it gained entry to the body by means

of the naso-respiratory tract. He further demonstrated

(1)

the extreme refractibility of the organism to ordinary
staining techniques and to the then known methods of
cultivation. Ehrlich (27) in 1882, was the first to

note the unique acid-fast character of g. tuberculosis.

 

His observations led him to propose a staining method
of identification which was subsequently modified by

Ziehl (93) and later by Neelsen (63).

This acid-fast property is also common to the other

members of the genus Mycobacterium, which includes many

 

other species, the majority of which are non-pathogenic
soil organisms. Some authorities have expressed the be-
lief that at an early period of animal inhabitancy of the
earth, one or several of these saprOphytes gained entry
into the body of hosts and through mutation and adapta-
tion became pathogenic. Jensen (43) presents an ex-
cellent review of the indications in favor of such adjust-
ment by these bacteria. Their function in soil is some-
what superfluous economically since the best known forms
seem to be primarily concerned with.decomposition of
hydrocarbons, a process, which for the most part, is not
important in ordinary agricultural soils.

I The Necessity of Culural Examination for y.

tuberculosis in Diagnostic Bacteriology

In the immediate period following the acceptance of

the tubercle bacillus as the causative agent of tubercu-

(2)

losis, medical science was to a great extent dependent
upon microscopic examination of sputa or other pathologi-
cal body fluids for the presence of this organism as a
means of diagnosis. The adaptation of chest x-ray films
in the years following 1907 proved to be a great aid to
detecting the insidious presence of this invader before
the disease had progressed to the exuditive stage, and
thus made it possible to arrest it at a much earlier

time when cure was relatively simpler (4).

However, it has been repeatedly observed that in
numerous instances the radiologist has been unable to
detect tuberculous infection long after the bacilli are
present in the sputa (59). Also there are many cases
wherein the questionable evidence of disease is present,
but apparently not to the point of cavitation, when
actually such lesions have been formed but are masked by

other anatomical structures.

Therefore, it is still essential that the radiographic
diagnosis be supplemented by several laboratory examin-
ations. Likewise in the control and arresting of tuber-
culosis, these laboratory findings prove valuable in
determining the lessening or increasing of the organism's

ascendancy over bodily defenses.

Microscopic examinations of stained smears of sputa

or other fluids during_such periods of the disease are

(3)

laborious and require the expenditure of a great deal of
time in order to be reliable. Smears must be carefully
made, properly stained, and observed through the micro-
scope for a considerable length of time. Various concen-
tration methods have proved to be an improvement over the
direct smear technique, but nevertheless a large amount of

sputa must often be studied to find a single acid-fast rod.

There has been in the past a great deal of controversy
as to the actual location of the majority of tubercle bac-
illi in sputa. It is the contention of some investigators
that the direct smear will often detect these organisms
present in the surface film while the sediment, upon con-
centration, will prove to be devoid of them. Because of
this, concentration methods have been developed which are
designed to recover all the material present in the sur-
face film as well as the sediment, through use of flota-

tion techniques.

In many instances the Sputum specimen itself will
prove to be extremely mucoid, thus making it much more
difficult to prepare representative smears. Some chem-
icals used for digestion of sputa have been shown to
exert a detrimental effect on the acid-fast nature of
the tubercle bacillus, while other agents act to pre-
cipitate certain constituents, consequently decreasing

the accuracy of the slide examination.

(4)

During periods of hemoptysis the presence of E.

tuberculosis will often be masked by the predominance of

 

erythrocytes in the sputum smears, making it impossible
to determine the extent of infection.

Another obstacle far too prevalent in clinical lab-
oratory examinations for tubercle bacilli is improper
staining technique. Precipitated stain or improper de-
colorization can often lead to many errors of varied
nature.

In the actual microscopic examination of smears,
several pitfalls exist. The presence of artifacts may
mislead the inexperienced laboratorian into mistaking
them for tubercle bacilli. Likewise scratches in old
slides will often simulate acid-fast rods due to the
failure of acid alcohol to penetrate into their recesses
and remove the retained stain.(73). Also it is possible
for tubercle bacilli to be rubbed off highly positive
slides and become lodged within the film of oil covering
the immersion objective unless care is taken to wipe the
latter following examination of each slide. Spores, many
of which are acid-fast, will sometimes appear remarkably
like a rod when they are grouped together in the presence
of other organisms and cellular debris. Even the most
experienced observer may sometimes mistake species of
mycobacteria for the tubercle bacillus, since they often

bear a very close morphological resemblance. Special

(5)

staining techniques for differentiation cannot be relied

upon to rule out these saprophytes in most instances.

Finally there exists the ever-present factor of chance
in slide examination. When but 1 or 2 acid-fast rods are
present in a smear, it is very improbable the microscopist
will discover them unless he happens, by chance, to observe
the fields in which they are present. Corper has estimat-
ed that there must be approximately 100,000 organisms per
ml. of sputum present before the probability of finding
them in smears becomes significantly large (11). To some
extent this is very likely due to loss of acid-fastness
of the organisms present as the result of physical trauma,

dissociation and/or senility (92).

Inoculation of guinea pigs with pathological material
is advantageous in diagnosis of tuberculosis since it offers
a means of determining the pathogenicity and virulence of
the organism, as well as indicating the presence of tuber-
cle bacilli when they occur in numbers so small that it is

almost impossible to detect them microscopically.

However, this method is slow. Most laboratories allow
a period of 4 to a weeks before killing one of these ani-

mals for histological studies.

(6)

Ddsadvantageous also is the expenditure of the time
and labor inoculating, grossly examining for lesions, per-
forming tuberculin tests, and preparing tissue sections

for histological examination.

The guinea pig is unfortunately extremely susceptible
to respiratory and intestinal epidemics. One such infec—
tion will often wipe out an entire colony in a matter of

hours, thus destroying valuable clinical data.

Therefore, for the reasons listed above as well as
several others, it is evident that neither the microscopic
slide examination nor the guinea pig inoculation is very
satisfactory when used alone as a means of detecting M.
tuberculosis in pathological body fluids. The advantages
of a satisfactory cultural method are numerous: Differen-

tiation of the tubercle bacillus from other Mycobacteria

 

may be made through colony morphology in many instances.
Non-staining bacilli will frequently proliferate upon a
suitable medium, and specimens containing but one or two
of these organisms may give positive cultures. Although
they may be masked by vast amounts of blood, growth will
be initiated. A reliable medium.which.would give rapid
cultural results would also reduce the time expended in
slide examination. The errors inherent in the latter
technique would be eliminated and the factor of chance

reduced. Likewise, a great deal of time would be conserved

('7)

if the routine inoculation of guinea pigs could be

rendered unnecessary in this fashion.

In an endeavor to achieve more satisfactory cultur-

al techniques for the isolation of E. tuberculosis, the

 

writer has studied and extensively modified media and
digestive agents which may be used in the cultivation of
this microorganism. This paper is concerned with one
phase of this work--the experimental investigation of
means of removing or retarding contaminants which.over-
grow culture media or otherwise restrict the value of

cultural methods.

(8)

lilll-Illlll.|ll lll'TllTlI‘lllll liliv

 

The Evolution of Inhibitory Agents in Cultured
Media and Digestion Techniques for the
Isolation of M. tuberculosis--
their Inadequacies

 

The cultural methods and media in use at present fall
short of the ideal goals as presented. Tubercle bacilli
have been recognized as being difficult to cultivate since
their discovery and original cultivation by Koch. Inso-
1ation and identification of this organism were hindered
until quite recently by the inability of investigators to

develop media which would grow it more rapidly.

For his first isolation Koch utilized bovine blood
serum, coagulated by inspissation. He found that it re-
quired 4 to 8 weeks for colony formation upon this medium.
He solved the problem of dehydration because of this pro-
longed incubation by sealing the cotton plugs of the
tubed slants with paraffin. For removal of other organ-
isms present in sputum specimens, this pioneer recommend-
ed concentration and digestion with 4 percent sodium
hydroxide, since he had observed that tubercle bacilli
appeared to tolerate quite alkaline environments if they
were not exposed for a prolonged period. Twelve to 24
tubes were inoculated to insure obtaining slants which
were not contaminated or overgrown with saprophytes

present in the specimens.

Immediately following Koch's successful cultivation

(9)

of these organisms, other investigators attacked the
problem of isolating tubercle bacilli from sputa while
still retarding the growth of contaminants. Nocard and
Roux (64), Smith (78), Raskin (75), Proskauer and Beck
(74), Wurtz (90), Dorset (21), Phisalix (71), Lubenau
(54), and Hesse (36), all subsequently formulated cultur-

al media for the cultivation of g. tuberculosis. Although

 

this organism could be successfully cultivated on all of
these media, some difficulty was experienced in primary
isolation from sputa even when 4 percent sodium hydroxide
was used as an inhibitory agent for saprophytic bacteria.
In spite of this treatment, contaminants often overgrew
the medium, or, in other instances, microscopically posi-
tive sputa failed to yield positive cultures. It was be-
lieved by many investigators that sufficient exposure to
the harsh alkaline effects of sodium hydroxide to destroy
the saprophytes often detrimentally affected the tubercle
bacillus to the extent that it was unable to resume multi-
plication in the artificial environment of the medium (7).

The introduction of "Antiformin" by Uhlenhuth and co-
workers (86, 28) represented a substantial improvement in
the search for an agent which would digest the mucoid por-
tions of sputa and also act to inhibit the growth of un-
wanted organisms. A commercial product of varying compo-

sition consisting for the most part of sodium hypochlorite,

(10)

"Antiformin" breaks down the undesired tissue and cellular
debris as well as destroying the majority of bacteria, but
does not disturb too seriously the morphology of acid-fast
organisms. Thus it was frequently used exclusively in di-
gestion and concentration of sputum specimens for micro-
scopic examination without an attempt at cultivation of
tubercle bacilli which may have been present in the speci-
mens being made. In such instances it was highly satis-
factory, compared to the relatively slow digestion of
extraneous material encountered with the 4 percent sodium

hydroxide.

Griffith (4), experimenting with "Antiformin", found
a time period at which uncontaminated cultures of tubercle
bacilli could be obtained without destroying all the viable
tubercle bacilli. Studies by Patterson (8) and by Brown
and Smith (9) indicated that it was considerably less
toxic to the tubercle bacillus than the high alkalinity
of sodium hydroxide. It was recognized, however, that
this agent still left much to be desired when utilized
in cultural work. Smith (80) demonstrated that it re-
quired at least 1500 times as many tubercle bacilli to
seed the then known artificial media after these organisms
had been exposed to antiformin as it did to infect a

guinea pig with untreated bacilli.

The literature indicates that much of the sodium

hypochlorite contained in commercial "Antiformin" is

(11)

susceptible to deterioration. Thus the solution may loss
over 50 per cent of its activity within 1 year (84).
”Antiformin" can, however, be tested for activity by a
simple method which yields a close approximation both of
the amount of sodium hypochlorite and of available chlo-
rine (6). In routine cultural examinations by digestion
and concentration methods where specific amounts of "Anti-
formin" should be employed, frequent standardization of

this reagent is imperative.

Several investigators, aware of these limitations of
antiformin, added various so-called stabilizing agents (10),
and experimented with shorter exposure times of sputa to
this digesting agent, only to find that these measures
resulted in a marked increase of contaminated culture (8).
Thus it was apparent that some other means of inhibition

of saphrophytes was urgently needed.

At least a partial answer to the problem was provided
by Petroff's introduction of a medium containing a 1 to
10,000 concentration of gentian violet, in addition to
glycerol, beef, and egg (70). This marked the first pub-
lished utilization of a dye in a medium devoted to the
isolation of tubercle bacilli from pathological material.
Studies conducted by Petroff and his associates (71, 90),

and by Corper (13), revealed that a shorter exposure time

(12)

 

 

 

IIIII‘ITII Illlll|l I ll

to antiformin was now possible if the resulting digested
material were planted on this medium. Other digesting
agents of a less toxic nature could now be feasibly utiliz—

ed with less chance of overgrown cultures.

The extraordinary resistance of acid-fast bacteria in
general to gentian and crystal violet had been investigat-
ed by Churchman (57). These dyes inhibit the growth of
Gram-pesitive bacteria, but, although mycobacteria retain
the Gram stain, most strains will tolerate relatively
large concentrations before their growth is seriously

retarded.

Following the successful utilization of the Petroff
medium, several workers reported formulation of new cultur-
al media containing gentian violet as an inhibitory agent
(11, 55, 17, so, 5, 85).

Corper reported that higher percentages of positive
cultures had been obtained using a medium.which actually
consisted of Petroff's enriched with peptone. It was sub-
sequently found by Twort (83) to be less toxic to tubercle
bacilli than was that of Petroff. He attributed this to
the richer content of nitrogenous nutrients available to
the organism which might act to partially overcome the
toxicity of the gentian violet. In the light of recent
discoveries by Dubos and Davis (23) and by Youmans (93) of
the protective effect of bovine serum albumin against agents

which might be toxic to the tubercle bacillus, it is prob-

(15)

able that the added peptone modified the detrimental
effects of this dye on all bacteria cultured with the

medium.

Meanwhile Petroff (71) and Koch (47) had studied the
effect of various other concentrations of sodium hydroxide

as digesting agents.

Numerous other agents for sputum digestion were re-
ported subsequently; including the digestive enzymes (30),
pyridine (31), potassium hydroxide (20), sodium carbonate
at moderately elevated temperatures (34), ammonium hydrox-
ide (8), 3 per cent hydrochloric acid (52), 6 per cent sul-
furic acid (52), sodium hydroxide-alum mixture (35), and
even acetic acid and distilled water. In some instances
it was recommended that digestion be followed by precipi-
tation or flocculation with aluminum, zinc, or iron salts.
Light benzene or gasoline products were used after hypo-
chlorite digestion to suspend tubercle bacilli in an inter-
mediate surface layer. Likewise chloroform (51), which
settles to the bottom, was used in an attempt to obtain
zonal separation. A few of these techniques remain in use
at the present time, but the majority were discarded due
to their complexity or because they were found unsatis-
factory either from the standpoint of toxicity to the tu-
bercle bacillus or since they failed to remove a large

enough.amount of contaminants.

(14)

Possibly, also, these methods failed to win wide-
spread acceptance due to the success of Lowenstein's gen-
tian violet medium (53) which was also introduced during
this period (1924). When used in conjunction with 3 per
cent hydrochloric acid or 4 per cent sodium hydroxide,
Lowenstein found that this medium gave a considerably
higher percentage of positive cultures and shortened the

period of colony development for many strains of bacilli.

Shortly thereafter, Petragnani (69) reported regarding
a glycerol-egg medium which contained malachite green as
an inhibitory agent. This dye has been demonstrated to be
considerably less toxic to M. tuberculosis than gentian
violet, yet it also possesses a strong inhibitory effect
on the majority of Gram-positive organisms. It was some
years following the publication of Petragani's paper before
his medium became widely known in this country, but at the
present time it, along with Lowenstein's (Jensen's Modifi-
cation), is probably the mostpopular of those utilized by
clinical laboratories. These are the only media, out of

20 studied, recommended by the American Trudeau Society.

Subsequently, Corper and Uryei in a series of papers
(l4, 13, 12, 15) studied the effect of numerous chemical
agents on tubercle bacilli and reported that 5 per cent
oxalic acid appeared to slow the growth of this micro-
organism the least. In their procedure a volume of the

oxalic acid reagent equal to that of the sputum specimen

(15)

(or sediment resulting from centrifugation) is added to
the latter and the resulting mixture is incubated at 37°C
for 30 minutes before inoculation of Corper's egg-yolk
medium. Van Vranken (89) reported that this technique
proved superior to treatment with oxalic acid and culture
on Petragnani's medium, or to treatment with many of the
previously discussed agents and subsequent inoculation

of Corper's and Petragnani's medium. Since the Corper
medium contains no dye, the accelerated and more luxur-
ient growth observed was attributed to lack of dye tox-
icity. Other reports have attested the value of oxalic
acid in sputum digestion, not only from the standpoint of
saprophytic inhibition but also by virtue of its rapid

action in dissolving mucus.

However, as with various concentrations of other
acids and with sodium hydroxide and "Antiformin", it is
necessary to limit the period of contact of oxalic acid
with a specimen. Van Vranken found that if the prepara-
tion incubates over two hours, the specimen is lost, since
all tubercle bacilli are usually either destroyed or in-
hibited. This, obviously, is an extreme disadvantage,
particularly in public health work where only one sputum

per patient is customarily available.

I

In 1939, "Tergitol" (sodium octyl sulphate) an organic
wetting agent, was introduced by Petroff and Schain (73).

It was claimed to possess a more rapid and complete action

(16)

than any other digestant previously utilized. The pro-
cedure, however, rapidly underwent changes as it was
studied by other investigators (66). One part of "Tergi-
tol penetrant 08", 1 part of 4 per cent sodium hydroxide
and 1 part of water was used for a time, but was subse-
quently replaced by equal portions of "Tergitol 08" and
Javelle water. Since the latter solution contains sodium
hypochlorite, it was unstable and required_frequent pre-
paration. Actually no controlled data have been presented
to substantiate the value of "Tergitol" in sodium hypo-
chlorite mixtures (42). Gradually its use in digestion
reagents has diminished, so that at present its place in
clinical laboratories is largely as a means of intensify-
ing the Ziehl-Neelsen staining of smears without use of

heat.

Another commercial alkaline sodium hypochlorite
solution, "Clorox", was studied by several workers
(63, 18, 83). In this concentration-digestion technique,
5 m1. of "Chlorox" (active ingredient 5.25 per cent sodium
hypochlorite) is added to each of 5-50 m1. screw-capped
tubes containing 5 m. of a single sputum specimen and the
tube capped and shaken for 5 minutes. It was found that
this reagent is satisfactory only for microscopic exam-
ination of concentrate smears, since it was shown to be
highly toxic, relatively, to the tubercle bacillus.

”Clorox" successfully eliminates such extraneous material

(17)

as: blood cells, fibrous tissue, and other bacteria

which might intensify the difficulty of detecting acid-
fast bacilli microscopically. If it is allowed to act

on a sputum specimen longer than 2 minutes, attempted
acid-fast staining of any tubercle bacilli present usually
fails (66). It has been claimed that 15 per cent more
positive results were obtained with this reagent than by

the smear method (66).

Meanwhile, in an effort to produce an effective di-
gesting agent which kills or inhibits contaminating mice-
organisms with relatively little effect on the acid-fast
bacilli, Corper and Stoner (16) reported great success
with trisodium phosphate (Na5.P04.12H20). These investi—
gators stated that a 23 per cent solution added in equal
volume to sputum specimens could remain in contact for as
long as 7 days at room temperature without perceivable
harm to the viability of any tubercle bacilli present,
and this amount and time, in most cases, destroyed all
potential contaminants contained in the specimens. Thus
the danger of loss of individual specimens is eliminated,
since sufficient time is allowed to render unnecessary
the usual cautious watch period necessitated by all re-
agents previously described where neutralization must be

performed within 1/2 to 2 hours at least.

Corper and Stoner recommended that the equal volume

of 23 per cent trisodium phosphate solution and the sputum

(ls)

specimen be incubated for 24 hours at 37°C. before centri-
fugation of the mixture and inoculation of a culture medium
with the sediment. Van Vranken (89) found this technique
to be superior to those previously described for the dos-
truction of contaminants, while yielding relatively high
percentage of positive cultures. Tarshus and Lewis (83),
who added a period of agitation of the reagent—sputum mix-
ture before incubation, also reported a greater number of
positive cultures using this technique, and confirmed
Corper's claim that neutralization of the digested speci-

men is unnecessary before seeding cultures.

However, subsequent papers by other workers have ex-
pressed varying opinions in regard to the value of the
trisodium phosphate technique. Mitchell and Jeffries (60),
following Corper's suggestion, placed the reagent in the
receptacles used for collecting the pathological specimens.
They reported that this prevents further development of
molds and contaminants, so that when the specimen was de-
livered to the laboratory a day of incubation at 37°C.
destroyed the remaining viable contaminating microorgan-
isms. This method was also used by workers at the Florida
State Health Department Laboratory (44), where it was found
that the extra day of incubation following receipt of the
specimen was usually unnecessary, and that a greater number
of positive cultures were obtained than with 4 per cent

sodium hydroxide digestion.

(19)

In a study of the most commonly used digestive
agents, Spendlove, Cummings, and Patnode (78) concluded
that sodium hydroxide and trisodium phosphate are most

suitable.

0n the other hand, Beattie (7) stated that the latter
reagent may adversely affect growth of tubercle bacilli.
Mullahy (62) observed in a study with trisodium phosphate
that nearly twice the number of contaminated cultures
resulted with this method than was obtained with the 4
per cent sodium hydroxide solution, but reported that the
former is superior in securing positive results by smear
from concentrated specimens. Hunter (40) also observed
greater contamination with trisodium phosphate digestion
than with sodium hydroxide, but to a much lesser extent.
In addition, he found that the former reagent yielded a

significantly larger number of positive cultures.

Kurung (48) reported that trisodium phosphate, along
with the majority of other digestion reagents, lacked the
ability to inhibit most varieties of fungi common to the
respiratory system and oral cavities of many long-term

chronic tuberculosis patients.

These and further studies of digestion-concentration
solutions were stimulated by the promise of more rapid
cultivation of M. tuberculosis offered through the announce-
ment by Dubos of a liquid synthetic medium containing a

polyoxyalkylene derivative of sorbitan monostearate

(20)

(Tween 60") in which he obtained rapid and submerged

growth of the H37Rv strain of this organism. Subsequent-
ly Dubos and Davis (22) described a liquid medium-includ-
ing a polyoxyethylene ester of oleic acid ("Tween 80") as
wetting agent, and bovine serum albumin , fraction V as
enrichment and protective agent against substances toxic

to the tubercle bacillus. Foley (29) reported that use

of this medium in diagnostic work had made possible iso-
lation of human strains from pathological material, follow-
ing 4 per cent sodium hydroxide treatment, in an average of
6 days, with 88 per cent correlation with guinea pig in-

oculation.

Dubos and Davis (24), however, after using it to cultivate
tubercle bacilli from sodium hydroxide-treated sputa, cau-
tioned against use of the medium in routine diagnostic work,
due to frequent contamination.

Dubos and Middlebrook (23) introduced a solid form of
this medium containing an oleic acid-albumin complex which

eliminated the toxicity of "Tween 80". Although growth of

 

‘M. tuberculosis on this medium was somewhat slower than
with the liquid form, Goldie (32) reported successful iso-
lation of this organism from sputa treated with ammonium
carbonate and penicillin. Middlebrook (59) obtained micro-
scopic growth on the solid medium, using penicillin as an
inhibitory ingredient, in an average of 13 days. Subse-
quently he recommended only the solid Dubos medium for

diagnostic work.

(21)

Mollov, Hill, and Oshinsky, (61) successfully util-
ized the liquid form to isolate tubercle bacilli from
pathological material. They treated sputa with wanm 4 per
cent sodium hydroxide (temperature not specified) in quan-
tities equal to the volume of each specimen, and agitated
the resulting mixture in a shaking machine for 10 minutes
before concentration by centrifugation. Fifty units of
penicillin was added to each 10 ml. of the medium as a
further protection against contamination. These workers
reported some difficulty in determination of growth in the
medium in the presence of cloudy or granular inoculum, and
thus recommended its use primarily in laboratories where
only a few cultures for tubercle bacilli are made, since,
frequent smears of the liquid are essential to confirm

multiplication of these microorganisms.

Numerous reports on the growth of mycobacteria in the
presence of penicillin (3, 76, 81) have stimulated wide
utilization of it as a selective inhibitory agent in vari-
ous culture media. Abbott (1) found it effective in this
respect when incorporated into Lowenstein-Jensen medium in
a concentration of 100 units per ml. He reported a marked
reduction in the total number of contaminated cultures by
alpha, beta, and non-hemolytic streptococci, as well as by
Gram-positive bacilli. Sula (82) claimed penicillin to be

superior to malachite green as an inhibitor of contamination

(22)

in liquid media due to less toxicity for the tubercle
bacillus. Unger and Muggleton (87) observed that peni-
cillin in low concentrations stimulates the growth of this
organism. Pramer and Heukelekian (74) added 5 units of
penicillin per ml. of solidified Dubos medium in a study

of survival of tubercle bacilli following sewage treatment.

However, McCulloch (57) had noted that the presence of
wetting agents in a liquid suspension of tubercle bacilli
caused these organisms to be much.more susceptible to vari-
ous disinfectants. Youmans and Ybumans (93) subsequently
warned against the use of antibiotics in culture media con-
taining the wetting agent, "Tween 80", since the dispersive
effect of the latter permits a more intimate contact be-
tween the antibiotic and the surface of the bacterial cell.
Other investigators concurred with this observation (2, 41,

19). It was claimed that partial lysis of M. tuberculosis

 

Occurs in Dubos medium in the presence of high concentra-

tions of penicillin (45).

I Advantages and Inadequacies of Contemporary
Digestants

Thus, the advantages and inadequacies of current con-
centration-digestion techniques and inhibitory agents can

be summarized as follows:

1. Various concentrations of sodium hydroxide are

(23)

relatively rapid in their digestive action. However, the
period of activity must be carefully controlled, since the
high alkalinity of sodium hydroxide will detrimentally

affect the tubercle bacillus.

2. Antiformin is rapid and efficient in destroying
not only contaminants, but also extraneous material
present in the sputum specimen. It has been shown to be

more toxic to M. tuberculosis than sodium hydroxide. It

 

deteriorates rapidly, and thus must be standardized fre-

qlently.

3. Crystal violet added to a medium in suitable con-
centrations, exhibits a selective bacteriostasis of nearly
all Gramrpositive bacteria excepting the tubercle bacillus.
It, too, is somewhat toxic to the metabolism of the latter

microorganism.

4. Hydrochloric acid, sulfuric acid, potassium hy-
droxide, and sodium carbonate are rapid and efficient
but they must be precisely timed, and properly neutral-
ized. Their toxicity is also relatively great.

5. Flocculation and emulsifying techniques are
complex and time consuming, and their value has never

been fully substantiated.

6. Malachite green is probably the least toxic to

(24)

tubercle bacilli of the selective dyes, yet it exerts

some inhibitory action upon their multiplication.

7. Oxalic acid in a concentration of 5 per cent
efficiently removes contaminants with little effect on

E. tuberculosis, but its period of action must also be

 

carefully limited and neutralization before culture is

essential.

8. "Tergitol" and "Clorox" are satisfactory only for
concentration preliminary to preparation of smears, since

they are too toxic to permit subsequently reliable cultures.

9. Twenty-three per cent trisodium phosphate may re-
main in contact with the tubercle bacillus for up to seven
days without serious toxicity being manufested. Neutrali-
zation is unnecessary. It may be added to sputum recep-
tacles before use to speed digestion. However it has not
proved very effective in the destruction of saprophytes
when used by several investigators. It does not inhibit
fungi, and one worker has expressed the belief that it

adversely affects multiplication of tubercle bacilli.

10. Penicillin has been shown to be an excellent
inhibitor of contamination when present in mlitable con-
centrations in culture media. Unfortunately, in the
presence of wetting agents which accelerate the growth of
tubercle bacilli through dispersal, it is markedly bac-

teriocidal for this microorganism.

(25‘

Experimental

I Use and Comparative Study of Sodium Hydroxide
and Other Chemical Agents as
Digestants of Sputa
During the course of investigating the possibility of
combining the attributes of Dubos' media (liquid and solid)
with those of a liquid medium developed some years previous-
ly by Mallmann (56), it was noted that the resulting modi-

fications were seriously hampered in the isolation of g.

tuberculosis, when sputa of tuberculosis patients were

 

cultured, by the problem of contamination resulting from
multiplication of the normal oral bacterial flora. It was
therefore concluded that before these media could be util-
ized clinically, it would be necessary either to treat
sputa chemically or to introduce into the medium some in-
hibiting ingredient which would prevent development of

these contaminants.

Since much of this preliminary study was performed in
the laboratories of the Calhoun County Health Department
where 6 per cent sodium.hydroxide was in routine use before
concentrates were inoculated on Petragnani slants, this

technique was adopted.

However, it soon became evident that several factors
were responsible for the wide variation in the growth-times

of different strains of tubercle bacilli isolated from Sputa.

(26)

Thus an attempt was made to standardize all conditions
under which the experimental work was done, including the

digestion-concentration process.

Further, it was believed that the relatively long
growth period of freshly isolated tubercle bacilli on ex-
perimental media compared with.that of small inocula of
stock strains was due to toxicity of the sodium hydroxide
digestant. Therefore a comparative study of sodium hy-
droxide in concentrations of 6 and 4 per cent, 5 per cent
oxalic acid, "Antiformin", and 5 per cent potassium hy-

droxide was made.
A. Materials and Methods

The technique followed at the Calhoun: County Health
Department was to permit spontaneous digestion of the sputum
specimen (customarily a seven-day pool) for from 1 to 8
days. A direct smear was made in the meantime and examined.
Then the sputum was centrifuged at 2500 r.p.m. for 10 min-
utes to obtain a concentrated sediment, and smears for
microscopic examination were prepared from it. The remain-
ing sediment was then diluted with an equal volume of 6
per cent sodium hydroxide, and the mixture stirred. At
intervals of 10, 20, and 30 minutes after unis dilution, a
tube of medium was inoculated with 6 loopfuls of the

mixture.

(27)

A similar procedure was followed with 4 per cent sodium
hydroxide, excepting that the intervals of treatment prior
to inoculation of the medium were extended to 20, 30, and

50 minutes.

with "Antiformin", approximately 20 m1. of the sputum
was mixed with 15 ml. of this reagent and 65 m1 of distill-
ed water.. The resulting mixture was incubated at room
temperature for one hour, following which it was centri-
fuged at 2500 r.p.m. for 10 minutes. The sediment was
washed 3 times with.physiological saline. A portion of it
was then used for preparation of smears, following which
6 loopfuls of the remainder were used to seed each tube

of cultural medium.

Oxalic acid in a concentration of 5 per cent was added
in equal volume to the sputum specimen and the container
was incubated at 37°C. for 30 minutes. The liquid was then
diluted with 10 ml; of 0.01 N sodium hydroxide and centri-
fuged. The sediment was used for preparation of smears,
then 6 loopfuls of it were introduced into each tube of

medium.

The 5 per cent sodium hydroxide reagent was utilized
in exactly the same fashion as was the 6 per cent sodium

hydroxide.

Twenty sputum specimens were obtained from patients

(28)

at the Arthur S. Kimball Sanatorium and pooled. The
homogeneous pooled specimen was then divided into 20
equal portions. Patients were selected whose sputa had
been negative culturally for at least 6 months, and whose
radiographic history indicated that they were nearly
arrested cases. Each of the 20 resulting specimens were
seeded with an approximately uniform amount of tubercle
bacilli from 1 pure culture and then divided into 5 equal

portions for treatment with the various reagents.

All cultures were incubated at 37°C. for a total of
6 weeks. The media used in this comparative study were
Petragnani's, and liquid and solid experimental prepar-
ations, media I, II, and III, respectively (appendix).
Three tubes of each medium were inoculated from each
sputum specimen. The cultures were examined micro-
scopically frequently, and if growth was detected smears
were prepared of the colonies and stained with the Ziehl-

Neelsen technique for microscopic examination.
‘B. Results

It was found that 6 per cent sodium hydroxide treat-
ed specimens were largely free of contamination when cul-
tured. In the few instances in which contaminants were
found, the microorganisms responsible were found to be
molds or extremely active proteolytic-type bacteria which

partially liquified the medium.

(29)

The potassium hydroxide treatment yielded a slightly
greater degree of saprophytic growth, as did 4 per cent
sodium hydroxide digestion. The contaminants present were

widely varied in Species and nature.

On the other hand, the oxalic acid reagent permitted
frequent saprophytic overgrowth of the liquid and solid
experimental media which, unlike Petragnani's contained no
selective inhibitary ingredients. In these instances it

was impossible to detect the presence of E. tuberculosis

 

by smear from the cultures.

"Antiformin", however, proved to be the most efficient
in destroying contaminants. Only 4 tubes of media yielded

saprophytic organisms, three of these being liquid media.

From the standpoint of recovery of E. tuberculosis, it.

 

was found that 4 per cent sodium hydroxide treatment gave
the largest number of positive cultures, with 5 per cent
potassium hydroxide digestion yielding a slightly smaller
number. The 6 per cent sodium hydroxide reagent was next
in efficiency in this respect, with 5 per cent oxalic acid
treatment producing a relatively poor yield of tubercle
bacilli, probably due to the excessive saprophytic growth

encountered.

The "Antiformin" technique, however, apparently des-
troyed most of the viable tubercle bacilli along with the

contaminants, since only 8 tubes of this liquid medium

(30)

contained these microorganisms at the conclusion of the

6 weeks incubation period.

An analysis of the relative time required for develop-
ment of macrOSCOpic colonies on the various media utilized
reveals that most rapid growth was obtained with oxalic
acid digestion. Potassium hydroxide treatment was second
in this respect, with 4 per cent sodium hydroxide, 6 per
cent sodium hydroxide, and "Antiformin" third, fourth,
and last, respectively. The latter reagent was extremely
poor from this standpoint, as 58 days were required for
colony development in the liquid medium, compared to 29
days for 6 per cent sodium hydroxide and 14 days for

oxalic acid digestion.

Detailed results of this study are presented in

Table l.

(51)

II Determination of the Relative Efficiency of
Trisodium Phosphate Digestion as Contrasted
with Other Agents
Van Vranken's report regarding the superiority of
25 per cent trisodium phosphate over any of the other
digestive agents which she studied, served to vastly in-
crease its popularity and bring it to the attention of
many investigators. Prior to this time, use of it as a
digestant was almost unknown in hospital and public
health laboratories, although Corper's introduction of

it predated Van Vranken's report by nearly two years.

Since sodium hydroxide, as well as the other reagents
previously studied had proved far from ideal, a compara-
tive survey of the efficiency of trisodium phOSphate, to
4 to 6 per cent sodium hydroxide, 5 per cent oxalic acid,

and 5 per cent potassium hydroxide was undertaken.
A. Materials and Methods

Trisodium phosphate (Na5P04.l2 H20) was prepared in
a concentration of 25 per cent by weight (equivalent to
10 per cent anhydrous salt). One portion of this solution
was utilized without further alteration as a digesting
agent, while to another portion, 5 per cent by weight of
"Triton X-lOO"(an alkylated aryl-poly—ether alcohol of
high.molecular weight which is a non-ionic surface active
agent, compatible with alkaline conditions-~manufactured

by Rohm and Haas, Philadelphia, Pa.) was added. The pur-

(32)

pose of this addition was to endeavor to more rapidly
eliminate contaminants in sputa sediments by reducing the
thickness of the mucoid film found in many specimens which

envelopes and serves to protect these undesirable bacteria.

It was realized that prolonged exposure of the tuber-
cle bacillus to a powerful wetting agent such as "Triton
x-loo" would be detrimental, as indicated by the work of
Youmans and Youmans which was previously reviewed. How-
ever, by conducting a timed study of the digestion process,
it was hoped that an average period of time could be ascer-
tained during which destruction of saprophytes would at
least partially disperse the latter bacteria so that more
rapid growth would result upon inoculation of media with

the digested sediment.

The 25 per cent trisodium phosphate and the tri-
sodium phosphate--"Triton X—lOO" reagents were utilized in
identical fashion: Equal volumes of the sputum sediment

and the digestant were mixed and incubated at 57°C.

Treatment of sputum specimens with 4 and 6 per cent
sodium hydroxide, 5 per cent oxalic acid, and 5 per cent
potassium hydroxide was performed in the same manner as

in the previous experimental study.

At intervals of 0.16, 0.34, 0.50, 1.5, 2.5, 6.0, 12,

24, and 26 hours following the start of the respective

(55)

treatments, one tube each of Petragnani's, and the liquid
and solid experimental media were inoculated with 6 loop-

fuls of the digesting sediment.

Seeded, pooled, and subsequently divided Sputum
specimens were again utilized as in the previous experi—

mental study of digestive agents.

All cultures were incubated at 57°C. The media were
contained in screw-capped culture tubes. Cultures were
examined frequently for macroscopic evidence of growth.
All apparent colonies were checked microscopically. Tubes
.of liquid medium were slanted during incubation, since
previous cultural studies had indicated that more rapid
and luxuriant growth is obtained under this condition.
These tubes were also shaken frequently to aerate the

medilm.
B. Results

Twenty-three per cent trisodium phosphate treatment
partially destroyed saprophytes in 24 hours, as evidenced
by uncontaminated tubes of solid experimental and Petrag—
nani media which were inoculated after this period of
digestion. However, the liquid medium planted at this
time was contaminated. Twenty-six hour treatment appar-
ently destroyed all contaminants, since all 5 media pro-

duced pure cultures of E. tuberculosis.

 

(54)

The trisodium phosphate--"Triton X-lOO" reagent
eliminated saprophytes sufficiently in 6 hours at 57°C.
to produce uncontaminated Petragnani and solid media.
Complete inhibition was not recorded until after 12 hours

treatment.

Sodium hydroxide in a concentration of 6 per cent
partially removed contaminants in 0,50 hours and complete-
ly in 1.5 hours. The 4 per cent concentration of this re-
agent completely destroyed extraneous bacteria in 1.5

hours, with no partial destruction evidenced.

Oxalic acid treatment brought about partial in 1.5
and complete destruction in 2.5 hours, while 5 per cent
potassium hydroxide produced the same effects in 2.5 and

6.0 hours respectively.

Trisodium phosphate reagent treatment for 24 hours
yielded growth of tubercle bacilli in the liquid medium
in 12 days, trisodium phosphate phosphate--"Triton X-lOO"
for 6 hours in ll days, 6 per cent sodium hydroxide for
0.5 hours in 20, 4 per cent sodium hydroxide for 1.5 hours
in 16, oxalic acid for 1.5 hours in 18, and potassium hy-

droxide for 2.5 hours produce growth in 18 days.

A complete summary of the findings of this study is

to be found in Table 2.

(55)

III Utilization of Trisodium Phosphate in Culture
of Routine Sputum Specimens

For a period of 4 months, all routine sputum speci-
mens from patients at the A. S. Kimball Sanatorium, as
well as those coming into the laboratory at that institu-
tion from out-patients, was divided into 2 portions. One
of these was digested with 25 per cent trisodium phosphate
for 24 hours, while the other was treated with the tri-
sodium phosphate--"Triton X-lOO" reagent for 12 hours at
this temperature. Total of 545 specimens were handled
in this manner. All were cultured on Petragnani and the
experimental solid media (5 tubes of each for each speci-
men). Of these, 126 were positive for tubercle bacilli
following trisodium phosphate digestion, while 144 were

positive after the combination reagent treatment.

In relation to amount of contamination, it was noted
at the conclusion of the period that neither digestant
proved effective in inhibiting the growth of certain fungi
which are present either in a parasitic or saprophtic
state in the oral and respiratory tracts of many tubercu-
lous individuals. Some of these contaminants may also
possibly enter the sputum specimens at intervals when the
patient has removed the top of the container. Since the
above sputa were, for the most part, collected over a

period of seven days, these molds had ample Opportunity

(56)

to enter in this fashion and even, in some instances, to

multiply within the specimen.

Twenty-three per cent trisodium phosphate with 0.1
per cent by weight "Tween 80" added was also used experi-
mentally as a digestant during this period. A comparative
study with the trisodium phosphate--"Triton X-lOO" reagent
indicated, however, that a greater toxicity to the tubercle
bacillus was occasioned, since a much lower number of cul-

tures of sputa with the former were positive.

Slower and more sparse growth of positive cultures
was also noted. At the same time, contamination by vari—
ous microorganisms was greater, particularly on the solid

experimental medium.

Experiments were conducted also in the addition of
various reagents in 5 ml. quantities to sputum jars be-
fore submitting them to sanatorium patients for collection
of specimens. Twenty-three per cent trisodium phOSphate,
trisodium phosphate--"Triton X-lOO", and trisodium phos-

phate—-"Tween 80" were utilized in this fashion.

Upon receipt of the sputum specimens in the labora-
tory, they were immediately placed in a 57°C. incubator.
Jars containing trisodium phosphate were incubated for 6
hours, trisodium phosphate-~"Triton X-lOO" for 4, and tri-

sodium phosphate--"Tween 80" for 8 hours.

(:57)

It was found that the trisodium phosphate—-"Triton
X-lOO" digestant procedure was very successful in digest-
ing the mucoid substance of the sputa and destroying con-
taminants when incubated for 4 hours. A longer incubation
time, however, was responsible for several negative cultures
from specimens which were positive microscopically by con-
centrate smear. Likewise sputa which were not brought
immediately to the laboratory upon completion of the seven
day collection period, also yielded similar falsely nega-
tive cultural results. A more rapid rate of growth and'
more luxuriant colonies were obtained from specimens of
patients who customarily raised sputa of a mucoid character,

but not necessarily more highly positive.

On the other hand, no advantage to this addition was
observed in the case of 25 per cent trisodium phosphate,
contrary to claims of Corper, Van Vranken, and others.

In fact, in most instances the 6 hour digestion period
proved to be inadequate, so it was Subsequently deemed
advisable to incubate these jars for 12 hours, and even
following this interval of treatment, considerable con-

tamination of cultures was encountered.

With the trisodium phosphate--"Tween 80" reagent
this technique was completely unsuccessful. The majority
of cultures were contaminated, and many were falsely

negative.

(58)

All three reagents permitted the multiplication of
fungus contaminants upon the culture media, despite vari-
ation of incubation periods. However, if the trisodium
phosphate--"Triton X-lOO" jars were incubated sufficiently

long to render all subsequent cultures for E. tuberculosis

 

negative, fungi, for the most part, were destroyed.

Due to much confusion on the part of patients in re-
gard to the liquid present in the specimen containers,
and the added hazard of more frequently spilled npecimens
resulting from the increased volume, this method of digest-

ing the specimen while collecting, was abandoned.

Sputa containing tubercle bacilli were heated to 56°C.
for one-half hour in an effort to destroy all contaminants.
It was found, however, that many strains of staphylococci
and molds are resistant to this treatment. Likewise, heat
treatment in conjunction with chemical digestion was found
to be unreliable, as the wide variation in character of
individual specimens apparently made it impossible to de-
vise a uniform technique which would destroy extraneous
microorganisms without seriously harming the tubercle

bacillus.
IV Study of Fungicidal Agents

In an endeavor to incorporate into the trisodum

phosphate--Triton X-lOO" digestion mixture an agent which

(39)

would prove non-toxic to M. tuberculosis, but would elimin-

 

ate, largely, the problem of fungus contaminants, a study
was made of the relative efficiency of fungicides and

mycostats.
A. Materials and Methods

Cultures of nine species of fungi were obtained (from
Dr. A. S, Kelner of the Westover Field Regional Station
Hospital, Mass.). Following consideration of various
chemical agents, "Dowicide A" (orthophenylphenol, sodium
salt), "Dowicide B" (2,4,5-trichlorophenol, sodium salt)
and"£bwicide F" (2,5,4,6-tetrachlorophenol, sodium salt)
were added, in concentrations of 0.001, 0.002, 0.005, 0.004,
0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 per cent by weight
to approximately uniform suspensions of each of the fungi

in 10 ml. of physiological saline solution.

The mixtures were incubated at 57°C. for 12 hours,
then centrifuged for 10 minutes at 2500 r.p.m. The super-
natant fluid was decanted and the sediment washed 5 times
with physiological saline, after which 2 Sabouraud dextrose
agar slants (Difco) were inoculated from each centrifuge

tube, 6 loopfuls being utilized as the standard inoculum.

The Sabouraud slants were incubated at 57°C. for a
total period of 12 days. At the end of this time, presence

or absence of typical colonial morphology was recorded.

(40)

B. Results

All three agents were found to be fungicidal in rela-
tively low concentrations (Table 5). "Dowicide F" appear-
ed to be the most effective against the common fungus
residents of sputa with "B" next in efficiency, and "A"

least.

Since these apparently efficient concentrations were
sufficiently low to be satisfactorily utilized in digestion
mixtures, efforts were subsequently made to develop suit-
able solutions in which they could be used in a study of
their toxicity for the tubercle bacillus. In the meantime
they were individually tested for toxicity to this micro-

-organism.

V Determination of the Relative Toxicity of
Dowicides for _I‘v_I. tuberculosis

 

A. Materials and Methods

Fourteen saline suspensions of an approximately uni-
form amount of tubercle bacilli obtained from the same
pure culture, were treated respectively, with the percent-
ages listed in Table 4 of "Dowicides" "A", "B", and "F".
The method utilized was identical to that employed in the
study of these agents against fungi as given above, except-
ing that 1 tube of Petragnani and 1 tube each of experi-

mental solid and liquid media were used for culturing the

sediment in each centrifuge tube. Cultures were observed

(41)

daily. All colonies which developed on the media were

smeared, stained, and examined microscopically.

B. Results

It was found that "Dowicide A" permitted growth of

M. tuberculosis in eleven days on Petragnani's medium in

 

concentrations up to and including 0.05 per cent, while
0.005 per cent "Dowicide B" was sufficient to prevent
colony development by this organism, as was also the case

with ”Dowicide F".

Table 4 records the relative degrees of inhibition of
each agent, and the variation in rate of growth which re—
sulted, both phases being influenced by the medium upon
which the sediment was cultured. "Dowicide A" allowed
multiplication of tubercle bacilli on the liquid medium
through all percentages tested, the highest being 0.15.
"Dowicide B" yielded results with the liquid medium
identical with.those obtained with Petragnani's. "Dowi-

cide C" permitted growth of E. tuberculosis up to a con-

 

centration of 0.01 per cent in the case of the liquid

medium.

Since 0.05 per cent "Dowicide A" failed to inhibit
this organism on any of the media utilized, and this con-
centration had previously been demonstrated to be fungi-
cidal for all Species of molds studied, it was selected

as the proper proportion of the agent suitable for use in

(42)

a digestion mixture.

Moreover, the tubercle bacilli treated with the 0.05
per cent concentration compared very favorably in growth
time with those which were untreated. These times were
identical for Petragnani's and the liquid medium, and

differed by only 5 days with the solid experimental medium.

The 0.001 per cent concentration which permitted

growth of E. tuberculosis with "Dowicides" B and F was

 

shown by the previous study to be insufficient to inhibit
the majority of the fungi tested. Therefore these com-
pounds were not added to various digestion mixtures for
further study, as was "Dowicide A".

VI Comparative Studies of Various Formulated
Digestion Mixtures

A. Materials and Methods

The search for more rapid and efficient sputum digest-
ants was continued through the preparation and testing of
several solutions, some of which had previously been util-

ized, and others which were new mixtures.

The agents tested were: 25 per cent trisodium phos-
phate, 25 per cent trisodium phosphate with 5 per cent
"Triton X-lOO", 25 per cent trisodium phosphate with l per
cent "Triton X-lOO", 25 per cent trisodium phOSphate with

0.1 per cent "Triton X—lOO", 6 per cent sodium hydroxide,

(4:5)

4 per cent sodium hydroxide, 5 per cent oxalic acid, 7 per
cent ammonium carbonate with 10 units of penicillin-G per
ml., 17 per cent sodium tripolyphosphate with 5 per cent
"Triton X-IOO", 25 per cent sodium phrophosphate with 5

per cent "Triton X-lOO", 10 per cent trisodium phosphate
with 1 per cent "Triton X-lOO", 7 per cent trisodium phos-
phate with 1 per cent "Triton X-lOO", 10 per cent trisodium
phosphate with l per cent "Triton X-lOO", and 25 units of
penicillin per ml., 7 per cent trisodium phOSphate with 1
per cent "Triton X-100" and 25 units of penicillin per ml.

Twenty culturally negative sputum specimens were
obtained and pooled. This large multiple specimen was
then inoculated with a phosphate buffer solution contain-
ing dispersed tubercle bacilli. To achieve this, these
organisms were removed in the colonial state from the same
pure culture and gently ground with a mortar and pestle in
the presence of 0.001 per cent "Triton X-lOO". When a
watery, apparently homogeneous paste was achieved, it was
transferred to 100 ml. of the phosphate buffer solution.
During 2 days observation at room temperature, only a
small fraction of these organisms settled t0 the bottom of

the buffer container.

Following the addition of approximately 50 ml. of this
buffer suspension to the pooled specimen, the latter was agi-

tated vigorously for 2 minutes and then divided into 14

approximately equal portions.

(44)

Direct smears from each portion were examined micro-
scopically and it was found that the number of tubercle
bacilli in each of the seeded specimens corresponded rough-

ly to IV on the Gaffkey scale.

Each of these was then transferred to a centrifuge tube
and centrifuged at 2500 r.p.m. for ten minutes. The super-
natant liquid was decanted, and an approximately equal vol-
ume of each of the reagents was added-~l reagent to l spec-

ific sediment.

During treatment of the sediment, the individual tubes

were incubated at 57°C. Two tubes of the cultural medium
were incubated at the end of the following periods: 0.16,
0.54, 0.50, 1.5, 2.5, 6.0, 12, 24, and 26 hours following

addition of the respective reagents.

Medium III was utilized for cultivation. It was incu-
bated at 57°C., and examined frequently for macroscopic

evidence of growth through a period of 42 days.
B. Results

The findings for 25 per cent trisodium phosphate,
tri-sodium phosphate-~5 per cent "Triton X-lOO", 4 and 6
per cent sodium hydroxide, and 5 per cent oxalic acid
were very similar to results obtained with these agents
in the previous study recorded in Table 2. Comparison of

the latter data with Table 5, which presents the findings

(45)

of the work under discussion, shows that the inhibitory
effects for saprophytes and relative toxicity for g.
tuberculosis proved to be almost identical. Since 5 per
cent potassium hydroxide was not used in the present study,
comparison cannot be made with the previous findings for

this reagent.

Twenty-three per cent trisodium phosphate--l per
cent "Triton X-100" proved to be as effective in all res-
pects as 25 per cent trisodium phosphate-~5 per cent "Tri-
ton X-lOO", while 25 per cent trisodium phOSphate--0.l per
cent "Triton X-100" appeared nearly as efficient. Since
24 hour digestion effected complete inhibition of sapro-
phytes with the latter reagent, it was considered to be
very promising as a digestion mixture ingredient, as it
had proved to be less toxic to the tubercle bacillus,

probably due to the lower content of wetting agent.

The ammonium carbonate--penicillin reagent was as
effective in removing contaminants as 25 per cent trisod-
ium phosphate with 5 and l per cent "Triton X-lOO". How-
ever it increased the incubation time of E. tuberculosis
by 2 to 5 days, thus indicating a greater toxicity for

this microorganism.

The sodium tripolyphosphate and sodium pyrophOSphate
reagents proved ineffective as digestants in this study.

Since the seeded sputa utilized were considered to be

(46)

representative of the characteristics of the great majority
of specimens encountered in routine cultural work, it was
concluded that these two chemicals are unsatisfactory as
digestion agent components. However, a further trial was

planned, using routine sputa from sanatorium patients.

Ten per cent trisodium phosphate with 1 per cent
"Triton X-100" and 7 per cent trisodium phosphate plus 1
per cent "Triton X-100" gave identical results in time
required for colony development, and in speed of removal
of saprophytes. In the latter respect, they were less
effective by 2 hours, than 25 per cent trisodium phosphate
with this concentration of the wetting agent. However,

they proved to be less toxic to tubercle bacilli.

The 2 trisodium phosphate mixtures containing peni-
cillin were almost as rapid in their removal of contamin-
ants as were the 2 concentrations of sodium hydroxide.

Toxicity to E. tuberculosis was at a minimum, thus indi-

 

cating that these agents were promising as digestion mix-

ture ingredients.

VII Use of Formulated Digestion Mixtures in
Routine Culture of M. tuberculosis

The same digestive agents utilized in the comparative
study of speed and efficiency of digestion of seeded sputa
(section VI) were used to treat 100 specimens from sanatorium

patients. These sputum specimens were not selected on the‘

(47)

basis of presence or absence of tubercle bacilli, but
instead were those received in the laboratory in the course
of several weeks. This afforded a better opportunity to
study the effect of the agents under routine hOSpital

laboratory conditions.

Each of the 100 specimens (7 day pools) was divided
into 14 approximately equal portions. Each portion was
then treated with l of the digestion solutions, using the
individual techniques previously described for each reagent.
Three tubes of medium II were then inoculated from each
digested portion. These cultures were incubated at 57°C.
with frequent subsequent examination for macroscopic growth.
The number of days which elapsed before the appearance of

colonies was recorded for each culture.

Results of this study are to be found in Table 6.
Twenty-three per cent trisodium phosphate plus 1 per cent
"Triton X-lOO", 25 per cent trisodium phosphate with 0.1
per cent "Triton X-lOO", 10 per cent trisodium phosphate
and 7 per cent trisodium phosphate each with l per cent
of the wetting agent, and 10 and 7 per cent trisodium"
phosphate with the "Triton" and penicillin again proved
to be the most promising in their action. Of these, the
2 tri—sodium phosphate-—penicillin solutions were most

effective.

The irregularity of results encountered in this ex-

periment, as compared with that of previous studies is

probably due to the diffiCulty encountered in preparing
homogeneous portions of each sputum specimen. The mucoid
character of some of these made it almost impossible to

divide them into equal fractions of identical properties.

A large percentage of the comtaminants encountered
were fungi, thus indicating that none of these agents is
universally effective against these microorganisms.

VIII Preparation and Study of Digestion

Mixtures Containing "Dowicide A"

Therefore, now that favorable results had been obtain-
ed with augmented trisodium phosphate solutions, an effort
was made further to develop digestion mixtures which would
inhibit fungi, as well as other saprophytes. "Dowicide A"

was added in varying concentrations to the more successful

digestion agents.
A. Study of Fungicidal Activity--Katerials and Methods

Liquid mixtures of the following composition were
formulated and prepared: I -- 25 per cent trisodium phos-
phate, 5 per cent "Triton X-lOO", IV -- 25 per cent tri-
sodium phosphate, 1 per cent "Triton X-lOO", 0.05 per cent
"Dowicide A", VII -— 10 per cent trisodium phosphate, 1
per cent "Triton X-lOO", 25 units/m1. of penicillin G,
VIII -- 10 per cent trisodium phOSphate, 1.4 per cent
"Triton A-20", 25 units/ml. of penicillin G, 0.05 per

cent "Dowicide A", IX -- 7 per cent trisodium phosphate,

(49)

1.4 per cent "Triton A-20", 100 units/ml. of penicillin

G, 0.05 per cent "Dowicide A".

"Triton A-20" is a 25 per cent aqueous solution of an
alkyl aryl polyether alcohol with non-ionic surface activ-
ity. It is at least as chemically stable under alkaline
conditions as is "Triton X-lOO". Although not as satis-
factory a wetting agent as the latter it was shown by
Dubos to disperse and subsequently accelerate growth of
tubercle bacilli in liquid media (26). It does not

"Tween 80" for this microorganism.

possess the toxicity of
Thus, after a series of experiments observing its action

on suspensions of E. tuberculosis, its efficiency as a

 

digestion mixture ingredient with that of "Triton X-100",
all of which were very favorable, "Triton A-20" was select-
ed as a more satisfactory component to provide wetting
activity in digestion than "Triton X-lOO". Since it was
found that it reduces the lag phase of tubercle bacilli
encountered when they are transferred from the digestion
agent-~sediment mixture to the culture medium, it was

utilized to obtain this stimulatory effect.

Twenty high Gaffkey sputum specimens were obtained
from sanatorium patients and pooled, This composite speci-
men was then divided into 65 approximately equal portions.
A small inoculum of each of the fungi used in the study
of fungicidal agents (section IV) was then added to por-

tions of the pooled specimen--l fungus to each group of

(50)

7 portions, thus making 9 groups of 7 sputa, each seeded

with a different fungus.

Each of 5 fractions in each group was then treated with
l of the digestion mixtures. Mixures I, IV, VII, VIII, and
IX were added to the sediment of sputa obtained as the re-
sult of centrifugation at 2500 r.p.m. for 10 minutes, mix-
ed and incubated at 57°C. for 24 hours. The treated sedi-
ments were then cultured on medium III at 40°C. Cultures

were examined frequently for macroscopic growth.

In addition the 6 per cent sodium hydroxide digestion
. technique was performed with 1 portion of sputum from each
group as a control, since it had been observed previously
that molds are not particularly susceptible to its action
within the limits of the time employed for treatment (50
minutes). As a further control, another portion from

each group was left untreated, although it was realized
that contamination from growth of Sputum saprophytes

would make recovery of the seeded fungi very difficult.

These sediments were also cultured on medium III at 40°C.
B. Study of Fungicidal Activity--Results

From the results of the study, reported in detail
in Table 7, it may be concluded that of those studied,
mixtures VIII and IX were most efficient in destruction
of fungi without appreciably harming E. tuberculosis,

with digestion mixture- IV proving nearly as effective.

Digestion mixture I proved almost completely in-
effective in checking the growth of fungi, since 6 of the
9 seeded molds were recovered by culture. Mixture VII also
permitted 6 of these organisms to multiply, but inhibited
their growth sufficiently to allow recovery of tubercle
bacilli in 7 instances, 4 of these in mixed culture with

the fungus.

The sodium hydroxide reagent proved to be a poor con-
trol, since it inhibited or destroyed only 3 of the fungi.
Its extreme toxicity for viable tubercle bacilli is again

in evidence, as it prevented growth of 5 of the cultures.

The untreated group of Sputum portions seeded with
fungi were largely overgrown by saprOphytic bacteria when
cultured. The fungus and tubercle bacilli grew together
on the same slant in only 2 instances, and these cultures
were highly contaminated by bacteria.

0. Study of Digestion Mixtures Containing "Dowicide A"

in Cultivation of y; tuberculosis from Sanatorium
Sputa-~Materials and Methods

 

Eighty-five specimens of sputa from sanatorium
patients, as they were received in the laboratory were
concentrated by centrifugation and a portion of the re-
sulting sediments examined microscopically for the presence

of tubercle bacilli. The remaining sediments were then

divided into thirds, 1 portion of each treated with digest

ion mixture IV, the second with mixture VIII, and the third

(52)

portion with IX.

All sediments were cultured on medium III; incubation

was at 4000.

D. Results

Thirty-eight per cent of the 85 sputum Specimens were

found to be positive for g. tuberculosis microscopically.

 

Forty-four per cent were positive by culture following
treatment with digestion mixture VI'I, 40 per cent follow-
ing treatment with digestion mixture IX, but only 20 per
cent of these exposed to mixture IV. Cultures treated with
digestion mixture IV were contaminated by saprophytes in
14 instances, VIII treated cultures in 4 cases, and IX in

only 1 instance.

From these results it was concluded that mixture IV
is much.more toxic to tubercle bacilli than VIII, and IX
is moderately more toxic than mixture VIII. However, VIII
treatment is more conducive to contamination of cultures
than IX, but only slightly so in comparison with treatment
by IV, which resulted in 10 more contaminated cultures than
did mixture VIII.
IX Formulation of Digestion Mixtures Containing
Terramycin and Determination of their
Relative Efficiency
Although digestion mixture VIII and, to a slightly

lesser degree, mixture IX were found to be more satis-

(55)

factory for digestion of sputa than any of the other agents
tested, the slight toxicity of penicillin to the tubercle
bacillus when used in conjunction with a wetting agent under
alkaline conditions, made desirable further efforts toward
preparing better mixtures. Therefore, a survey of liter-
ature was undertaken, attempting to find a satisfactory
substitute for penicillin which could be used in digestion
mixtures. At this time terryamycin hydrochloride was
suggested by Dr. S. A. Yannitelli, of the A. S. Kimball

Sanatorium.

Available literature revealed that this antibiotic is

ineffective against the tubercle bacillus i5 vivo and in

 

zitgg, and that it is fairly stable under acid and aklaline
conditions (38). It is, however, active against most of
the Gram—positive bacteria susceptible to penicillin, as
well as several Gram-negative microorganisms resistant to
the action of the pioneer antibiotic. It had been noted
by Bobby (58) that terramycin is excreted in high concen-
tration in the sputum and appears to exert a marked effect
on the nature and consistency of the sputum, as well as
killing most of the normal resident bacteria of the mouth
and naso-respiratory tract (59). In fact, it was found by
this investigator that when terramycin hydrochloride was
administered to far advanced tuberculous patients, their
sputa were found to yield on culture, in most cases, only

tubercle bacilli, other bacteria having been inhibited or

killed.

(54)

~Terramycin also had been found effective against bac-
terial species which had acquired resistance to penicillin
(37). Therefore, since, in the course of routine use of
digestion mixture VIII, g slightly greater number of cul-
tures contaminated with staphylococci had been encountered,
it was believed that terramycin might prove more effective
than penicillin as an inhibitant of some of the more trouble-
some saprophytes which reside in and on the human body and
thus may have recently been exposed to penicillin therapy

in any of several ways.

The following digestion mixtures were subsequently
formulated: X -- 0.1 per cent terramycin hydrochloride,
1.4 per cent "Triton A-20, 0.03 per cent "Dowicide A";

XI -- 0.1 per cent terramycin hydrochloride; XII -- 0.1
per cent terramycin hydrochloride, 1.4 per cent "Triton
A-20", 0.03 per cent "Dowicide A", 7 per cent trisodium
phosphate; XIII -- 100 units/ml. of penicillin G, 1.4 per
cent "Triton A-20", 0.03 per cent "Dowicide A"; XIV --

100 units/ml. of penicillin G; XV -- 0.1 per cent terra-
mycin hydrochloride, 0.03 per cent "Dowicide A"; XVII --
100 units/ml. of penicillin G, 0.03 per cent "Dowicide A".

Digestion mixtures X and XIII were designed to compare
the action of penicillin and terramycin. The remaining
constituents of each are identical. Mixtures XI and XIV
were formulated to compare the efficiency of the 2 antibi-

otics when used alone in supposedly equivalent amounts.

(55)

Mixture XII duplicates the ingredients of IX with the ex-
ception of substitution of terramycin for penicillin, and

XV differs from XVII also only in these two antibiotics.

Before utilizing these digestion mixtures with sputum
specimens from sanatorium patients, cultures were made from
several buffered saline suspensions of recently isolated
tubercle bacilli, to each of which one of the mixtures had
been added. The number of bacilli in these suspensions
was roughly standardized by direct smear microscopic counts
made after the suspension had been shaken thoroughly.
Tubercle bacilli were recovered on culture from all the
treated suspensions. Four other digestion mixtures, also

studied in this fashion, inhibited E. tuberculosis, so

 

they were discarded.

Digestion mixtures X, XI, XII, XIII, XIV, XV, and
XVII where then used to digest sputa in the following
manner: .Nine microscopically negative Sputum specimens
were pooled, allowed to stand for 24 hours, then equal
amounts were transferred to 9 containers. These were
individually seeded with tubercle bacilli from 1 stock
culture, concentrated by centrifugation, and each sedi-
ment treated with l of the given mixtures for 24 hours.
Then the sediments were cultured on medium III (2 tubes

inoculated/ sediment) at 40°C.

Tubercle bacilli multiplied on cultures from each

of the sediments, but cultures of sediments treated with

(56)

I‘I‘“ '
V

 

mixtures XI, XIV, XV, and XVII were contaminated by

Saprophytic bacteria and, in l instance, by molds.

Treatment with digestion mixtures XI and XVII result-
ed in macroscopically visible colonies after 10 days incu-
bation, while digestion with XII and XIII produced colonies
in 11 days. Mixtures XIV and XV treated material cultured
tubercle bacilli only after a much longer period of incu-
bation (28 and 19 days, respectively) probably due to al-

most complete overgrowth of the medium by contaminants.

It thus appeared that terramycin was at least as
effective as penicillin as an ingredient of digestion
mixtures,although neither was satisfactory when used

alone, the former proving slightly better in dhis respect.
A. materials and hethods

The degrees of efficiency revealed by each antibio-
tic in this limited study, as digestive agents and as
components of mixtures, was then checked on a larger scale,
using 50 sanatorium sputa. These specimens were pooled

and seeded with E. tuberculosis, and then divided into 50

 

approximately equal portions.

Digestion mixtures IV, VIII, IX, X, XI, XII, XIII,
XIV, XV, and XVII were added to the sediments of these
portions--l mixture to each of 5 portions of sediment.
One tube of medium III was inoculated from each treated

portion at the end of the following periods of digestion

(57)

at 37°C.: 1, 1%, 2, 2%, 5, e, 12, 24, and 48 hours. The
cultures were incubated at 40°C., and were examined fre-

quently for evidence of macroscopic growth.
B. Results

It was found, as shown in Table 8, that digestion
mixtures IX, XII, and XIII exhibited the most rapid inhib-
itory action for contaminants, each proving effective
after only 8 hours digestion. Mixture VIII was next in
efficiency in this respect, as it yielded uncontaminated.
cultures after 12 hours treatment of the sediments. The
poorest mixtures were found to be XI, XIV, and XV, as some
cultures were still contaminated which had been planted
after the inoculum had undergone a 48 hour digestion
period. The remaining digestants all proved effective

when permitted to act for this extended time interval.

As indicated by the relative time in days required
for visible colony formation on the cultures, digestion
mixture VIII manifested the least toxicity for E. tubercu-
lpgig. Mixtures IX, XI, IV, and XVII were next in this
respect, and X, XII, and XIII treated sediments required
only 1 day more of incubation on medium III to produce
macroscopic growth than did the former group. Material
digested with XV required 16 days, while that exposed to
XIV failed to grow tubercle bacilli after 42 days incuba-

tion. Since both of these groups of cultures were con-

(58)

taminated, the long incubation period in one instance and
the failure to grow tubercle bacilli in the other does not
necessarily indicate high toxicity of these mixtures to E.

tuberculosis. One may conclude, however, that the mixtures

 

are unsatisfactory as inhibitants of saprophytes.

Again it appeared that terramycin and penicillin were
nearly equivalent in value as ingredients of digestion
mixtures. Mixtures X and XIII both satisfactorily removed
contaminants after 24 hours treatment of sediment, and growth
of tubercle bacilli was obtained from these sputum sediments
after 10 days incubation of their cultures. Digestion mix-
tures XI and XIV, in Which terramycin and penicillin, res-
pectively, were the only active ingredients present, both
failed inhibit saprophytes completely. However, XI, seemed
more satisfactory of the 2, since growth of tubercle bacilli
took place in 9 days, while XIV yielded no growth on cul-
ture of the treated Sediment, due to marked contamination

of all of the tubes planted.

Mixtures IX and XII also were very similar in their
action, since saprophytes were inhibited after 8 hours
digestion of sputum sediments, in each instance. Sediments
treated with IX (containing penicillin) subsequently cul-

tured M. tuberculosis in 8 days. Those treated with XII

 

required a 10 days incubation period to produce visible

colonies. Thus, it appears that, in association with

Ir:n\

trisodium phosphate, "Dowicide A", and "Triton A-20",
penicillin is slightly more effective as a digestive mix-

ture ingredient than terramycin.

This was also the case when XV and XVII were compar-
ed. Xixture XVII, containing penicillin, gave uncontamin—
ated cultures after 48 hours digestion, while the terramycin
mixture, XV, failed to do so. Growth times of tubercle
bacilli exposed to the 2 cannot be compared, since the
cultures from material treated by XV were contaminated.

X. Comparison of Efficiency in Routine Hospital

dork of Terramycin Digestion Mixtures with
those Containing Penicillin

These digestion mixtures were used to treat routine
sanatorium and out-patient sputum Specimens, which were
divided into fractions for separate addition of the in-
dividual mixtures. However, it was impossible to divide
the majority of specimens into 10 equal portions. In some
instances the volume of the sputum was so small that only
1 digestion mixture could be used. Generally, however,
each specimen was treated with at least 2 digestion

mixtures.
A. Materials and Methods

The sputum specimens were centrifuged at 2500 r.p.m.
for 10 minutes, and treated with the digestion mixtures

for a period of 24 hours at 37°C. Four loopfuls of each

(60)

sediment were then used to inoculate each of 2 tubes of
medium III and 2 tubes of Petragnani medium. The latter
medium was utilized because it is the policy of the Kimball
Sanatorium laboratory to use it to culture all routine
specimens examined for tubercle bacilli. All cultures were
incubated at 40°C. for a total period of 6 weeks, and were

examined macroscopically frequently throughout this time.

Considerably larger numbers of sediment were digested
with mixtures VIII and IX, as the majority of the specimens
were received before the study reported in Section IX was
completed. Other sputa were received at later dates when
preliminary results indicated that these 2 mixtures were

more advantageous for routine hospital utilization.

As was customary with all cultures, colonies develop-
ing on the media used were checked microscopically. The
time in days required for development of macroscopically
visible colonies was recorded for all sediments cultured on
medium III, as was the number of tubes of medium found to be

contaminated.
B. Results

Table 9 reports the time required for macroscopically
visible colony formation of specimens which were positive

microscopically for E. tuberculosis. The microscopic find-

 

ings are divided into three classes; high and low Gaffky,
and positive only by concentrate. The percentage of con-

tamination cultures for all Sputa treated with each mixture

(61)

is also recorded, as well as the total number of specimens
treated. The average elapsed time before macroscopic
colonies were noted is recorded for each class of the

positive sputa.

0f the digestion mixtures studied, VIII, IX, X, XVII,
XIII, and XII, (in the order of their efficiency) were
found satisfactory for treatment of sputa sediments. The
remainder of the mixtures failed to inhibit saprophytes
sufficiently and also exerted a more toxic effect on the
tubercle bacillus. Although 2 per cent of the cultures
from VIII digestion were contaminated, while none of the
mixture IX cultures were, the former mixture was consider-
ed more effective since it did not manifest the high degree

of toxicity for M. tuberculosis that was displayed by the

 

latter, as shown by the difference in growth times for this

organism.

Digestion mixture X, which is the same in composition
asXIII except for the substitution of penicillin for terra-
mycin, proved more efficient than mixture XIII, XI and XIV
each consisting of l of the 2 antibiotics as the sole in-
gredient were unsatisfactory. Mixture IX, identical with
XII except for the use of penicillin the former and terra-
mycin in the latter, was found to be more effective than
mixture XII, especially with respect to toxicity for tubercle

bacilli in Sputa positive microscopically by concentration.

(62)

Table 10 records the per cent correlation between
concentrate microscopic examination and culture of the
sediment following treatment with one of the digestion mix-
tures. The following values in terms of per cent of total
Sputa are given: specimens with both positive concentrate
and culture, Specimens with negative concentrate but posi-
tive culture, those with positive concentrate but negative
culture, sediments with positive concentrate but contamin-
ated culture, those with negative concentrate and culture,

- and specimens with negative concentrate and Petragnani cul-

ture, but positive culture with medium III.

The last named category was included to further indi-

cate the lower toxicity for M. tuberculosis of digestion

 

mixture VIII, as compared with.mixture IX. As the mala-
chite green present in the Petragnani medium is slightly
inhibitory for this organism, tubercle bacilli which pre-
viously have been subjected to adverse conditions during
digestion will be less likely to grow on the medium. Mix-
ture VIII treatment resulted in 4.14 per cent cultures
positive only on medium III, as compared with 1.72 per cent

for mixture IX.

Mixture VIII proved superior to the other digestants
in all respects. A greater recovery of tubercle bacilli
from microscopically negative sediments was achieved, and
sediments containing tubercle bacilli treated with this
solution subsequently gave positive cultures. Contamina-

tion was very low--mostly due to bacteria with only rare

(65)

recovery of fungi.

Mixture IX, as discussed previously, was more toxic

to M. tuberculosis than VIII. However, its terramycin

 

counterpart, digestion mixture XII, exhibited greater
toxicity with respect to recovery of tubercle bacilli on

medium III and not on Petragnani's.

Terramycin mixtures in general proved unsuccessful
due to the high degree of contamination of cultures plant-
ed from material digested by these solutions. Their rela-
tive toxicity to the tubercle bacillus was greater than
their penicillin counterparts, as indicated by fewer
positive cultures from negative concentrates, and by
failure to give any positive medium III cultures when all

Petragnani cultures were negative.

(64)

Discussion

The experimental eVidence indicates that 2 concen-

iod of digestion with these rea eats is care

L.
ed, it was found that they possessed high toxicity for h.

tuberculosis, evidenced by the slow growth of this or-

go
0)

 

ganism iollowin ex osure to them. Even the protective

014)
D (:3

DO

effect of mucus failed to reduce their adverse eiiect.

Lower concentrations of sodium hydroxide were not
studied, since it was found that this digestive agent
precipitated the protein material of Sputa sediments.

It was believed that the presence of this precipitate
surrounding the individual tubercle bacilli would further
increase the period of adjustment of each to the unaccus-
tomed environment of the culture medium. Twenty-three per

cent trisodium phosphate proved less toxic in preliminary

studies and did not require carefully timed periods of

Q.-
H.
OH

CD

stion. It also showed less tendency to form precipi-
tates with sputa constituentsa Therefore sodium hydrox-
ide was used in subsequent experimental work only as a

standard for comparison.

It was not utilized in conjunction with wetting agents
because sodium hydroxide "Triton" mixtures were found to

be less stable over several months observation than is

(65)

trisodium phosphate in various concentration. Although

it Was not demonstrated experimentally, it would appear

that the toxicity of sodium hydroxide would be multiplied
considerabley by dispersal of the naturally occurring clumps
of tubercle bacilli brought about by wetting agents. Other

1
7

moderately toxic caemicals have been observed by Dubos and
Youmans to be markedly bacteriocidal to this organism when

dissolved in surface active agents, as reviewed previously.

Sodium hydroxide was considered unsatisfactory as a
digestive agent also due to the resistance of many sapro-
phytic fungi to its action. It shared this shortcoming
with all of the agents described by previous workers which
were studied by the author. Attempts to use it, in several
concentrations, in the presence of "Dowicides" A, B, and F
were unsuccessful, since such combinations proved extreme-

ly toxic to the tubercle bacillus.

One might speculate that addition in this way of more

sodium ions caused decreased ionizat*on of the Dowicide
into sodium and phenate ions by shifting the equilibrium

to the left to produce a more COHCBHtFited solution of the
un-ionized sodium phenate compound. This might subsequent-
ly, due to its insolubility, while passing through the
colloid state, be absorbed on the lipid coating of tubercle
bacilli and act directly upon these organisms, resulting has
substantial increase in toxicity. The possibility of this

occurring is Opposed by observation of the apparent stabil-

(66)

ity of these "Dowicides" in a 6 per cent solution of
sodium hydroxide, and by the lack of such an increase in
their toxicity when in trisodium phosphate solutions.

The latter compound also yields sodium ions when dissolv-

ed in water.

However, it was found that degree of alkalinity had
little effect on the extent of toxicity observed with the
"Dowicide"--sodium hydroxide mixtures. when 6 per cent
concentrations were partially neutralized by addition of
1 normal hydrochloric acid, the resultant increase in hydro-
gen ion concentration did not diminish it. These pH values
ranged from 7.5 to 10.0. The pH of 25 per cent trisodium

phosphate is approximately 10.5.

"Antiformin", which proved even more rapid than sodium
hydroxide in the destruction of contaminants, was found to
be much too toxic to tubercle bacilli for routine digestion

of Sputa prior to culture.

Potassium hydroxide in the 1 concentration utilized
(5 per cent) proved approximately equivalent in efficiency
to the sodium hydroxide solutions studied. Oxalic acid
treatment of Sputum sediments did not effectively inhibit
saprophytic growth on culturing the digested Sputum sedi-
ments. It was, however, the least toxic of the chemical

agents studied in this group (section I).

(67)

The success of cultural examinations in the labora—
tory diagnosis of tuberculosis depends on the preservation
of tubercle bacilli and the elimination of contaminating
microorganisms._ The selection of techniques and reagents
will vary with local factors such as the populace from
which specimens are received, general character of speci-
mens, and the number of tubercle bacilli usually encounter-
ed. A sanatorium, whose beds are occupied by far-advanced
tuberculosis patients, will more readily be able to use, in
its laboratory, a harsh digestive agent which will rapidly
dissolve mucus than a public health laboratory which ex-
amines specimens collected from the general public. In the
latter instance the risk of secondary contamination is in-
creased, and many types of bacteria are able to multiply
before the specimen reaches the laboratory. The rate of
contamination is relatively high, while the number of
specimens in which tubercle bacilli can be detected is low.
Thus the average health department laboratory requires a
digestive agent which will prevent saprophytic contamination
of cultures but will not harm the few acid-fast bacilli that

may be present in specimens.

In View of the experimental results obtained, sodium
hydroxide, "Antiformin", potassium hydroxide, and oxalic

acid do not entirely fulfill the requirements of the average

sanatorium nor those of the public health laboratory.

Trisodium phosphate, however, proved to be a step
nearer the ideal digestive agent. Treatment of sputa sedi-
ments for 26 hours with this reagent destroyed all contamin-
ants. It was found to be relatively non-toxic to tubercle
bacilli since it yielded growth of these microorganisms in
12 days incubation of cultures inoculated with the treated
sediments. The trisodium phOSphate method does not require
neutralization of the digested material before culturing,

nor does it have to be carefully timed.

However, it did not rapidly break down mucoid Speci-
mens to a homogeneous suspension, nor did it prevent growth

of fungi from sputa which contained these microorganisms.

Therefore the wetting agent fiTriton X-100" was added
to various strength trisodium phosphate solutions to en-
deavor to more rapidly reduce the mucoid film surrounding
and protecting undesirable bacteria. By means of timed
digestion experiments, it was found that periods of action
for various relative concentrations of solutions of these
2 agents could be established whereby action was fairly

rapid, but the tubercle bacillus was not affected adversely.

In routine work with sputa, some disagreement in re-
sults from study to study can be noted with respect to time
required to remove or inhibit saprophytes and also the time
required for positive cultures to develOp. The significance

of this should not be exaggerated, since routine clinical

specimens may be irregular at times so far as the distri-
bution of small numbers of tubercle bacilli is concerned.
The lack of homogeneity in sputa accounts for the fact that
the more tubes planted, the more positive cultures will re-
sult, a phenomenon nearly independent of the digestion

agent.utilized.

The inadvisability of adding trisodium phosphate--
"Triton X-100" solutions to sputum containers before they
are given to patients for collection of specimens was
demonstrated by difficulty in standardizing digestion mix-
ture ingredient concentrations because patients often fail-
ed to return the jars promptly after collection of the
specimen. The confusion on the part of the patient in re-
gard to this liquid, and the hazard of more frequently
spilled specimens due to the increased volume, also con-
vinced the author that this means of accelerating digestion

was unreliable.

The periods of digestion studied were selected as
those which would be feasible for adaption to the routine

of the public health or clinical laboratory.

Fungi in specimens treated for the maximum time were
not inhibited by the trisodium phosphate--"Triton" mixtures.
Thus the study of the action of "Dowicides" A, B, and F was
undertaken. "Dowicide A", which was found to be effective

as a fungicide in suitably low concentrations and is least

INA\

toxic of the 3 to E. tuberculosis, has been recommended by

 

the United States Department of Agriculture as a disinfec-
tant for surfaces contaminated by bovine tubercle bacilli.
The apparent contradiction is difficult to explain, except
on the basis of differences in cellular chemistry between

bovine and human tubercle bacilli.

"Dowicides" B and F inhibited E. tuberculosis in the

 

concentrations in which they were fungicidal. The tubercle
bacillus greatly resembles the pathogenic fungi in many of
its characteristics. It also customarily produces deeply
seated infections beneath the surface epithelium so that
tissue must be removed or destroyed to combat it effective-
ly. Under the right conditions it will assume filamentous
forms resembling the molds. It also is fairly resistant to
an adverse environment which would kill most bacteria.
Colonies of virulent tubercle bacilli on the classical

media are hard, dry and brittle like those of many fungi.
Thus it is difficult to find fungicides which will not harm
this bacterium. "Dowicides" B and F are effective against
fungi in higher dilutions than "Dowicide A". It, therefore,
is not surprising that the former chemicals should prove too
toxic to the tubercle bacillus for use in digestion mixtures,
while the latter, through some unexplained selectivity, is
satisfactory for inhibition of fungi present in sputa

sediments.

More complex digestion mixtures were formulated as

attempts were made to achieve solution which would efficient-

(r71\

1y destroy contaminants in a convenient length of time.

The concentrations of trisodium phosphate and wetting agents
were therefore varied, and antibiotics and "Dowicide A" were
added in various concentrations to improve the selectivity

of the mixtures.

The ammonium carbonate--penicillin reagent used by
Goldie in cultivation of tubercle bacilli from sputa proved
more toxic to this organism than trisodium phOSphate--"Tri-
ton X-lOO". Nevertheless, the value of penicillin was
demonstrated by its presence in the mixtures studied, and
it was hoped that it would prove sufficiently stable for
use in digestion mixtures. It was subsequently found to
possess greater stability over several months time than

terramycin, with which it was compared.

"Triton A-BO" appeared to reduce the lag phase of the
tubercle bacillus which occurs during the period of the
organisms adjustment to the cultural environment, since it
reduced the incubation time necessary for macroscopic colony
formation. Like "Dowicide A", it had never previously been
used in digestion mixtures formulated by other workers.
Dubos, however, had found it an improvement over "Tween 80"

as a growth stimulant in liquid media.

Terramycin hydrochloride was also an innovation as an
ingredient of a sputum digestant. It appeared promising

in limited preliminary trials, but subsequent use routinely

on a large scale revealed its lack of stability and slight-

(72)

ly greater degree of toxicity to tubercle bacilli, when
compared with penicillin. Terramycin's relative incompat-
ibility to alkaline environments, as well as its instabil-
ity probably account for the greater degree of contamination
of cultures resulting from its routine utilization over a

period of several months.

It should be realized that the number of specimens
treated with terramycin mixtures is much smaller than that
of those digested with some of the solutions containing
penicillin. Thus it would doubtless be advisable to con-
duct more extensive routine studies with the former antibi-
otic, keeping the digestion mixtures refrigerated when not

in use.

Digestion mixture IX proved more toxic to these organ-
isms than VIII, thus indicating that 100 units/ml. of peni-
cillin G is not only unnecessary but also reduces the

efficiency of digestion.

Digestion mixture VIII, containing 25 units/ml. of
penicillin G, 10 per cent trisodium phosphate, 1.4 per cent
"Triton A-20", and 0.05 per cent "Dowicide A", was found to
be the most effective of all the agents and mixtures studied.
Penicillin, in the presence of a wetting agent is slightly
toxic to tubercle bacilli, so this mixture does not com-
pletely solve all problems involved in Obtaining fl. £3233-

culosis in pure culture from Sputa. However it does in-

(7:5)

\

hibit the great majority of saprophytes (including fungi)
and the toxicity of penicillin may be partially overcome
by the stimulatory effect of "Triton A-ZO". Therefore
when used to treat sediments for 24 hours at 57°C.,
digestion mixture VIII will prove more efficient than any
of the other digestants studied.

('74)

Summary

The relative efficiency of sodium hydroxide,
potassium hydroxide, "Antiformin", oxalic acid, ammon-
ium carbonate--penicillin, and trisodium phOSphate in
Specific concentrations as digestants of sputa prior to

culture for E. tuberculosis was studied experimentally

 

using specimens from tuberculosis patients. Twenty—three
per cent trisodium phosphate proved most effective. It

was subsequently modified extensively through addition of
other chemicals, and these modifications used as sputum
digestants. Fungi were effectively inhibited by 0.05 per
cent "Dowicide A", which did not prove toxic to tubercle
bacilli in this concentration. Surface active agents were
also used in the formulated digestion mixtures to permit
more rapid action of the other ingredients. "Triton A-ZO"
was found to be the most effective of these when digestion
periods were standardized. Prolonged exposure to digestion
mixtures containing wetting agents is toxic to tubercle
bacilli. Penicillin G and terramycin hydrochloride were
used as digestion mixture ingredients. Penicillin G was
superior in efficiency and stability. Of the several
digestion mixtures formulated and/or studied, VIII, contain-
ing: 25 units per m1. penicillin G, 1.4 per cent "Triton
A-20", 10 per cent trisodium phosphate, and 0.03 per cent
"Dowicide A", was least toxic to E. tuberculosis and most

effective in reducing cultural contamination.

('75)

Culture media utilized in experimental studies:

‘5.
V
.

-
,

u 1|" I
vsb-L

.. L)

Petra gnazii Egg hcdium

o
\

(Lemon modification,

Potato (p-ec‘ei and cxt into sua 11 pieces) 250. 0 g.
Distilled water 330. ml.

7, a s,— . .

Lix and eats ”1 ve at 121°C . TOP n9 minutes. brain, did
make up potato water to ”SJ ml. Add
BaCtO-Skjin milk (DifCO) Lick; 3.
Potato starch 1:.C g.
Proteose peptone ho. 5 (Pifco) 5.0 3.

Stir to a szgzooth past and heat in a d ublo boi c
2 hours, with frequent stirrizg. Cool to 59°C. Add the
following mixture:

Eéjs (whole) 12

P * yolks 5

Jl‘yfcerol 35.0 ml.

Lalacrite leen (2.0g aqueoqs) 30.0 ml.
Lix t'arvigbly and filter t?rnigi sterile gauze into

a sterile distrib1t1n= fuqnel. Distrib1te as aseptically

as possible into sterile screw-capped Cl-tl?8 t1bes. Place

is a slanted positio. in an inspissator or _ariold and heat

at 90°C. for Ej minutés the fir t day as: 2 }1ours, or mole,

the second nay.

Protease iLIILLlc o. 5 (b.’CJ) 2.0 g.
Disodium p aspha‘e 2.5 g.
goicpotisslum L esp ate 1.0 g.
Kagneniuu 3‘lfate 0.7 g.
Ferric am.3uiim citrate 0.1 3.
Yeast extract (Difco) 2.0 f.
Sodiim silicate (meta) 0.2 3.
Triton A-20 10; solution) t.o m1.
Distilled water 997.0 ml.

Dispense in screw-capped calture tubes in 10 ml.

. . 9 ‘ * _o f‘ ( f‘ . ,
portIst. Lt rllize by autoclavlng at lml)p. for 20
niqute: Then add aseptically 1 ml. of the following

Lacto-penase (Difco) 0.1 r1.
Serum albumin (bovine fraction) 5.0 ml.
Sodiun chloride 0.35 3.
Di tilled water 100.0 ml.

Sterilize by filtration through Seitz or porcelain
filter. Final pH of medium: 7.0-7.2.

me :3 ium II .1:

ixoerimental Solid hedium

Protease peptone lo. 5 (Difco) 2.0 g.
Disodium phosphate 2.5 ;.
Eonopotassium phosphate 1.0 g.
magnesium sulfate 0.? g.
Ferric armonium citrate 0.1 5.
least extract (Difco) 2.0 3.
Sodium silicate (meta) 0.2 3.
Agar 16.0 5.
Distilled water 973.0 cl.

Adjust pH to 7.2

'l-fi . r— 0 Q o o .

1:119 at 35 0., suspense in sterile screw-capped
culture totes is 10 ml. portions. S eri_ize by autoclav-
ing at 12100. for 20 minutes. Add aseptically 1 ml. of

the following solution to each tzbe:

Triton A-20 (10} aqueous solution)
Eacto-penase (Difco)

Oleic acid-albumin complex 1
Glucose
Distilled water

erilize hy filtration thruur? Seitz or porcel in

1 a
. ine tubes are slanted for the medium to cool.

*SUJ
('1’

Q

The oleic acid-albumin complex is pre 3 ed as follows:

.-

l

we 0.12 ml. 05‘ oleic acid (0.1 {To

) is 10 rl.
n nydroxide by shaking with a rota

ry motion

a
) Add 5 m . of this solition to 95 ml. of a neqtral 5
r cent solution of bovine serum fraction V (albumin) in
35 per cent salin
) Incubate at 56

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(l)

(2)

(3)

(h)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

Literature Cited

Abbott, J. N. l9h8 The comparative study of Sula,
Dubos, and Lowenstein media. 'In New York (State)
Department of Health. Division of Laboratories and
Research. Annual Report, 23-2h,

Abbott, J. N. 1951 Effective use of penicillin to
reduce contamination in sputum concentrates to be
examined for tubercle bacilli. Am. J. Pub. Health,

g1, 287-291.

Abraham, E. P., E. Chain, C. M. Fletcher, and H. W.
Florey 1 #1 Further observations on penicillin.
Lancet, 1, 177-188.

Anonymous l9u9 X-rays and You. Eastman Kodack
Company, Medical Division, Rochester, New York.

Andrus, P. M., and H. E. Mac Mahon l92h The use
of volatile hydrocarbons in the concentration of
tubercle bacilli. Am. Rev. Tuberc.,‘9, 99.

Association of Official Agriculture Chemists 1945
Official and Tentative Methods of Analysis. Wash-
ington, D.C. 6th edition. 53.

Beattie, M. l9h9 Cultivation of Mycobacterium
tuberculosis. J. Lab. and Clin. Med., QR, 733-738.

Braver, Kurt 1918 Ein neues Verfahren zur An-
reicherung von Tuberkelbacillen im Sputum. Deutsche
Med. Wehnschr., g4, 266-267.

Brown, L., and D. Smith 1910 The cultivation of
tubercle bacilli directly from Sputum by the use of
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