Streptomycin

Streptomycin ~hwulConsiderations,

Tests for Bacterial

oJ hfeasurement bT.\i.~ .\(:P: E.

of Streptomycin in Body Fluids

HEIWIL,

hf.D.

and

*

ROCXIXI’ER.

T

IIE antibiotic

agent,

was first described and

Waksman’

Sensitivity arid A4ethod.~

I<.

HEIl.hf.w,

hl.1).

*

MINNESOI‘A

inhibitory

streptomycin,

by Schatz? Bugie in January,

FORDKE

effect on Mycobacteriuin

culosis in vivo was first reported and Hinshaw.”

1944.

tttbcr-

by Feldman

Thes.e and other studies led

‘I‘hc substance was produced by an actinomycetc which had been discovered and

to a rather clinical trial

dcscribcd

ment of tuberculosis. The results of such studies will be discussed in other articles in

some years previously

by Waks-

man. This actinomycete was subsequently placed in the genus Streptomyces by Waksman and Henrici” and is now known as “Strcptomyccs griseus.” The newly discovered 1hc

and

antibiotic was consequently “streptomycin.” Schatz,

IlarllC

Waksman

suggested

that

this

the present symposium. Further studies by the investigators at Kutgcrs University6 revealed that strcpto-

given Bugic

mycin was effective in the treatment of certain experimental infections owing to Salmonella schottmiilleri, Pseudomonas ac-

anti-

ruginosa,

I)actcrial agent possessed properties which inight make it useful in the treatment of disease caused by certain gram-negative

such microbes

as Eschcrichia

clinical

ahortus. to-ials

to be of outof clinical in-

fections due to the organismsjust mentioned. It was evident from the in rlitrnand in r~iwt studies reported by one of us (Heilman)‘,” that streptomycin possibilities in the

evident from the original reports that in the group of organisms which might be inof streptomycin

subsequent

have not proved streptomycin standing \ralue in the trcatmcnt

organic base. It is rather thermostabilc. It is not soluble in ether or chloroform but it is soluble in water and dilute acids. It was

by thr action

fowl typhoid and Brucrlla

Unfortunately,

as well as some gram-positive pathogens. Streptomycin behaves chemically as an

hibited

intensive experimental and of streptomycin in the treat-

due and

were

coli, Bacillus

possessed thcrapeutit treatment of infections

to Pasteurella tularensis (tularcmia) infections due to organisms of the

Friedlander group (Klebsiella). carried out at the Mayo

From studies Clinic”
subtilis, .2erobactcr aerogenes, Proteus vuland some species of Salmonella. saris I ,ikrwisc, it appeared that streptomycin possessed a limited suppressive effect on

elsewhere’O it was evident that streptomycin possessed antibacterial activity against Hcmophilus influenzae. The studies reported

I’seuclomonas aeruginosa. ‘That LIycobacterium tuberculosis was scnsitivc to the action of streptomycin in ilitr0 was suggested by the reports by Waksman. Rugie and Schatz” and by Schatz and Wa ksman. i That streptomycin exerted an

by Hegarty, Thiele and Verwcy” iudicatcd likewise that streptomycin possessed value in the treatment of experimental infections owing to Hemophilus pertussis. While streptomycin has been used satisfactorily in the treatment of clinical cases of infection

* From hblPKL,:.,S

the Division

[OT_‘RNAL

OF

of Medicine MEDICINE

and the Section

on Ractwiolog~-,

421

Mayo

Clinic,

Rochester,

Minn.

422

Heilman

Streptomycin-Herrell,

due to Hemophilus its possible reported

influenzae,

use in pertussis

studies

on

have not been

at the time of this writing.

Streptomycin the growth microbes.

has been

found

in vitro of a great In this article

number

of

which

lus

paratyphi

enteritidis

dysenteriae,

to inhibit

we are concerned

only with those organisms

Salmonella monella

Pseudomonas

pyocyaneus),

(some strains), abortus

are con-

With

(some

Sal-

Shigella

aeruginosa

(Bacil-

Staphylococcus

Brucella

and Brucella certain

strains),

(some strains),

aureus

melitensis,

Brucclla

suis.

exceptions,

the sensitivity

of

sidered pathogenic

for man. The organisms

organisms

which

may

in vitro studies can be used as an index of the

at

practical

present

be considered

purposes to be sensitive

for

and which

are pathogenic for man are listed in Table I. It should be emphasized, however, that TABLE I ANTIBACTERIAL

ACTION

OF

sensitivity

of

these

organisms

probable

effectiveness

treatment

of

as determined

by

of the antibiotic

clinical

infections.

It

in was

evident from even the earliest experimental studies that the variation in sensitivity of different strains of the same organisms to the action of streptomycin was of consider-

STREPTOMYCIN

Organisms Sensitive to Streptomycin Escherichia coli Eberthella typhosa Salmonella paratyphi (some strains) Salmonella enteritidis (some strains) Shigella dysenteriae Proteus vulgaris Aerobacter aerogenes Pseudomonas aeruginosa (Bacillus pyocyaneus) Klebsiella pneumoniae Hemophilus influenzae Hemophilus pertussis Staphylococcus aureus (some strains) Mycobacterium tuberculosis Brucella melitensis Brucella abortus Brucella suis Pasteurella tularensis Pasteurella pestis

the

to streptomycin

able importance from a therapeutic of view. Different strains of the bacterial

species

may

vary

markedly

their sensitivity to streptomycin. mediately suggests the importance the

sensitivity

of the

strain

point same in

This imof testing

isolated

from

the patient before and during treatment. This necessitates a close collaboration between the clinician and the laboratory worker in the management of patients suffering from infections in which streptomycin may be used as a therapeutic Two other important

varies

greatly. It should be emphasized further that the inclusion of an organism in this list does not mean that streptomycin has proved effective in the treatment of clinical

be

mentioned

therapeutic definite

in

connection in

the

should

with

use of streptomycin.

variation

agent.

considerations

the

One is the

absorption

and

the

excretion of streptomycin by different patients or by the same patient at different

purpose of the discussion the organisms might well be listed in two groups: (1) those that are rather highly sensitive and (2) those that are moderately sensitive. In the group of organisms which could be considered rather highly sensitive are placed Pasteurella tularensis, Pasteurella pestis, Hemophilus influenzae, Hemophilus pertussis, Klebsiella pneumoniae, Escherichia coli, Aerobacter aerogenes, Proteus vulgaris and Mycobacterium tuberculosis. The organisms which could be considered only moderately sensitive are Eberthella typhosa,

evaluation of the antibiotic agent difficult at times. Second, but of no less importance, is the ability of certain pathogens to develop resistance to streptomycin. Some strains and species of organisms may develop resistance to streptomycin with incredible rapidity. This has been demonstrated repeatedly both in vitro and in vivo. This observation is exceedingly important from a clinical standpoint. Buggs and his associates12 have pointed out? however, the difficulties which may be encountered in

infections

due

to that

organism.

For

times. This variation

AMERICAN

may make the clinical

JOURNAL

OF

MEDIClNE

studying

this problem

clinically.

cult at times to determine the same organism

or not

is being isolated

at dif-

ferent times from a given patient. organisms ~JUL

may

develop

develop

in the body. From a clinical i tnportant

to

Moreover,

resistance

will not necessarily remember

in uitro resistance

standpoint that

Much has been learned concerning

It is dish-

whether

it is

organisms

sorption,

diffusion It

mycin. made

was

by

evident

readily

from

following

intravenous

the

reports

its

injection,

-‘!‘I that

intramuscular diffuses

rather

bodv tissues. Followink; streptomycin c;3miot administration,

oral

into

t’he ab-

of strepto-

investigators”’

various

streptomycin, or

and excretion

most

\vliich easily can be made resistant to strepto-

be detected

inycin in vitro at times may retain their sensi-

blood stream.

~i\,ity in patients although the patient has received repeated courses of streptomycin. All of the previously mentioned facts

biotic is not destroyed in the Aastl,ointt.stirlaI tract and large portions of the rriaterial administered

entphasize lal)oratory

the feces. It exerts an antibacterial eff’ec~t on the intestinal flora and this ot)scrvation

the importance of adequate methods of assay and adequate

in significant

orally

suggests

its use when

bacterial

content

Furthermore,

Satisfactory streptomycin

ously stated, certain adopted in the clinical

of the reasons

previ-

dictums have been use of streptomycin.

Because of the development of resistance, it is rsscntial that bacteria be eradicated as cornplctely as possible in the shortest possiLyle time if good clinical results are to be obtained.

This rule implies

the administra-

in tht* tht, anti-

can t)c rec~o\~c~recl from

methods for testing bacterial sensitivit\ which will be discussed later in this paper. because

amounts

On the other hand,

a reduction

in the

of the bowel is desired.

therapeutic will appear

concentrations 01 in the blood and

urine following intermittent intramuscular or subcutaneous

intra~~enous, adrninistra-

tion. Approximately 60 to 80 per (~‘rit ol’ streptomycin injected is excreted b\- the kidneys and may be recovered in the urine. It should be pointed out, however. that

tion of large doses of streptomycin from the onset of treatment. It also calls for frequent recoursr to the laboratory for determination

streptomycin at times may accumulate to toxic levels in the blood stream of patients

of the sensitivity if the infection

appears

of the organism especially is not responding satis-

factorily to treatment. The removal 01 foreign bodies and the eradication of foci before or soon after by surgical means treatment

is

begun

is

important.

'l‘hC

presence of foreign bodies or foci favors the continuation of infection and thereby favors tile possibility of the development of resistance on the part of the infecting organism. In the treatment of infections in which stasis and obstruction play a r61e, such as in infections of the urinary tract, it is important that these two factors be eliminated. Since it is known that streptomycin exerts its maximal antibacterial effect in the presence of an alkaline medium, it is suggested that the urine should be kept alkaline.

who have poor renal function.

to diffuse into the peritoneal

in substantial early

amounts

peritonitis.

diffuse

readily

amounts spinal

Streptomycin

appear

Streptomycin

readily When

into the

of meningitis.

to diffuse through

thereby

reaches

the

It does not appear

into empyema to be

streptomycin

tracheobronchial

cavities. excreted

the frtal

to diRusc Strcptomy-

in

is introduced

the into

bile. t trr

tree by means of ncbuliza-

tion, it is not absorbed in significant

not Auid

diffuses into the tissues oi‘ the

and

appears

of

therapeutic

to diffuse readily

eye and also appears placenta

does

however.

fluid in the presence

circulation.

c.avity

in the presence

into the ceretJrospina1

of normal individuals;

tin

Strcptoniycin

amounts.

into the blr~od stream

424

Streptomycin-Herd,

When

streptomycin

the unit of potency

was first introduced,

potency

activity.

was based

material

required

a given

strain

known

as

the

Recently, adopted

in

streptomycin. streptomycin

on that

The

to inhibit “S”

unit

metric One

coli. of

with

minimal

1 Gm. to at present

dosage of

of pure

equivalent 1,000 S

l,OOO,OOO S units. indicates that the should

infection, as much as 5 Gm. per day may be given. For intermittent intravenous or intramuscular injections, the total daily dose is dissolved in 16 cc. of physiologic saline water. An average of is injected every three

hours. In some instances, satisfactory results may be obtained by making larger injections of four or six hours. The recom-

mended daily dose of streptomycin for oral administration is 2 to 4 Gm. in four divided doses.

For

streptomycin

intrathecal

quantity

administration

it is recommended

mg. of streptomycin 10 cc. of physiologic

that

of 100

be dissolved in 5 or saline solution. This

may be administered

every twenty-

four to forty-eight hours. For nebulization, the concentration recommended is, as a rule, 50 mg. per cc. of physiologic saline solution. For local application, concentrations of the drug which have been used vary from 10 to 100 mg. per cc. CLINICAL

menin-

of the

urinary

fever. Streptomycin

used locally

wounds infected with organisms sensitive

to its action.

also

in the treatment

of

known to be

A detailed

discussion

articles

been

be 1 to 3 Gm. (l,OOO,OOO to 3,000,OOO S units). In the treatment of overwhelming

at intervals

tract and undulant has been

influenza1

infections

has

daily dose of streptomycin

solution or distilled 2 cc. of this solution

peritonitis,

tularemia,

of these results will be dealt with in separate

microgram

is approximately

of

It was

gitis,

Waksman.

to 1 S unit; 1 mg. to approximately units and Experience

of

of the

the growth

system

connection

unit

amount

of Escherichia

the

endocarditis,

was defined on the basis

of its antibacterial

Heilman

TRIALS

In recent years streptomycin has been subjected to rather extensive clinical trials by a host of investigators. These trials have been limited, for the most part, to tuberculosis, bacteremia and subacute bacterial

in this

experiences variety

symposium.

FOR

THE

clinical

in the use of streptomycin

of bacterial

the Mayo Clinic elsewhere.21-2”

TESTS

Some

infections have

SENSITIVITY

been

OF

GROWTH-INHIBITING OF

in a

treated

at

reported

BACTERIA

TO

EFFECT

STREPTOMYCIN

The activity of streptomycin in a bacteriologic medium is affected by the pH and by the presence of cysteine, sodium thioglycollate and other reducing substances. 3*26-28 In a medium highly favorable to bacterial growth more streptomycin may be required to inhibit growth of a given strain of bacteria than in a medium of deficient nutritional value.2g,30 Since streptomycin becomes active if the substrate is acid or is in a duced state, tes,ts of sensitivity should carried out in mediums containing

less rebe no

fermentable sugar and adjusted to a pH close to neutrality, and under aerobic conditions. There are several methods of testing the sensitivity of bacteria to streptomycin. In one of these the test is carried out in a series of tubes, containing liquid medium suitable for growth of the organism, to which various amounts of the antibioticl’ have been added. The liquid medium in the tubes is inoculated with a drop of a dilute suspension in broth of the organism and incubated for eighteen hours or until good growth appears in the control tube. The lowest concentration of streptomycin which completely inhibits growth is recorded. Since the end point of growth may be difficult to determine by inspection, a loopful of material from each AMERICAN JOURNAL

QF

MEDICINE

of

the tubes

naar

may

be

completely

to determine

in

then is recorded.

the end point

strc*akrd on an agar plate

which tubes growth has or has not occurred. Prolonged opment point.

‘I’hc

inoculum contain

incubation

of resistant

may allow the devel-

forms and alter the end

larger

thr greater

the

some of the more rrsistant

thr plates in an atmosphere

in determining

nutrient agar adjusted to pH 7.2 IO 7.4. Blood agar is used for more fastidious organisrns. Scv~i plates are prepared in which by careful

mixing,

prc=vious to solidification of the agar, SO, 25, 1l?.i, 0.75, 3.12, 1.56 and 0 units (micrograms) of strrptomycin per cc., respWtively. In order that sevFra1 different strains of baclrria may be tested on one set of plates, c*ach platra is divided into several sectors by marking marking

the back with a pencil used fol glass. ‘l’he sectors on each plate are o11

abortus tension

of carbon

dioxide.

Placing

a stock strain sensitivity is sectors of each strain is a trst the plates and decision as to

whether or not a patient is to be treated with streptomycin often rests on the results of this test of sensitivity. The inoculated plates are incubated at 37”~. overnight or until there is ,good growth of the test strain on the control plate containing no streptomycin. The lowest concentration of streptomycin which

in-

ol’ 10 per cent

carbon dioxide lessens (he activity ofstreptomycin

in the. test plates,

1owPring

the

11f.1 of

prcsuinat)Iy

thr

nirdillln.

I,) Al‘lcr

scvrral subcultures, the carljon dioxidt* requiremrnts of such strains arc* dccrcased somctimrs.

‘l’he or,yanisni then will grow in

an atmosphere

of 2 to 3 per W~I carbon

dioxide which concrntration will not ,alter the pH of medium SignificantI?, and, thereforr, will not alter the results of thr test 01 srnsitivity to streptomycin. ‘1‘he standard

solution

of strcptornyc+lj

used in making thcl dilutions for thr prrparation of the test plates is stable and ma:y t)(a kept in a refrigerator in a sterile corked I.ubc for several weeks without significant loss of titrr. Simpler

methods,

mcasuremcnts of an organism able.

Plates

tomycin able

less

to streptomycin,

seeded various

in

are avail-

with the organism.

on

cups filled with solutions concentrations

Schleicher

clipped

accurate

of scnsi tivi ty

or disks of blotting

from

York)

giving

of the degree

containing

suspension. A suspension of of staphylococci of known always strcakcd on one of the plates. ‘l’hc use of this stock of the proper preparation of is 01’ importance since the

of

an

organisms

and a

to

strains

requircb

which are placed

to be tested are prepared

may

which

nurnbcrc*d. IAlutc suspensions in nutrirnt Ijroth (which contains no sugar) of the loopful of each suspension is streaked on each platcb over the sector assigned to that

strain

the sensitivity isolated

or,e-anisms

on agar plates containing various amounts of streptomycin. ‘l’hp plates are prepared fro111

incorporated

in testing of frrshly

creased

streptomycin arc carried out routinely 11); srrraking a dilute suspension of the organism

ha\,ch Ixwl

be encountered streptomycin

that it will

tile end point of yrowlh in liyuid mcdiullls, in our laboratory tesLs for sensitivity (0

of eaclr

If thr plate method is used, difficulty

Rrucella

which are usually present in an)- cultural. ljcscause 01‘ the difficulty

growlh

bacterial

primary

the chance

inhibits

such

paper

and

of strep(ohtain-

Schuell,

solutions

prepared. ‘I’ A4fter incubation,

New

may

be

inhibition

of

growth around the cups or disks is noted and compared plates

with

seeded

sensitivity.

similar

with an organism

Another

method

ping a disk of blotting containing

of knobvn

paper

in a solution

it on a plate of nutrient

per CC. agar.

to be tested are streaked outward

from the periphery distance

011

consists in dip-

20 units of streptomycin

and placing Organisms

preparations

from

of the paper disk and thr

the disk that

growth

ii; irl-

426

Streptomycin-Herd,

hibited

after

incubation

is an

index

of

sensitivity. OF STREPTOMYCIN

IN BODY

FLUIDS

Streptomycin

in serum,

urine

body fluids is often measured material

or other

by noting

volume

of such

added

to a liquid

inhibit

the growth of a test organism.

which

bacteriologic

is measured

the

which

is preserved

in

mycin

It also various

test by adding

are that

the

body fluid to be tested must be sterile; the end point of growth in liquid mediums is often difficult to determine; different amounts of body fluid may have different growth promoting properties for the test 4

organism, and end points between the dilution intervals used cannot be detected. In our laboratory, a method of using cups on agar plates which is similar in principle to that described by Stebbins and Robinson3” is favored. This method does not accurately measure concentrations of streptomycin of less than 1 unit per cc. but since weaker concentrations are of doubtful therapeutic effectiveness, the method is sufficiently sensitive for general use. The test organism is a strain of Staphylococcus aureus which on agar gives relatively sharp margins at the edges of the zones of inhibition. The test organism is maintained in nutrient broth by daily transfer. Since growth of the organism for prolonged periods on broth has yielded variants which have lessened

the

The test is carried out in a system adjusted

to

Such methods are somewhat more sensitive than the cup-plate method and simpler to disadvantages

slant culture refrigerator.

medium

various amounts of the serum or other fluid to be tested to liquid mediums containing the test organism have been described.15!32’33

Their

at the end of a week

to pH 8. This pH is chosen because

by making

the

is discarded

and a new series is started from a stock agar

be

with a test organism and after incubation measuring the zone of inhibition of growth around the cups. Methods of making

in broth

of the edges of zones of inhibithe cups, the series of cultures

must

dilutions of the serum, placing these dilutions in cups on an agar medium inoculated

perform.

the sharpness tion around

MEASUREMENT

frequently

Heilman

is more

neutrality.

active

All dilutions

in a sterile

tenth-molar

pared from potassium

at

pH

strepto-

8 than

at

in the test are made pH 8 buffer, phosphate

pre-

(KHzP04

and KzHP0.J. Nutrient agar adjusted to pH 8 is used as the test medium. A commercial dehydrated medium with the pH already adjusted and known as “Streptomycin Assay Agar”

(Difco)

is satisfactory

for the

purpose. To furnish a perfectly flat surface for the test 12 cc. of unseeded melted agar is placed in each of a series of Petri dishes and allowed to harden. A second portion of melted agar is cooled carefully in warm water to 44” to 45”~. and inoculated with a broth culture of staphylococci which has been incubated for six hours. One cc. of a 1 in 100 dilution of this broth culture in a buffer solution is used for each 9 cc. of agar. A final dilution of lop3 of the staphylococcal culture is obtained. The inoculated agar is agitated to distribute the organisms With a warm, widemouthed pipette the seeded agar is distributed

evenly. 5 cc. of

over the sur-

face of the first layer of agar on each plate while the plate is rotated so that the seeded agar forms an even layer. Sterile beveled porcelain or glass cylinders, such as are used in the assay of penicillin (sold under the trade name of penicylinders), are warmed slightly in a flame and placed on the surface of the hardened agar. These cylinders should be just warm enough to seal the beveled surface in the agar. Four or five sterile cylinders are placed on each plate. Four or more serial 1: 1 dilutions in buffer of the samples of body fluids for assay are prepared, the AMERICAN JOURNAL

OF

MEDICINE

Streptomycin-Herrebl, numhpr

of

pxpectcd

dilutions

depending

concentration

For thr standard,

of

dilutions

in huffcr arc prepared of the trst

iq pJacPrl in ii separate I)ipcttc;

which contain sample

1, 2, ?I

Each of the and

standard

with a capillar).

cup

each cup is nearly filled.

tclsts arc set up on a srparate all of the, samples

the

of streptomycin

and 4 units per cc., respectively. dilutions

on

streptomycin.

IIuplicatc

set of plates fol

as well as the standard.

‘l’hr Petri dishes arc covered

with unglazed

Heilman

are averaged

to give the concentration

in thr

body fluid. Specimens

contaminated

with

may bc assayed by this method.

bac,tc-ria

‘Two cc. of’

a sample is required for an assay. With assays of urine, thr

plates

incubator rcfrigcralor

if it is desired may

directly

without preliminary

test,

in thr

storage in t hr

sit1c.r urine diffuses rapidly front

the cups. Samples being

to hurry thr

bc placed

assayed

of the original

should

sper~mrn

be preserved

in thr

from

cold in cast the assay has to bc rrpca tcbd at

c~ondcnsed wa trr and placed in thr rcfrig-rrator overnight to allow the material to dif-

higher dilutions. Disks of blotting paper dipped in the trst fluid or mrasurrd drops of

porcelain

tops

to prevent

dripping

fuac from the cups into the agar. Following this the plates are incubated for twentyfour

hours

at

30’~.

Then

the

cups

arc

test

fluid

placed

directly

agar are used in place workers.36s37 If only

an

estimate

on

the

of cups of the

seeded

by some

amount

of

removed from the plates and the diameter of the zones of inhibition of growth of the staphylococci is estimated to the nearest 0.2 mm., preferably by means of a colony

streptomycin is desired, the test may bc simplified.. Several cups placed on a platr of seeded agar arc filled with various dilutions

counter equipped 10 lines to the

in saline :solution of the sample to be tested and the plates are incubated at XI” or 37”~:.

with a glass plate centimeter. Such

ruled ruled

class plates arr available from commercial sources. ‘l’he diameters of the zones from duplicate c:ups arr averaged. A curve is drawn on arithmetic graph paper by plotting the diamctcr of the zones of inhibition of the cups containing the standard streptomycin solutions

on the ordinate

against

the con-

c.entrations in units of streptomycin per cc. of fluid on the abscissa. For a model, one of sevrral

references

rnay

be

consulted.3”“7

From the standard curve the concentration of the druy in the sample under test can be read by noting the concentration of streptomycin on the abscissa of the standard curve whic.h corresponds on the ordinate to the diameter of the zone of inhibition around the samples. This reading should be multiplied by the dilution of the body fluid used in the CUPS. If sizes of the zones of two different dilutions of the sample fall within the range of the standard curve, the concentration of each dilution is calculated and the results

overnight. The extent of the zones of inhibition is a rough measure of the conc.cntr;l tiorl of streptomycin. REFERENCES 1. SCHATZ, ALBERT, BUGIE, EI.IZABE.I.H and WAKIMAN, S. A. Streptomycin, a substance rxhibiting antibiotic activity against gram-positive and gramnegative bacteria. Pm. Sm. Exfm. Riol. & Med., 55: 66-69, 1944. 2. WAKSMAN, S. A. and HENRICI, ‘2. .I‘. ‘The nomenclature and classification of the actinomyl:~tcs. 3. Burt., 46: 337-341, 1943. 3. WAKSMAN, S. A., BUGIE, EMYABETH and SCHATZ, ALBERT. Isolation of antibiotic substances from soil micro-organisms, with special rrferencr to streptothricin and streptomycin. Pm. Staf. ~,IcPI., May0 Cl&., 19: 537.-548, 1944. 4. SGHATZ, AI,BERT and WAKSMAN, S. A. Effect of streptomycin and other antibiotic substances upon .Uycobacterium tubercuhis and related organisms. Pm. Sac. Exfier. Rlol. 63 Med., 57: 244&248, 1944. 5. FELDMAN, W. H. and HINSHAW, H. C. Effects of

streptomycin on experimental tllberculosis in guinea pigs: a preliminary report. Proc. St& Meet., .Mayo Clin., 19: 59s-599, 1944. 6. JONES, DORIS, METZGER, H. J., SCHATZ, ALBERT and WAKSMAN, S. A. Control of gram-negative bacteria in experimental animals by streptomycin. &i-;ence. n.s., 100: 103-105, 1944. 7. H~II.MAN, F. R. Streptomycin in thr~ trratnwlt vf

428

Streptomycin-Herrell,

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