The isolation of tubercle bacilli from contaminated tuberculous materials

December, 1927]

ISOLATION OF TUBERCLE BACILLI

115

AMERICAN SECTION. Under the Editorship of Dr. A. K. Krause.

THE ISOLATION OF TUBERCLE BACILLI FROM CONTAMINATED TUBEIWULOUS MATERIALS.' By H. J. CORPER, M.D., Ph.D., and NAO UYEI, M.S., Ph.D. SINCE Koch first isolated tubercle bacilli from tuberculous materials in 1882, two significant advances have be·en made in the technique of isolation of the bacilli from contaminated materials. The first important improvement was the proposal of the antiformin method of Griffith L1] and the other the introduction of the sodium-hydroxide method of Petroff [2]. A comparison of these two methods [3J has shown Petroff's method to be decidedly superior to the antiformin method, and the former has now been universally accepted as the most satisfactory method for obtaining primary cultures from contaminated sources. . In spite of the original claims that it was possible to obtain successful primary cultures in a high percentage of cases (129 out of 135 positive sputnms, or 95'5 per cent.), an exhaustive evaluation of Petroff's method by other investigators shows a decidedly lower percentage of primary cultures obtainable. Mitchell and Simmons [4J obtained primary cultures from 28 sputa out of 35 specimens (80 per ceut.), Keilty [5] 12 out of 18 samples (66'6 per cent.), Stewart [6] 24 out of 37 samples (64'8 per cent.), and Corper, Fiala and Kallen [7J 144 out of 526 samples (27'3 per cent.). 'I'hese figures indicate that there is still ample room for improvement over the Petroff method of isolation. Recently Dold [8] suggested the possibility of isolating tubercle bacilli from tuberculous materials by using urea to destroy contaminators, and Lowenstein [9] proposed a new acid method for isolating them. The advantages and disadvantages of these latter methods over Petroff's method have not yet been fully appraised. In the following study are reported the results of: (1) Investigating the possibility of substituting the more uniform and standard crystal violet or methyl violet now obtainable for the gentian violet of uncertain composition used in Petroff's medium; (2) an endeavour to find a better reagent to replace sodium hydroxide as no bactericide for the contaminators usually present in sputum i and (3) an attempt to find a better culture medium for the isolation of tubercle bacilli. (1) COMPARISON OF THE VALUE OF CRYSTAL VIOLET TO REPLACE GENTIAN VIOLET IN THE CULTURE MEDIUM. The purpose of the addition of gentian violet by Petroff to an eggmeat-infusion medium was to inhibit the growth of such contaminating micro-organisms as were not destroyed by the previous sodium-hydroxide treatment. Another advantage of the use of the dye in the medium consists in the possibility of early detection of the growth of the tubercle bacilli as a result of the contrast between the cream-coloured colonies on the violet background of the medium. Prior to the World 'War, the Griibler Company, or Griibler and Holborn, I

Research Department, National Jewish Hospital, Denver, Colorado.

116

TUBERCLE

[Decem bel', 1927

of Germany, were practically the sole distributors of the biological stains used in America. In 1922 the Commission on Standardisation of Biological Stains was organised in America, with H. J. Conn as ChairmaJ?' to .meet the post-\yar unst.aL!e conditio.Ds of. the ~iological ~yes available 1D America. 'I'his Commission found Itself 1D a difficult position in defining the dye or dyes termed " gentian violet," since in pre-war days the term "gentian violet" was not applied to any pure or definite dye but to a poorly defined mixture of violet rosanilines. However, the Commission, after investigations, defined gentian violet as either pentamethyl or hexa, methyl pararosaniline, or as a mixture of methylated pararosanilines, composed primarily of the two compounds just named and having a shade at least as deep as that recognised in the trade as methyl violet 2B [lOJ. The Commission further recommended the use of crystal violet as a more uniform substance in place of gentian violet. . As far as a search of the available literature was possible, no study of the action of crystal violet or related dyes, sold under the name of gentian violet on the American market, on the growth of tubercle bacilli was found. It was reasonable, therefore. to question whether these dyes could be used in place of gentian violet, and in the same concentration as in Petroff's medium, for the primary isolation aud subculturing of tubercle bacilli. '1'0 the bacteriologist and pathologist, using Petroff's method for isolatin~ tubercle bacilli, this question assumes importance. In studying this problem four samples of crystal violet and one sample of methyl violet, all sold as gentian violet on the American market, were used, in order to note their effect upon the growth of tubercle bacilli . The dyes tested were as follows :Sample 1. Griibler's gentian violet. II. Crystal violet obtained from the National Aniline and Chemical Company and sold by them as gentian violet. III. Crystal violet from the Empire Bio-Chemical Company and also sold as gentian violet . • IV. Crystal violet from the Hartman Leddon Company and also sold by them as gentian violet . V. Methyl violet from Coleman and Bell and also sold as gentian violet. VI. Crystal violet from Coleman and Bell. The dyes were dissolved in 95 per cent. alcohol and introduced into Dorset's egg medium in concentrations of (a) 1 : 1000, (b) 1 : 5000, (c), 1 : 10000, and (d) 1 : 50000. Four strains of tubercle bacilli, namely. "Gluckson" (a virulent human bacillus), " Human ., (an avirulent human bacillus), II Bovine Virulent" (a virulent bovine strain) and" Bovine D .. (an avirulent bovine strain), were seeded by the suspension method (using three to four drops of a fine suspension containing 75 mg. per c.e.) on four tubes each for each concentration of the dye, and incubated at 37°C. As a control Dorset's egg medium without the dye was used. Table I gives the results of an experiment with Grubler's gentian violet, which may be taken as the standard for comparison with the action of the other dyes on the growth of tubercle bacilli. As seen in Table 1. Griibler's gentian violet in 0'1 per cent. concentra. tion exerts a slight inhibitory action on the growth of tubercle bacilli, and it is obvious that this concentration is unsuitable in the egg medium used for the primary isolation of the bacilli, while a concentration of 0'02 per cent. or less permits growth of the tubercle bacilli unhampered when

December, 1927]

117

ISOLATION OF 'L'UBERCLE BA.CILLI

heavy seedings are used. In order to ensure absence of inhibition a 0'01 per cent. concentration was adopted by Petroff in his original method. All the other dyes studied in this experiment were found to act about the same as Grubler's gentian violet, the only difference being that they exerted 11 slightly greater inhibitory action on the growth of the tubercle bacilli in concentration of 0'1 per cent., and in some cases still exerted a faint inhibitory effect in concentration of 0'02 per cent., while at 0'01 and TABLE J.--THE EFFECT OF GUiiBLEU'S GEN'rrA~ VIOLET UPON THE GROWTH OF TUBERCLE BACILLI ON EGG MEDIA.

Control on egg medium alone 8truill of bacilli tested

"-

~ I~

----------------Gluckson (virulent human bacilli)

..

Gentian violet (Grubler's) in egg medium

I

0'\

01)2

per cent.

\>el' cent. ---

r

~

~

I" 2 13

21 3

3

2

.. , 0

....~

fj:

co I .... _1 _ _' -

Human avirulent (avirulent human 1 bacilli) Bovine virulent (virulent bovine bacilli) 1

T.

~

: ~1

I:

01

-'"

1

2

2 I1 I

1

0

"

co

....

2

3

1

I

2

,

1

2

0'002

0'01

per cent.

per cent. - -----

., ""'"'" I~~ ...~'"'" "". """'" ""It'" ...t1'" ...il:'" '" It '"

-s:

~

I

3

I ""'" ... e, "'" '" T.

II

""

ee

"

It .....

2

8

I

2

3

1

1 12

8

1

1

;

....,

-"

.,'"

-"l

t1

~

"

~.

2 1

3

1

2

3

1

2 11

2

8

0

2

3

I

01

2 8 8 0 2 8 0 2 3 1 1 1 • The growth of bacilli is recorded from" 0" = no discernible growth to "4," a profuse or excellent beavy growtb. The figure recorded is an average of the reading of four tubes in each case, fractions being omitted and the nearest wbole number being recorded. Tbe tubes were seeded witb 3 to 4 drops of a fine suspension of the bacilli containing 75 mg. of young, actively growing bacilli (about one to two months old) per cubic centimetre of liquid. Bovine D (avirulent bovine bacilli)

TABLE

n.-THE

1

1

1

2

1

BACTERIOSTATIC ACTION OF CRYSTAL VIOLET (EMPIRE BIOCHEMICAL COMPANY) D! EGa MEDIA,

------------:----;--------------stmin of tubercle hacilli

".,'d

Crystal vlolet In egg medium Controlon \ elH; medium ---~-,--------------. nlone 0'\ I 0'02 ! 0'01 0'002 _. __ per cent~ per cent. I per cent~ pel' cent.

I

~

];~ ~ I~

..

"II<

I<

"

2.3

0

e tl~ ~ ~

~I~ ~ ~ ~ ~;~ ~ e ~ s ~ :E

itt

I-I

4)

~

~

t~

l1I

~ ~1

I<

0

1

1

2

8

1

2

8

1

2

8

1

8

1

2

8

1

2

3

_____________ ~~\..:....; ~ _~ -=Gluckson (virulent human bacilli) ..

~ iO " ~ ~ jo ~~ ~ ~ ~

il=

I

..:..

~

Human avirulent (avirulent human 1 I 2 bacilli) Bovine virulent (virulent bovine bacilli) 11 122

3

0' 0

o

0

a

0

0

1

1

a

1

\I

8

1

3

Bovine D (avirulent bovine bacilli) ••

8

0

0

1

1

8

1

\I

8

1

2,3

I

i

• The numerals have the same significance as in Table 1.

0'002 per cent. concentration growth occurred equally well to that in the

control. The slightly more efficient bacteriostatic action of the crystal violet or methyl violet over gentian violet would appear to bear out the results of other investigators with other bacteria. These results would make it appear safe to use any of the dyes tested, in 0'01 per cent. concentration, to replace Grubler's gentian violet in Petroff's egg medium for primary isolation of the tubercle bacilli from

118

TUBERCLE

[December, 1927

contaminated materials. As an example of the bacteriostatic action found, Table II records that for crystal violet obtained from the Empire Biochemical Company. Before concluding a consideration of the action of the dyes it may be well to point out that a bacteriostatic action of these dyes occurs in far greater dilution in glycerol-agar medium and the use in 0'01 per cent. concentration applies only in Dorset's egg medium. In glycerol-agar media tubercle bacilli failed to grow even in so Iowa concentration as 0'001 per cent. of crystal violet (National Aniline and Chemical Company). (2) A COMPARISON OF V ARIOUS REAGENTS FOR PRELIMINARY TREATMENT OF POSI'l'1VE f::)PUTA TO DESTROY UNDESIRABLE CONTAMINATORS.

For the purpose of comparing the value of different germicides to be used for isolating tubercle bacilli, the following criteria were considered of importance in choosing the most suitable substance for tbis purpose :(1) It must be an efficient germicide for all contaminators and not be injurious or detrimental to the growth of tubercle bacilli. . (2) It must be of low toxicity for tubercle bacilli in a wide range of useful concentrations. (3) It must be easily obtainable on the market and of reasonable purity. (4) It should require no special ability in preparation or use and but a minimum amount of manipulation, thus making it available to the average technician in successful primary cultivation of tubercle bacilli. . It is almost impossible to 'select a reagent satisfying all the foregoing ideal requirements. In general, if a substance possesses a strong germicidal action upon contaminators it also exercises a more or less toxic action upon the tubercle bacilli as well. For the sake of simplicity, operations such as incubation, neutralisation and centrifugation, and washing should be obviated as far as possible without impairing the efficacy of the method for isolating the tubercle bacilli. With the foregoing considerations in mind, a standard procedure was outlined and used, adhering in general to the technique prescribed by Petroff; and the action of other reagents in destroying contaminators was studied, with the idea of finding more suitable ones for isolating tubercle bacilli in contaminatedmaterials. . The standard procedure used was as follows :One cubic centimetre of microscopically positive sputum was introduced into a sterile 15-c.c. centrifuge t~be, together with an equal volume of the reagent (in case of a liquid reagent) or a weighed amount of the substance (in case of a solid reagent) under investigation. The contents were thoroughly mixed and, after incubation for a definite interval, the contents were diluted to about 15 c.c. with sterile water, mixed and centrifugated. The supernatant liquid was poured off, and three culture-tubes, containin~ Petroff's gentian violet egg medium, were planted with the residual fluid. The tubes were then placed in an incubator at 37° C. Weekly examinations of the tubes were made for evidence of growth of tubercle bacilli or contaminators. In case of evidence of growth of tubercle bacilli confirmatory tests were made by staining smears from the colonies by the Ziehl-Neelsen method (11). Throughout the experiment, four different time intervals of incubation

December,_1927]

119

ISOLATION OF TUBERCLE BACILLI

were used-fifteen minutes, thirty minutes, one hour, and two hours-to evaluate the action of the reagent. In calculating the percentage of contamination or of isolation of tubercle bacilli, the total number of tubes inoculated for each interval was taken as a basis if not otherwise stated. If, for example, four sputa were used for testing one reagent and three tubes had been inoculated for each sputum for the fifteen-minute period, and in two tubes the tubercle bacilli grew, the percentage of successful isolations would be 2 out of 12, or 17 per cent. Contaminators detectable only after the fifth week were not counted as contaminators, since these were mostly due to moulds finding their way through the cotton plug into the culture tube, while growth of contaminators detected within the fourth week was designated as due to contamination not destroyed by the reagent, TABLE III.-EFJ'ICIE:-lCY OF SODIUM HYDROXIDE I:-l DESTROYING CONTA)UNATORS IN THE SPUTUM WHEN USED AS A REAGENT FOR THE ISOLATION OF TUBERCLE BACILLI.

I Coneentra· tion of NaOH used

._~--

Per cent.

2

3 4

11)

sominutes

minutas

I

1 hour

2110UrR

-----Percentage Percentage I

Percentage Percentage Percentage of positive Percentage of positlve Percentage or postttve I Percentage of positive of eentarn- Isolations of con tam I· isolations of eontarni- isolations i of Ion tum I· laolatlon. of tubercle of tuberel e inabions nattons of tubercle nations of tubercle natlona bacilli bacilli bacilli baciill ,

------ ----- ---- - - - - - - - - ----,---, 0 0 0

I

58 58 33

67 41

8 8 8

67

8 0 8

33

,

41

17

67

0 0 0

8 41

TABLE IV.-POWDERED UREA AS A REAGENT FOR DESTROYING CONTAMINATORS IN THE ISOLATION OF TUBERCLE BACILLI. 30 minutes

}[, minutes Concentra· tration" of urea

I hour

I

:! hours

Percentage Percentage Percentage Percentage Percentage of positive Percentage of positive Percentage uf positive Percentage of positive ufeontaml. Isolation. ef eontami- Isolnbions ofcontami· i.olatlonl ot conteml- 18olatlons nations of tuberele nattons of tubercle nations of tubercle nations of tubercle bacilli bacilli bacilli bacilli

I r;;:-;,;;t.- -----1---- ----.----,---- ----1---- ---so " 100

33 W 33

I

0

41

IOU 33

33

0 0 0

I

I

41

ro 0

0 I 0 0:

50

U

33

0 0 0

• The concentration of urea recorded 10 this table Indicates the concentration of urea added and contalned in the mixture of urea and sputum. Thus, 75 per cent. urea concentration indicates a mixture of 1 c.c, of sputum and 0'75 gr. of urea.

regardless of whether it was due to the inefficient germicidal action of the reagent or to faulty technique. As a standard for comparison, sodium hydroxide, in the concentrations of 2, 3 and 4 per cent., was used on four microscopically positive sputa for 'each concentration. Examinations for growth were continued to the tenth week, or in some cases to the fourteenth week. The results with sodium hydroxide are recorded in summary in Table III. -If the growth of tubercle bacilli, in anyone of the tubes inoculated from the. residual fluid after a certain interval of incubation with one of the concentrations of reagent, is taken as a proof of positive isolation, the percentage. of positive isolations may be considered to be 92, since 11 out of 12 sputa used in the experiment gave growth. However, the sputa used were heavily laden with bacilli.

120

[December, 1927

TUBERCLE

(A) Urea as a Reagent Jor Destroying Contaminators. In order to study the possibility of using crystalline urea as a reagent or isolating tubercle bacilli, as recommended by Dold, powdered urea was introduced into a centrifuge tube containing positive sputum, and the standard technique, as previously used for sodium hydroxide, was followed. Four heavily positive sputa were used for each concentration of urea studied. The results are recorded in Table IV. Urea possesses an advantage in the ease with which it can be mixed with sputum, as well as in its solvent action on mucus-containing substances, and in that the resulting mixture is not so stringy as that resultiuc from the use of sodium hydroxide. But, as seen from Table IV, urea i~ practically valueless in the primary isolation of tubercle bacilli, since there results with its use a high percentage of contaminations, as well as only a small percentage of primary cultures of tubercle bacilli. TABLE V.~-THE ACTIO:" OF SODIUM CARBONATE (ANHYDROUS) ON THE CONTA)UNATORS IN TUBERCULOUS SPUTUM. •

I

},j

30 minutes

miuntes

1 hour

2 hours

Coneen- , Number f - - - - - . , - - - - - - + - - - - - ; - - - - - - , - - - - - - - f - - - - - - ; - - -

tration of reagent in the sputum

l'er

of

sputa used

I

I

Percentage Percent- Pt'lee~tage l'ereent- PelCeJ1t~gf" Psrcent- or posltl , e age of of POSIt!"" age or or po.~tlve 8g~ of, iaolattons contami- isolations ceutaml. isolations con .aml- of tubercle of tubercle of tubercle nations bacilli nations hacilli nations baeillt

!f-I--- :- - ~~ '-I~-TABLE

VI.

~~ --~--i--

\ .- --g-

Percent-

c:~~a~i. nations

-\~:

Percentage

or positive isolations of tubercte bacilli

-.

~

~·TH}~ ACTION OF A)[MONIUM HYDIIOXIDE ON THE CONTAMlN.\,rORS IN

TUBERCULOUS SPUTU)[.

0'1 1'0 3'5 7'0

2 4

]00

0

100

0

100

17

0

0

6

0

0

4

0

0

0 6 8

0 6 17

0

0

0 0 0 0

100 0 0 0

0 0 0 0

(B) Sodium Carbonate and Ammonium Hydroxide as Reagents for Destroying Contamina tors in I solating Tubercle Bacilli.

The exact reason for the slight to only moderate germicidal action of urea upon contaminators is not known, but it is probably, to some extent, associated with its alkaline reaction in concentrated solutions. For this reason, it was decided to determine the value of a few weakly basic substances, such as sodium carbonate and ammonium hydroxide, on the contaminating bacteria. The results of these studies are recorded in Tables V and VI. Table V reveals that sodium carbonate, even in concentration as high as 40 per cent., does not prevent the' development of the contaminators. This is probably accounted {or by the fact that it has a coagulating rather than a solvent effect upon the soluble substances (mucus) in the sputum which prevents an efficient germicidal action on the contaminators. Aside from permitting a large percentage of contaminations to occur, sodium carbonate also gave only a small percentage of primary cultures of tubercle bacilli, which makes it an unsatisfactory reagent for isolation. On the other hand, an outstanding feature of ammonium hydroxide towards bacteria in the sputum is its strong bactericidal action in concen-

December, 1927J

121

ISOLATION OF TUBERCLE BACILLI

trations of 1 per cent. or above. A comparison of Table III with Table VI indicates that this action of ammonium hydroxide is equal to that of sodium hydroxide. However, ammonium hydroxide yielded no positive cultures c:f tubercl~ ba~ilJi, whil~ with sodium hydro,xide a large percentage was obtained. ThIS difference IS probably due to differences in the degree of resistance of tubercle bacilli to the action of these two substances. Tubercle bacilli are highly resistant to the action of sodium hydroxide; for instance, Corper [12] and Soparkar [13J have reported that nearly all of the tubercle bacilli survived when in suspension in saline solution, mixed with an equal volume of a 5 per cent. sodium hydroxide, and incubated for one hour at 37° C. In contrast to this, the bacilli are highly sensitive to ammonium hydroxide, as is apparent from Table VII. A fine suspension of a young and actively growing culture of tubercle bacilli in physiological salt solution was mixed with an equal volume of ammonium-hydroxide solution of definite strength and, after incubation for a determined length of time, the mixture was diluted with five volumes of sterile distilled water TABLE VII.-THE ACTION OF AMMONIUM HYDROXIDE ON SUSPENSIONS OF HUMAN TUBERCLE BACILLI (" HUMAN ") IN SALINE SOLUTION,

Concentration ofNB. In the reagent

Specimen of bacillary suspension

Time of incubation of bacilli in ammonia xolut ion

Control wrthout ammoma treatment

ID minutes

I

I

30 minutes

I

1 hour

21lOurB

lneubntlon, in weeks, of cultnre tubes before being read ~--

1

2

3

1

2

3

A B

O· 0

1 1

2 2

0 0

0 0

0 0

A

0 1

2 2

3 3

0 0

0 0

1

-Per---cent. 3'3 1'0

B

2

1

I

2

3

1

o

0 0

0 0

0 0

o

0 0

0 0

1 0

0 0

0 0

--

2

i

3

1

2

S

o' 0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

-I-

• The readings recorded are the average of the readings of three culture tubes seeded. readings are graded from 0 = no visible growth to 4 = a profuse and luxuriant growth.

The

and centrifugated, and the centrifugate was seeded on Petroff's gentian violet egg medium. It is to be noted from Table VII that the time factor plays an important role in the action of ammonium hydroxide upon suspensions of human tubercle bacilli. Since the reagent was found to possess an efficient germicidal action toward the contaminators, it was thought that the elimination of incubation entirely might possibly lead to success in isolating tubercle bacilli. In order to determine this, two sets of experiments were performed. In one (series 1) only the incubation of the sputum reagent mixture was eliminated, the remainder of the technique being carried through as in the original standard procedure described above. In the second set (series 2) a few drops of an indicator (alizarin red) was first introduced into the centrifuge tube and, immediately after mixing the sputum and the reagent, the contents were neutralised with concentrated hydrochloric acid, after which the standard procedure was again adhered to. In both series ammonium hydroxide in concentrations of 1, 3'5 and 7 per cent. was used on four sputa for each concentration. In series 1 contaminations were rare, while in series 2 they were common, but in no 9

122

[December, 1927

'1'UBEUCLE

case was a detectable growth of tubercle bacilli obtained even after incubation .Ior ten weeks. (C) A mmonium Carbonate as a Reagent to Destroy Contaminators. The marked toxicity of the ammonium hydroxide for the tubercle bacilli was considered the main drawback to the use of this reagent, and it was reasoned that if a less toxic substance could be found that would nevertheless retain its strong germicidal action toward the contaminators, this reagent might prove highly satisfactory for the primary cultivation desired. Since ammonium carbonate is a salt formed by the union of a weak base and a weak acid, it hydrolyses readily in water, to yield free ammonium ions, and might possess the strong germicidal power of ammonia toward contaminators, yet be less toxic toward the tubercle bacilli. Therefore ammonium carbonate was tested on suspensions of "Human Avirulent" tubercle bacilli and all bacteria in sputa. As suspected, it had little toxic effect upon suspensions of tubercle bacilli up to the addition of an equal volume of a 20 per cent. solution (or 10 per cent. TABLE

VIII.-THE Eb'FECT OF

ACID UPO)[ SUSPENSIONS (lC HUlIlAN ").

SlJLPHURIC

TUBERCLE BACILLI

Period or incubation

Control without H,80,

trPlltrnen_t _ _ L')

COllcclltratiOlI ora,HO, used as rengent

20

\I

ith sulphuric aci1

--., - - - , - - - -\ - - - - - - - - ; - - -

millllte~

30 minlltt.!4

.~--

2 hours _

I_h_OI_lr_-'---

Specimen Pel iod in weeks aftel Beeding- when cultures were road

-Per- ---- cent. 10

1 - --

OF HUMAN

i

A B A B

1

O· i

0

0 ~ 0

-

2

-

1

:1

-

1 1

2 2

0 0

1 1

2 2

0 0

• The numerals have

t~e

.,

-- -I- - - !0 1 2 1 2 ~

1

3

3

1 _.-

~

--

3

-

-

-

2

3

--

1

2

0

1

2

0 0

1 1

2 2

0 0

1 1

2 2

0 0

1 I 0 2 I 0

0 0

1 1

0 0

0 0

1 0

0 0

0 0

0 0

\

I

same significance as In Table VII.

actual strength of ammonium carbonate) when the mixture was incubated for thirty minutes or less. 'With longer incubations of the same or higher concentrations its toxicity was greatly increased. Contaminating microorganisms in sputum were not killed by the addition of an equal volume of a 5 per cent. solution of ammonium carbonate (2·5 per cent. actual strength in the mixture) when incubated for thirty minutes or less, while longer incubation with this concentration destroyed most of them. With higher concentrations of ammonium carbonate (20 I?er cent.) most contaminating organisms were killed after even shorter Incubations, but no primary cultures of tubercle bacilli were obtained from sputa, and the margin of rermitting growth of contaminators without affecting the tubercle bacilli 10 sputum was nil, which could not be disclosed from the studies on suspensions of bacilli. (D) Sulphuric Acid as a Reagent for Destroying Contanunators. Of all the substances tested for destroying contaminators, sulphuric or hydrochloric acid proved to be equal, if not superior, to sodium hydroxide in the primary isolation of tubercle bacilli from contaminated sources:

December, 1927J

123

ISOLATION OF TUBERCLE BACILLI

Sulphuric acid, as recorded in Table VIII, does not inhibit growth of the tubercle bacilli when added in equal volume to suspensions of bacilli up to 20 per cent. (or 10 per cent. actual concentration in the mixture). In this concentration, no inhibitory action is noted when the mixture is incubated for thirty minutes or less, but after longer incubations a detrimental effect upon the bacilli results. 'With sputa most of the cultures were contaminated after treatment with 3 per cent. sulphuric acid, while with G per cent. or higher concentrations little contamination resulted. Since the preliminary experiments with positive sputa treated with sulphuric acid or hydrochloric acid gave encouraging results in isolating tubercle bacilli, a comparison of these two reagents with sodium hydroxide for the isolation of tubercle bacilli was undertaken. Table IX presents the results of a comparative study with sulphuric acid and sodium hydroxide. In order to make the comparisons as accurate l1S possible, the same sputa were used for both sulphuric acid and sodium hydroxide, and the inoculated culture tubes were kept in the same covered tin box in the same incubator in order to avoid variations in temperature and humidity. TABLE

IX, --A

COMPARISON OF SUI,PHURIC ACID AND SODIUM HYDROXIDE AS REAGENTS FOR THE IdOLATION OF TUBERCLE BACILLI.

Period or incubation of the sputum-reagent mi x ture

XumlJer

Reagent used

of

sputa te sted

15 min ute s

30 uunutes

1 hour

2 hours

r----;~-__t----;----t---__;_--+--~-­

Percent-

Percent- Percent- Percent- Percent . Percent - Percent- Percent-

eontam l-

Pf~~,:ve contami- p~·,V,e contami- p~RI~l\e contami-

.~

uations

p;;~~H.SO:- -4" - ---S3

~~

~~

.~

.~

tiOJ~:

nations

t~O a-

nation s

41

33

41

17

60

.~ ~i

&.

4

6 per cent. II.SO. 2 per cent , NaOn

6

22 22

50

5 11

10 per cent H~SO. 2 per cent NaOH

4 4

8

25 17

8

25

o

8

8

17

o

25

25

G

~~

Pj'slllVe

toa-

-r/- 91-- -~-R-IG7--I_~~8_ -G7--~~~-

3 2 per cent. NaOH

25

.~

nat.ions

GG

G6

17 17

i i

58

8

50 40

5 0

~

68

I

44 60

I 2g

The findings recorded in Table IX indicate that 3 per cent. H2S04 cannot be employed to destroy the contaminators because It is too weak a germicide in this concentration, while 10 per cent. sulphuric acid is not suitable because the percentage of resulting primary cultures is too low. In contrast to these an equal volume of G per cent. sulphuric acid gave good results in that the percentage of primary cultures obtained was slightly higher than that with sodium hydroxide and the first visible evidences of growth of tubercle bacilli occurred earlier than after the use of sodium hydroxide. Sulphuric acid possesses an added advantage over sodium hydroxide in that mucoid substances in the sputum are slightly soluble in strong acids but not in dilute acids. 'l'herefore, upon dilution of the sulphuric 'acid solution the liquid substances in the sputum coagulate and the coagulated materials on centrifugation materially assist in bringing down the tubercle bacilli to the bottom of the tube. Sodium hydroxide, on 't h e other hand, completely disolves the mucoid substances, and on dilution no precipitation of the dissolved substances takes place. A slight disadvantage exists in using sulphuric acid because a longer time

124

[December, 1927

1'UBERCLE

is required forra thorough mixing of the acid and the sputum; this slight disadvantage is, however, amply repaid by the greater advantage of sulphuric acid over sodium hydroxide in giving a larger percentage of primary cultures and resulting in an earlier development of the colonies. (E) Hydrochloric Acid as a Reagent for Isolating Tubercle Bacilli {rom Contaminated Materials.

On the whole hydrochloric acid acts like sulphuric acid, the main difference being a narrower range of serviceable concentration in which the hydrochloric acid can be used. Incubation for thirty minutes or longer of a mixture of equal parts of a suspension of tubercle bacilli and 10 per TABLE X.-THE ACTION OF HYDROCHLORIC ACID UPON

SUSPENSIONS OF HUMAN

TUBF:RCLlil BACILLI.

Period of Incubation with hydrocbloric acid Control not treated 1.'j

Concentration

ofHCI in reagent

minutes

I

30 minutes

I hour

hours

----

Time in weeks Letween seeding and when cultures weore read

~

1

1

:l

I ~ i

1

:1

2

3

I

1

I ~

-- - - - .--- - --1---1-- - - _ . - . 1I - - - Per cent. 10

l'

G

°

3

~

I

o

o

IIi ~ ----1--

0 0 j 0 0 1 2 1 0 011 2 ! 0 1 , 1 0

2301010 1 2 1 I 2 1 2 0 1, 2

°\

°

I

3

3

o 0 i 0 10 201 1

° ° °2

• The numerals have the same significance as in Table VII. TABLE XI.-COMPAIUSON OF HYDROCHLORIC ACID AND SODIUM HYDROXIDE AS REAGENTS FOR THE ISOLATION OF TUBERCLE BACILLI.

Period of incubation of the sputum-reagent mixture

15 minutes

, I 1.---;----+----,----+---_--,-_ _

Reagent used

, Percent-

age of

contamiI

nations

--------_. ---3 per cent. nci 2 per cent. NaOH

6 per cent. lICl 2 per cent. NaOH 1 per cent. HCl 2 per cent. NaOH

--------------------30 minutes i 1 hour I 2 hour.

..

..

.... ....

Percent- Percent- \ Percent- Percent- Percent. I Percent- Percent.~ .~ .~ .~ ·~I·~ .~ positive eoutamt. positive eontami- positive ecntami- positive isolations ~~I~'~~ ~~ ~tionH I ~i~ ~~

8 8

83 75

8 33

50 58

67 25

33 50

25

° °8

17 33

75 67

°

58 25

67 50

8 0

17 67

83

50

58 67

41

8

°

I

33 88

33 58

8

8 83

25 25

75 58

cent. hydrochloric acid completely ~estroys the. via~ilit.y. of the bacilli, while after the use of 6 per cent. acid only a slight inhibitory action on the growth of tubercle bacilli is discernible after incubation of from fifteen to thirty minutes. On sputum the optimum concentration which can be used for the isolation of tubercle bacilli was found to be about 3 per cent. W"ith 1 per cent. contamination.s ~ere frequ~nt, while with 6 per cent. no primary cultures of tubercle bacilli were obtained. In Table II are recorded the results of hydrochloric acid on positive sputa. as compared to a 2 per cent. sodium hydroxide.

December, 1927J

125

ISOLATION OF TUBERCLE BACILLI

(3) THE MEDIA USED FOR THE ISOLATION OF TUBERCLE BACILLI. Petroff's gentian violet egg medium has been considered the most satisfactory medium in most cases for the isolation of the tubercle bacillus from tuberculous materials. In this study an endeavour was made to improve on this medium, if possible, and also to evaluate it more accurately. Calmette's 1 potato glycerol bouillon medium [14J was found to be far superior to Petroff's when the tubercle bacilli were present in small numbers in the inoculum. Its only and a main disadvantage was found to be that TABLE XII.-GROWTH OF TUIlERCLE BACILLI ON DIFFERENT MEDIA WHEN SEEDED WITH DIFFERENT DILUTIONS OF SUSPENSIONS OF TUBERCLE BACILLI.

Millij(rams of bacilli per c.c,

Kind of medium

Type of bacilli

.c

------1 Glycerol Lon~'s - agar Gluckson

1

Petroff's Dorset's Calmette's..

Long's .. Glycerol agar Human ~ Petroff's .. Dorset 's ( Calmette's ..

I

Bovine virulent

0"

"'

9

~

~

§'

s

§

~ c;~ ~

§ 8 §

i

§

g g gig g g

1:"1l"""'lr-40000000000

+-?? ? 0 0 0 0 0 + + ? ? ? 0 0 0 0 + + + + ? ? 0 0 0 + + + + + + + + -++ + + -+- + -+- + + -+-+-

+ -++ +

+

+ + + +

?

? ?

0 ?

0 0 0

0 0 0

0 0 0

0 0 0

010 0 0

I 0

I 0?

+: + ?

0 0 0

I

0 0 0

0 0

0 0

0 0

0 0

0 0 0 0

0 0 0 0 ?

-+-

+ + ? ? + + -+- + -+- + ? ? ? + + + + + + -+- + + +

?

~~;;~:ol agar t t t ~ ~ I g g g g g g g g .. -+- + + + + -+- -+- + + ? ? 0 0 f Petroff's Dorset's -t- + -+- -+- -+t- -+- + -+? ? ? 0 t

Calmette's..

-+-

-+-

+ -+- -+-

-I-

+ + + + + + -+-

+ + ? 0 0 0 0 0 0 0 0 0 0 + -+- -+- ? 0 0 0 0 0 0 0 0 0 Bovine D + + -+- -+- -+- -+- + + + ? ? 0 0 ++ \-+1- + -+- -+- + I + ? ? ? ? ? 0 + -+- I-+-+ + + I + + + + + The markings recorded indicate the followmg: + = growth occurs in all the tubes seeded; LODg'S •• Glycerol agar Petroff's .. Dorset's .. Calmette's ..

•

?

= growth occurs only

in a few spots in some of the tubes; 0 = no growth of a.ny kind.

it permitted a greater number of contaminations when used for the actual isolation of tubercle bacilli from sputum. However, the addition of crystal violet to the medium removes this undesirable feature and makes it more satisfactory for the isolation of the bacilli. In order to compare the relative efficiency of various media in the primary cultivation of tubercle bacilli, the suspension method of seeding the media with graded quantities of bacilli has been found most suitable. The details of this method were published recently [15]. The method consists essentially of making fine suspensions containing different amounts of tubercle bacilli, from 50 to 0'000,000,001 mg. This method of study gives a fairly definite idea of the efficacy of a given medium for the growth I

tion

Thepotato-cylinder medium was called Oalmette'a Potato Medium, 'since its deseripWILS obtained

from Calmette's recent book.

12G

TUBERCLE

[December, 1927

of any tubercle bacilli present in a known concentration. The method consists in weighing out a definite amount of pure tubercle bacilli in a graduated centrifuge tube, and, after a thorough grinding and emulsification with a definite quantity of sterile physiological salt solution, different dilutions are made, and the bacilli in these diluted suspensions are seeded on the surface of various media. The media used in this study were Long's non-protein synthetic medium [16J, 5 per cent. glycerine-agar medium[17J, Petroff's gentian violet egg medium [18J, Dorset's egg medium [19J, and Calmette's potato glycerine bouillon medium [\:l0]. 'I' he results are recorded in 'l'able XII. Care is necessary in selecting youug and actively growing bacilli, in using fresh media that are not dried out or otherwise changed, and in making a satisfactory and fine suspension. In a recent paper [21] it was reported that tubercle bacilli failed to grow on Long's medium when seeded with a 10 mg. per c.c. suspension, which probably occurs only when excellent, fine suspensions free from clumps are used. Further tests showed that growth may take place in some of the culture tubes with suspensions of a bacillary concentration as low as 0'1 rug. per c.c.; although the growth in such low concentrations occurs in only a few isolated spots. With concentrations of 0'01 mg. per c.c., or below, growth never occurs in any of the tubes, even after twelve weeks' incubation. On 5 per cent. glycerine-agar all the tubes seeded with 1'0 mg. per c.c, or above (except strain "Gluckson in which case 10 mg. or more per c.c, was required) resulted positively, while 0'001 mg. or below gave no visible growths in auy tube. With Petroff's, Dorset's and Calmette's media the minimum concentration for growth and the maximum concentration for absence of growth have not yet been accurately determined, but the results thus far indicate that with Petroff's medium a bacillary concentration of 0'1 mg. or above per c.c, results positively, while with a concentration of 0'0001 mg. or less the transplants fail to grow in any tubes seeded with human bacilli (including both virulent and avirulent strains). With bovine bacilli (including both virulent and avirulent strains), the same medium yielded growth in a eoncentration as low as 0'0001 mg. pel' C.C., while at about 0'000,000,1 mg. growth does not take place, and with intermediate concentrations growth mayor may not .occur. With Dorset's and Oalmette's media these points of growth and extinction have not yet been established with certainty, but the" growth concsn; tration" (the smallest concentration with which all tubes seeded give positive results) and the "extinction concentration" (the greatest concen; tration of bacilli at which no growth is detectable in all tubes) for Dorset's medium seem to lie far below those for Petroff's medium and far above those for Oalmette's medium. When Dorset's and Calmette's media are compared where bacilli are in small numbers in the inoculum, growth on Calmette's medium is always visible one or two weeks earlier and always more luxuriantly than on Dorset's medium, showing the superiority of Calmette's over Petroff's and even over Dorset's medium. /I

(A) Comparison of Petroff sand Calmettes Media in the Isolation

01

Tubercle Bacilli from Sputum. In order to evaluate them further, a comparison of Petroff's and Calmette's media was made in the isolation of tubercle bacilli from positive sputa. Sixteen sputa were treated with 6 per cent. sulphuric acid and eight with 3 per cent. hydrochloric acid, and incubated for thirty minutes, after which three tubes each of Petroff's and Calmette's media were inoculated

Decem bel', 1927J

ISOLATION OF TUBERCLE BACILLI

127

from the sediment. The results were 55 per cent. of isolations and 12 per ce!lt. of contaminations ~n Pe.troff's medium after the 6 per cent. sulphuric acid and 56 per cent. of isolations and 25 per cent. of contaminations on Calmette's medium; while, after 3 per cent. hydrochloric acid, there were 46 per cent. of isolations and 21 per cent. of contaminations on Petroff's medium, and 21 per cent. of isolations and 67 per cent. of contaminations on Calmette's medium. At first glance these figures give the impression that Petroff's medium is superior to Calmette's, because the percentage of isolations appears to be greater or equally great, and the percentage of contaminations is lower. If, however, we analyse the findings with regard to the earliest appearance of growth, Calmette's medium proves superior to Petroff's and the colonies are always much more luxuriant on it. Of 48 tubes, seeded from 16 sputa after 6 per cent. sulphuric acid, and planted on Calmette's medium, there were 11 with growth appearing in the second week, 4 in the third, 7 in the fourth, 3 in the fifth and 2 in the sixth week, while on Petroff's medium 2 showed growth in the second, 6 in the third, '2 in the fourth, 2 in the fifth, 8 in the sixth, 1 in the seventh and 2 in the eighth week. Of 24 tubes seeded from 8 sputa after 3 per cent. hydrochloric acid and planted on Calmette's medium. there were 3 with growth in the third week, and 2 in the fourth, while on Petroff's medium growth appeared on 3 tubes in the fourth week, 3 in the fifth, 1 in the sixth, 1 in the seventh, 1 in the eighth and 2 in the eleventh week. These results make it evident that both contaminators and tubercle bacilli grow better on Calmette's medium and that contamination lowers its efficiency in isolatin~ tubercle bacilli as compared with Petroff's medium. If a satisfactory antiseptic could be incorporated in Calmette's medium to inhibit the growth of the contaminating micro-organisms, in which capacity gentian (or crystal) violet serves in Petroff's medium, it might prove superior in isolating tubercle bacilli. (B) Tests ioitl: Modified Calmette:e Media for the Isolation of Tubercle Bacilli. In an attempt to improve on Calmette's medium and adapt it for isolating tubercle bacilli from contaminated sources, media were prepared containing ground potato with varying proportions of glycerine and bouillon. These gave an excellent growth of tubercle bacilli when the latter were present in suspensions containing 1 mg. or more per c.c., but with decreasing concentrations of bacilli growth was absent. The next trials consisted in preparing potato cylinders, as in the original Calmette's medium, and soaking them in 1 per cent. sodium-carbonate solution containing different concentrations of crystal violet (National Aniline and Chemical Company). The potato cylinders, thus treated, were then set into 5 per cent. glycerine bouillon in suitable culture tubes. A preliminary study of the growth of tubercle bacilli on this crystal violet potato medium, seeded with graded suspensions of tubercle bacilli, showed that the higher concentrations of the dye inhibited growth, while in the lower concentrations the bacilli grew equally well if not better than on the original Calmette's medium. It is noted from Table XIII, a composite table of three experiments, that medium V (which was prepared by soaking potato cylinders in 1 per cent.. sodium-carbonate solution having a d.y~ concentration of 0'01 1,>er cent.) IS better for the growth of tubercle bacilli than Petroft's, but this concentration of dye exercises a slight inhibitory action on bacilli as compared with growth on the original Calmette's medium.

128

[December, 1927

TUBERCLE

In media VI and VII, prepared by soaking potato cylinders in 1 per cent, sodium-carbonate solution and containing, respectively, concentrations of O·~O~ and 0'000] per cent. of crystal violet, the bacilli grew as well as in the orIg!nal
Concentration of suspenslon used for "edill!:

-~;~~~p~~.~~ I --;~;::--l-O~~~g. per c.c.

Culture medium used

Intervals in weeks before cultures were read

_____.

.

I 4_1_ 5_

.

, 1* ' 3 Calmette's Petroff's •• I o ! 3 V. Potato cylinder with 0'01 per cent. 0 4 crysta.l violet 4 VI. Potato cylinder with 0'001 per cent. 1 crysta.I violet 4 VII. Potato cylinder with 0-0001 per cont. 1 crystal violet

I

~

" 4_ !_6 :_

, 8_ 1_4__ ~ I__

4 1 ~4 4 '02 a 4001000 4001001 4

4

1

o

4 4

4

o

2

a 3

I

• The numerals have the same significance as in Table VII.

per cent. isolations on potato plus 001 per cent. crystal violet medium with no contaminations, 75 per cent. isolations on potato plus 0'001 per cent. crystal violet with 17 per cent. contaminations, and 50 per cent. isolations on potato plus 0·0001 per cent. crystal violet with 25 per cent. contaminations. These findings again emphasise the importance of concentration of the dye in the development of contaminators, the weaker concentrations being associated with more contaminations. In another set of experiments with positive sputa it was found that the best concentration of crystal violet for Impregnating the potato was 0'003 per cent., or about 1 to 75.000 dilution. (C) A New Method for the Primary Isolation of Tubercle Bacilli from Contaminated Sources. The foregoing experiments have led us to propose the following new method for the primary isolation of tubercle bacilli from sputa or contaminated materials :Preparation of the llIedium.-Fresh, clean, large potatoes, preferably without surface defects, are cut into cylinders of 3 in. or more in length with a cork-borer of a. diameter of about i in. The cylinders are then cut longitudinally into halves. These are then soaked in 1 per cent. sodiumcarbonate solution, containing crystal violet (or gentian violet) in concentration of 0·003 per cent. (1 to 75,000) for one to two hours.' At the end of this time the potato cylinders are gently wiped with a towel to free them from the excess of liquid, and are then introduced into a sterile culture tube (1 X (j in.) containing 1'5 c.c, of 5 per cent. glycerine bouillon. The tubes 1 The dye solution must be added to the sodium carbonate solution immediately before use, since prolonged sta.nding causes a decolorizing of the crystal violet.

December, 1!:J27]

ISOLATION OF 'fUBERCLE BACILLI

129

are plugged with cotton and sterilised in the autoclave for thirty minutes at 15 lb. pressure. . Treatment of Sputum. and Seeding.-The sputum or contaminated material is first thoroughly beaten to a homogeneous pulp, and 1 c.c. of this is introduced into a sterile centrifuge tube. One cubic centimetre of 6 per cent. sulphuric acid (or 3 per cent. hydrochloric acid) is added, and the contents are thoroughly mixed. Too much emphasis cannot be laid upon the thoroughness of mixing, since the opportunity for destroying the contaminators depends upon it. The tube is stoppered with a sterile cork and incubated for thirty minutes at 37° C. The contents of the tube are then diluted to 10 c.c. with sterile normal salt solution and mixed. After centrifugating at moderate speed, the supernatant liquid is decanted, leaving 1 to 2 c.c. of residue. This is stirred up with a sterile pipette, and three to four drops of the mixture are spread over the surface of the potato crystal violet medium. After cotton-plugging and paraffining the plug with a tinfoil cap, the culture tubes are incubated at 37°0. 'Within two to five weeks a luxuriant growth of tubercle bacilli may be expected from the positive sputa. SUMMARY AND CONCLUSIONS.

(1) Crystal violet, accepted by the Commission on Standardisation of Bioloaical Stains,' to replace the pre-war gentian violet of uncertain compositi~n, is a suitable material for incorporation in egg media, as recommended by Petroff to inhibit the development of contaminators during the primary isolation of tubercle bacilli from contaminated sources. The concentration of crystal violet required is about the same in egg media as that recommended for gentian violet. (2) Of a large number of reagents tested (urea, sodium carbonate, ammonium hydroxide, ammonium carbonate, sodium hydroxide, and sulphuric or ~ydrochloric ';l-cids) to.destr?y the rapidly gr~n,:ing micro-organisms in contammated materials for isolating tubercle bacilli, sulphuric acid was found most serviceable. It possessed a wider range between its being innocuous for tubercle bacilli and its being capable of destroying undesirable contaminators than any of the others tested. Hydrochloric acid and sodium hydroxide also proved satisfactory for this purpose, the former slightly more so than the latter ; while urea, sodium carbonate, ammonium hydroxide and ammonium carbonate proved entirely unsatisfactory. (3) In quantitative seeding tests with suspensions of tubercle bacilli, Calmette's potato medium was found to be superior to Dorset's egg medium, Petroff's gentian violet egg medium, glycerol-agar, and Long's non-protein medium, in favouring the growth of the bacilli when present in small numbers. The other media were found serviceable in this respect in the order ~iven above. (-if Crystal violet potato medium, in which the potato cylinders were treated with 0'003 per cent. crystal violet solution, proved suitable for the isolation of small numbers of tubercle bacilli in sputum. This medium proved superior to Petroff's gentian violet egg medium, and possessed an advantage over plain potato media, in being less favourable for the growth of contaminators, without detrimentally affecting the growth of tubercle bacilli. (5) A new method for the isolation of tubercle bacilli from contaminated 1 The st ains used in the se experiments were kindly furni shed by Dr. II. J. Conn, Chairman of the Commiasion, to whom we are grateful.

130

TUBERCLE

[December, 1927

s0!lrces is proposed, using 6 per cent. sulphuric acid at 37°0. for thirty minutes in the preliminary .t reatment or the tuberculous material, and crystal violet potato medium for culturing the bacilli . The authors are grateful to Mr. Harry Ogintz for assistance III some of the technical work of this study. REFERENCES. [IJ GUIFF!TH, A. S. "Further Investigations on the Type of Tubercle Ba.cilli occurring in the Sputum of Phthisical Persous," Brit. Med. [ourn., 1914, 1,1171. [2] PgTHOFF, S. A. "A New and Rapid Method for the Isolation and Cultivation of Tubercle Bacilli directly from the Sputum and Freces," Journ, Exp . Med., 1915, 21, 38; "Some Cultural Studies on Tubercle Bacilli," Bull. Johns Ilopkins Hosp., 1915, 26, 276. [3] LUUIE, M. B. "A Comparison of the Sodium Hydroxide and Antiformin Methods for Cultivating Tubercle Bacilli," Amer. Rev . Tuberc., 1923, 7,332. [4] MlTCHELL, Q. W. II., and SIMMONS, R. R. II Isolation of Bacillus Tuberculosis from Sputum by Method of Petroff," JOUnL . Amer. Med, Assoc., 1915, 65, 245. [5 J KEILTY, R. A. "A Study of the Cultivation of the Tubercle Bacillus directly from tbe Sputum by the Method of Petroff," J OIlTn . Exp . ..lIed., 1915 22,612. ' [6] STEWAUT, F. C. "A Note on Petroff's Cultural Method for the Isolation of 'l'u.bercle Bacilli from Sputum and its Application to the Study of Milk," iua., 1917, 26, 755. [71 CORPEH, II. ,T., FlAU, L., and KALLKN, L. "The Routine Cultivation of T~bercle Bacilli Irom the Sputum by Petroff's Method," JOUnt. Infect. DIS., 1918, 23, 267. [tjJ DOLD, II. "Beitriige zur Frage der Wirkung des II arnstoffes auf Bakterien,' Centralbl, j. Bact., Oriq., 1923·4, 91, 268; "Ein neues Verfahren zur Homogeoisierung von Sputum, Anreicherung und ReinzGchtung von Tuberkelbacillen," Beitr. z. Kli». d. Tuberk., 1924, 58, 335. [9] LOWENSTEIN, E . "Direct Cultivation of Tubercle Bacilli," TVien. klin, Wochenschr., 1924, 38, 231. [10] CONN, II. J. "Biological Stains," Geneva, N.Y., 1925, 69. [11] CORPER, II. J . "Methods of Staining Tubercle Bacilli," JOUnt. Lab. and Olin . Med., 1926, 11, 503. [12J Idem. .. The Virulence of the Tubercle Bacilli isolated from the Sputum," JOUr/t. Infect. Dis., 1918, 23, 493. [13] SOPAHKAR, M. B. "The Cultivation of Tubercle Bacillus directly frOlu Sputum and Post-mortem Material," Indian Jousn , Med, Research, 1916-17,4, 28. [14J CALMETTE, A. ".L'infection baeillaire et la tuberculose chez l'homme et chea les animaux," Masson & Cie, 1922, p. 32. [15] UYEI, N. "Food Accessory Substances and Tubercle Bacilli. 1. Do Tubercle Bacilli Contain or Produce Growth Promoting Substances?" Jaunt. Infect. Dis., 1927, 40, 423. [16] LONG, E . R. "A Review on some Recent Studies on the Metabolism of the Tubercle Bacillus and on the Nature of Tuberculin," Tubercle, 1924,6, 128. [17] NOCUID, M. M. , and Raux. "Sur 1110 culture du bacille de 1110 tuberculose," Ann. de l'Inst. Past. 1887,1, 19.

[18] Loc. cit.

[19] DOUSET, M. "The Use of Eggs as a Medium for the Cultivation of the Bacillus Tuberculosis," Amer. Med. (Philadelphia), 1902, 3, 555.

[20] Lac. cit. (21] Loc. cit.