THE INFLUENCE OP IRON ON THE METABOLISM OF ACTDTOICCES WITH SPECIAL REFERENCE TO THE HATE OP

PROTEOLYSIS I I MILK

Winnifred Odetta Hicks

A Thesis submitted i n P a r t i a l Fulfilment of

The Requirements for the Degree of

MASTER OF SCIENCE IN AGRICULTURE

i n the Department of.

AGROKOM

The University of B r i t i s h Columbia A p r i l , 1941.

AGE¥OWLl.I)GIffiM!

The writer wishes to acknowledge the assistance

received, from the National Research Council during

the year 1939-40o Laboratory f a c i l i t i e s were provided

by The University of B r i t i s h Columbia throughout the

period of investigation* The writer ^also wishes to

express her deep appreciation to Dr. D. G-. Laird for

invaluable advice and c r i t i c i s m during the course of

the study*

TABLE OF CONTENTS Page

IHTRODUCTIOF 1

REVIEW OF LITERATURE... 2

EXPERIMENTAL .................... 5

Part A P r o t e o l y t i c Studies 5

Media...............*.. 5 Preliminary Experiments. V Procedure... . 8 Stimulatory Effect of Iron. 9 S t a b i l i t y of Actinomyces on a S o i l Medium. 14 Effect of S o i l Ash 18 Effect of Trace Elements.......... 20 Discussion and Summary - Part A. 24

Part B - Studies on Oxygen Requirements 28

Methylene Blue Studies. 28 Influence of Iron under Anaerobic Conditions........ 35 Discussion and Summary - Part B..................... 38

Part C ̂ Enzyme Studies. 41

Influence of Iron on the Proteolytic Enzyme Complex® 41 Discussion and Summary - Part C...................v" 45

GMERAL SUMMARY * 47

BIBLIOGRAPHY 50

THE INFLUENCE OF IKON ON THE 1ETABOLISH OF ACTINOHICES - WITH SPECIAL REFEEENCE TO THE RATE OF

PROTEOLYSIS IN MILK

I. p.TRpPUOTIOg :

A s t r i k i n g p e c u l i a r i t y of the Actinomyces, as commonly observed, i s

t h e i r v a r i a b i l i t y in morphology and cu l t u r a l characteristics. Whether

or not t h i s i s an inherent character of the members of the group, or mere-*

l y the result of the a r t i f i c i a l conditions under which the cultures are

grown i n the laboratory, has never been made clear. I f the l a t t e r i s the

case, the lack of some essential factor might well he the cause of the

variation encountered.

It i s reasonable to suppose that i f the different species are

naturally unstable, they would remain so under a r t i f i c i a l conditions i n

the laboratory* I f , on.the other hand, they are normally stable, i t

should be possible to determine the factor which would maintain them at a

high and consistent l e v e l of efficiency*

In an endeavor to s t a b i l i z e the actinomycetes, the various factors

which might conceivably exercise an influence on t h e i r proteolytic

a c t i v i t y were reviewed. Recalling that the s o i l contains a wide variety

of metallic elements, many of which stimulate the physiological a c t i v i t y

of certain microorganisms, and recalling that iron i s essential for the

formation of the respiratory pigments i n both plants and animals, i t was

decided to study the influence of trace elements with particular reference

to iron.

Since the decomposition of protein i s one of t h e i r most important

functions i n nature, proteolytic a c t i v i t y was selected as the basis for

determining their efficiency and response to trace elements*

I I . REVIEW OF LITERATURE.

The genus Actinomyces has i n the past been given many appellations,

such, for instance, as Streptothrix, Nocardia, Ray Fungi, Oospora,

Discomyees, and Cladothrix. Oohn (10), who i n 1875 f i r s t isolated an

Actinomyces, referred to i t as Streptothrix Foesteri. The name "Actinomyces

was f i r s t employed by Harz (23), who isolated and described Actinomyces

bovis in 1877. Trevisan (47) applied the term "Nocardia" to a saprophytic

form, but according to Waksman (53), there i s no authority for separating

the p a r a s i t i c and saprophytic forms. Domec (14) and Drechier (15) made

morphological studies on the oasis of which the l a t t e r c l a s s i f i e d them

with the Hyphomycetes. They showed that the terms "Oospora", "Streptothrix"

or "Discomycetes" cannot oe applied since the actinomycetes are u n i c e l l u l a r .

Waksman (50) agreed with Isiadson (35), who suggested that they form a

special group of fungi to be c l a s s i f i e d separately. Olaypole (9) con­

cluded that this group should be looked upon as representing the ancestral

type of both the higher fungi and the true bacteria, while i n 1939

Newcomer (36) demonstrated nuclear morphology by the Feulgen technique

which showed a s i m i l a r i t y to the fungi rather than to bacteria.

The saprophytic forms of Actinomyces were more or less ignored

prior to 1900. Beijerinck (4) i n 1900 studied actinomycetes i n nature

and investigated th e i r a b i l i t y to form quinone. Muntor (33) and Fousek

(19), Hiltner and Stormer (28), Conn ( l l ) and Krainsky (32) investigated

t h e i r numbers and di s t r i b u t i o n throughout the s o i l p r o f i l e , Glaypole

(9) and Sanfelice (42) were perhaps the f i r s t to record the great v a r i a b i l

i t y i n morphology and cultural characteristics of these microorganisms,

while Krainsky (32), Waksman and Curtis (57), and Conn (12), (13), came to

the conclusion that pigment production was variable and hence an i n ­

sufficient basis for characterization of the species. Skinner (44), (45),

i n 193S and 1939 discussed the tyrosinase reaction of the actinomycetes

i n relation to pigment production, and i n the same year Kedrovskii (31)

concluded, i n agreement with the earlier workers, that pigmentation

should not constitute a basis for classification,, Katznelson (30) des­

cribed a thermophilic strain which was subject to autolysis, while

Hassegavm, et a l (24) suggested mutation i n t h i s group of microorganisms.

Waksman (56) i n 1940 revised his system of c l a s s i f i c a t i o n , which i s now

based on morphology rather than on a wide variety of cu l t u r a l character­

i s t i c s .

The physiological requirements and responses of actinomycetes have

been studied q u a l i t a t i v e l y by many workers with a view to c l a s s i f i c a t i o n ,

while quantitative measurements have been rarely reported. Waksman (52)

in studying the proteolytic action of these organisms i n milk with a

view to di f f e r e n t i a t i o n between the species, measured amino nitrogen

production using the micro-apparatus of Van Slyke and the F o l i n aeration

method, and came to the conclusion that the variations encountered,

although often very s t r i k i n g , were of a quantitative rather than a

qualitative nature.

The temperature requirements of the actinomycetes have oeen more

or less f u l l y investigated. According to Waksman (55), the optimum for

most species i s 25° to 28° C, with a maximum of 40° C and a minimum

"between 18° and 20° C. Krainsky (32) reported that most species grew

oest at 30° C, while a few showed optimum growth at 35° C and A. citreus

has an optimum at 26° 0. Domec (14) and Foulerton and Jones (18)

studied the thermal death points of the spores and mycelial growth*

Giloert (20) i n 1904 isolated a thermophilic species with an optimum tem­

perature of 55° 0.

The oxygen requirements of the species do not appear to he well

understood. The earlier workers believed the pathogenic types to be

anaerobic and the saprophytic types, aerobic. Beijerinck (4) c l a s s i f i e d

them as facultative anaerobes, while Musgrave, Clegg and Polk (34)

stated that the actinomycetes are neither s t r i c t aerobes nor s t r i c t

anaerobes. Waksman (50), (53) concluded that they are not s t r i c t

anaerobes, but that some may be able to thrive under semi-anaerobic

conditions. A search of the l i t e r a t u r e has f a i l e d to reveal any direct

information regarding the oxidation-reduction potentials of the saprophy­

t i c forms, but Hagan (22) described the formation of hydrogen peroxide

by A. neerophorus on exposure to a i r .

Among the papers of. a widely diverse nature which have appeared i n

recent years i s that of V/aksman and Woodruff (61), who reported the

production of bacteriostatic and bactericidal substances by s o i l

actinomycetes, and of Patrick, et a l (.40) who described a species of

Actinomyces which attacked xylan strongly, but which did not attack

xylose or any of the common carbohydrates or alcohols.

ITo publication has appeared, as far as the writer i s aware, re l a t i v e

to the u t i l i z a t i o n of iron i n the metabolism of the actinomycetes.

f • : : " ? I " ' ' '

'I n • • • if • " • •

- 5. -

However, the importance of the heavy metals i n the n u t r i t i o n of fungi

and certain bacterial species has Deen known for a considerable period

of time. Gottheil (21) as early as 1901 used traces of metallic iron for

the c u l t i v a t i o n of s o i l bacteria. lyem, et a l (29) stated that the addi­

tion of iron oxide to the s o i l caused an increase i n the numbers of bac­

t e r i a , fungi, and actinomycetes present. Foster (17) i n 1940 ably

reviewed the l i t e r a t u r e on the necessity of the heavy metals i n the

n u t r i t i o n of the fungi, while Steinberg (46) showed that certain cations

are essential for bacteria associated with nitrogen f i x a t i o n , and Roberg

(41) indicated their importance for the optimum growth of the green

algae. The considerable volume of l i t e r a t u r e dealing with the necessity

of the heavy metals for the pathogenic bacteria i s considered outside

the scope of t h i s study.

The increasing interest i n the heavy metal n u t r i t i o n of a l l types

of microorganisms, and the fact that iron i s of prime importance i n many

l i f e processes of plants and animals, prompted an investigation of the

influence of t h i s element on the physiology of representative cultures

of Actinomyces.

I I I . EXPERIIMTIL

Following considerable preliminary investigation proteolytic a o i l i t y

appeared to be the most suitable measure of a c t i v i t y . Actinomycetes

representative of species found i n the upland g l a c i a l s o i l of coast

regions of B r i t i s h Columbia were used for this purpose.

PART A - PROTEOLYTIC STUDIES

Media Skim milk was chosen as the oasic medium for measuring the rate of

proteolysis, since, despite i t s heterogeneity of constitution, i t i s a

convenient source of native protein and supports good growth of a l l

strains of Actinomyces (59), (60)»

Although freshly skimmed milk from the University farm was f i r s t

employed as the medium, i t s use was abandoned early i n the study when

the nitrogen content was found to vary somewhat from time to time. Skim

milk powder from the Fraser Valley Milk Producers" Association plant

in- Vancouver was substituted, and used at a concentration of 10 grams per

100 ml. of d i s t i l l e d water. The nitrogen content of milk prepared i n

t h i s v/ay has remained r e l a t i v e l y constants

While there i s considerable variation i n the analyses of milk from

individual cows, the average composition as presented by the Associates

of Rogers (2) i s ;

Water 87.27 per cent Casein 2.95 " " Albumin .52 " " Fat 3.66 ,f " Lactose 4.91 " " Ash .69 " n

According to Babcock (3), the ash has the following constitutions

KgO 25.02 per cent CaO 20.01 " 11

l a 2 0 10.01 " " IfeO 2.42 " " FegQg .13 ". '* . P 20 5 24.29 " " CI 14.28 " " S0 3 3.84 " "

Of the trace elements, copper ranges between 0.2 to 0.8 ppm. of whole

milk, zinc between 3.6 and 5.6 ppm., s i l i c a about 2 ppm., and Iffig, 3 to

4 ppm. In addition s traces of aluminum, manganese and iodine are said

to occur. The non-protein nitrogenous constituents include urea, amino

nitrogen, creatin, creatinin, and u r i c acid. Traces of thiocyanic acid,

choline and methyl guanidine have also been reported.

While i t i s admitted that milk i s not a perfect medium, due to the

presence of iron, trace elements, and the so-called growth factors, yet

i t appeared to be quite suitable for the problem under investigation.

Stock cultures, isolated in. the autumn of 1937, have been carried

continuously on sodium asparaginate glycerol agar of the following

constitutions

Later i n the study a duplicate set of stock cultures was

transferred to and maintained i n s t e r i l e s o i l tubed i n convenient

quantities to which s t e r i l e water was added as required to maintain the

a c t i v i t y of the cultures.

Preliminary Experiments

In preliminary experiments, which have oeen reported i n unpublished

data (27), a comparison of the a c t i v i t y of various soluble iron, salts

resulted i n the selection of f e r r i c n i t r a t e as the source of iron for

use i n subsequent studies. In the case of a l l salts tested, f e r r i c iron

gave stronger stimulation than the corresponding ferrous s a l t , and f e r r i c

nitrate was more active than the sulphate, chloride, or ammonium c i t r a t e .

The optimum concentration of the f e r r i c ion was shown to l i e between 40

and 50 gammas per ml. of milk. Since the concentration curves f a l l off

gradually, either there i s no definite t o x i c i t y l i m i t , or the iron i s

Sodium asparaginate Dipotassium phosphate Dextrose Glycerol Agar Water, d i s t i l l e d pH

1.0 gm. 1.0 " 1*0 "

10.0 " 15.0 " 1000 cc. approximately neutral

rendered insoluble through combination with some component of the milk.

Since Waksman (53) has pointed out the influence of the type of inoculum

used i n quantitative studies of the actinomycetes, various methods of

inoculation were compared, and i t was found that the most consistent

results were obtained when 0.5 ml* of a water suspension prepared from

seven-day slope cultures were used* In later work, the influence of

the incubation temperature on the rate of hydrolysis of milk protein was

studied, and although Waksman (52) reported that the greatest ammonia

production, measured with brom cresol purple as recommended by Clark

and Lubs |7), (8) took place at 37° G, the cultures under study showed

the greatest color change at 28° C*

In l i g h t of the above findings, the standard procedure, as described

below, was evolved*

Procedure

Skimmed milk prepared from milk powder was put up i n 500 ml.

quantities in l i t e r conical flasks and s t e r i l i z e d at 12 pounds pressure

for 25 minutes. Stock solutions of iron i n d i s t i l l e d water were pre­

pared containing 1 mg. of the metallic ion per ml. of solution. The iron

enrichments were added to the milk prior to autoclaving, precautions

being taken to secure uniform dispersal i n the medium before subjecting

to heat.

Each culture as required was seeded on several slopes and incubated

at 28° C for seven days. A t y p i c a l culture was then selected, 3 ml. of

s t e r i l e water were added and the growth carefully worked off with a loop

to form a uniform suspension. 0.5 ml. of the water suspension was used

as inoculum for each flask.

The foregoing milk cultures were incubated at 28° C for seven days.

At the end of that time t o t a l nitrogen was determined on 5 ml. aliquots of

the control, after the method of Orla-Jensen (39)• The decomposition of

protein was measured as t r i c h l o r a c e t i c acid soluble nitrogen, or non­

protein nitrogen, after the method of Eagles and Sadler (16), a modi­

f i c a t i o n of the procedure of Wasteneys and Borsook (62)• The procedure

for the non-protein nitrogen determination i s as follows? Add 8 drops

of.formalin to the 300-ml. culture; treat with 40 ml. of 20 per cent

t r i c h l o r a c e t i c acid and allow to stand for one hour. F i l t e r off the

precipitate and take two 70-ml. aliquots of the f i l t r a t e . Heat the 70-ml.

quantities i n a water bath for three hours, cool, f i l t e r , and make up

to volume. Using two 10-ml. aliquots from each f l a s k , determine nitrogen

content by the Kjeldahl method.

A l l data are presented as non-protein nitrogen expressed as per

cent of t o t a l nitrogen.

Stimulatory Effect of Iron

An experiment was set up i n such a manner as to enaDle the procuring

of information respecting (l) the variation i n proteolytic a c t i v i t y

within a species which may be expected from time to time; (2) differences

between the species; and (3) the influence of iron on the rate of pro­

te o l y s i s .

That the twelve cultures of Actinomyces used i n the study are

di s t i n c t species cannot be stated with absolute assurance, but each does

at least represent a pure l i n e . For purposes of discussion they may

be considered as species. They were isolated from the s o i l eight months

prior to the commencement of the experiment.

- 10 • -

The data as presented i n Table I, while inconclusive i n some

respects, set forth certain s p e c i f i c information. A study of each

species i n "milk alone" over an eighteen months' period reveals that,

while some species, notably A-5, A-7, A-8 and A - l l are r e l a t i v e l y con­

sistent i n the production of non-protein nitrogen, others, p a r t i c u l a r l y

A-l and A-2, vary over a wide range. A-7, for instance, varies only from

7.5 to 8.4 per cent while A-E shows the maximum variation of 7.6 to 37.7

per cent. That t h i s indicates the extent of variation which may normally

be expected i n a laboratory study of actinomycetes i s very doubtful for

reasons which w i l l be apparent l a t e r .

It i s interesting to observe that a l l the species with the exception

of A-3 and A-7 show a reduction i n proteolytic a c t i v i t y over the period

of the experiment. This decrease i n a c t i v i t y i s most noticeable i n the

case of cultures A - l , A-2, A-6 and A-10, which exhibited strongest proteo­

l y t i c a c t i v i t y at the time of the f i r s t determination, while A-5, A-8, A-9,

A - l l and A-l2 show a small but significant decrease. Thus, the hydrolytic

a b i l i t y of the species i n Experiment 5 i s on the average at a d i s t i n c t l y

lower level than i n Experiment 1. This decrease i s more pronounced

between Experiments 1 and 3 than between Experiments 3 and 5. I f the

average percentage increase i n non-protein nitrogen i s calculated for

a l l cultures on the basis of the control, i t w i l l be seen that a decrease

from 146 per cent to 49 per cent has taken place during the period of

time elapsing between Experiments 1 to 5. Although certain i r r e g u l a r i t i e s

are seen to occur i n the data, i t would thus appear that as the cultures

are carried on an a r t i f i c i a l medium, their proteolytic a c t i v i t y tends

to decrease u n t i l i t reaches a more or less constant le v e l where the

a c t i v i t y of each species i s at a minimum. In the l i g h t of t h i s data i t

Table I

Response of Species of Actinomyces to Iron over a Two-Year Period *

Species .Ho.

:Experiment 1 8 months' cultures Milk Milk + Fe

Experiment 2 18 months* cultures Milk Milk + Fe

Experiment 3 20 months' cultures Milk . Milk + Fe

1 • ! .T

Experiment 4 23 months* cultures Milk Milk * Fe

Experiment 5 26 months'cultures Milk Milk + Fe

Average i n

Milk

A v e r -i n

Milk + . Fe

i i v e r -age % Stimu

lation A - l l 24.5 48.0 19.6 16.4 8.0 10.9 10.0 18.4 6.9 7,8 . 13.8 20.3 47.1 A-2 • 37.7 36.6 7.6 10.4 12.5 15.0 XX * 2 19.5 9.3 9.1 15.6 18.1 16.0 A-3 7.9 8.0 8.7 8.7 12.0 12.0 10.2 8.7 8.6 9.6 9.5 9.4 — A-4 10.7 14. 8 8.4 11.6 8.0 13.3 11.4 .8.1 14.1 9.3, 13.4 44.1 A-5 12.0 22.6 * 11.8 15.1 9.8 10.0 11.4 12.9 9.1 12.3 10.8 14.5 34.2 A~6 20.1 25.5 19.1 12.6 11.4 11.8 12.4 14.1 10.7 10.6 14.7 14.9 1.3 A-7 7.6 9.5 8.4 7.6 7.5 7.9 7.5 8.7 7.7 . 6.7 7.7 8.1 5.1 A-8 13.2 11.8 11.8 11.8 17.8 13.2 18.5 10.6 16.6 11.8 16.1 36.4 A-9 12.5 23.5 .. 10.1 14.8 7.6 8.8 8.7 7.5 10.3 9.6 9.8 12.8 30.6 A-10 19.2 17.3 16.3 10.6 7.8 9.7 9.2 12.7 8.8 11.1 14.2 12.3 -A - l l 8.7 15.3 8.3 10.6 6.3 6.9 6.6 6.8 6.9 9.2 7.3 9.7 32.9 A-12 12.0 16.0 8.3 24.8 6.4 12.4 12.2 13.4 9.2 18.9 9.6 17.1 78.1 Average 15.5 21.5 11.3 12.9 9.1 11.4 10.3 12.8 8.8 11.3 11.0 13.8 27.1 Q 6.3 6.3 5.8 5.8 6.0 6.0 6.1 6.1 5.9 5.9 Average % In­crease •: 146.0 241.1 94.8 122.4 51.6 90.0 68.8 109.8 49.1 91.5 over G • -SJ

* A l l figures represent non-protein nitrogen as per cent of t o t a l nitrogen. ** Control, uninoculated.

appears reasonable to suppose that had the experiment been undertaken when

the microorganisms were freshly isolated from the s o i l , greater proteolytic

a c t i v i t y would have been noted, resulting i n a wider range of v a r i a b i l i t y

within the species than has been observed above.

It i s to be noted too that i n Experiment 1 the cultures i n "milk

alone" differed markedly i n their a b i l i t y to hydrolyse milk protein. For

instance, A-7 showed 7.6 per cent non-protein nitrogen, whereas the

corresponding figure for A-2 was 37.7 per cent. At the conclusion of

the experiment there was r e l a t i v e l y l i t t l e difference between the species,

as i s apparent from the extremes A-l and A - l l with 6.9 per cent each and

A-6 with 10.7 per cent. When the average for non-protein nitrogen pro­

duction i s considered the spread i s 15.6 to 7.3 per cent, as compared

with 37.7 to 7.6 per cent i n Experiment 1. The species which showed the

highest i n i t i a l a c t i v i t y , Ar-1, A-2, A-6 and A.-10 are s l i g h t l y higher than

the remaining species, while A-7 and A - l l are the least active. A-3 i s

peculiar i n that on the average i t has increased s l i g h t l y i n protein

hydrolysing a b i l i t y .

One might conclude from the data presented that, while A - l , A-2,

A-6 and A-10 are extremely active, some of the species, notably A-3 and

A-7 are weakly proteolytic in nature as they appear i n t h i s experiment.

Such a conclusion, however, i s scarcely warranted at t h i s time, since,

as has already been stated, the stock cultures had been carried on a

laboratory medium for some eight months pr i o r to the commencement of the

study. I t i s suggested that, during that time A-3, A-7 and l-ll may

have largely lost t h e i r protein hydrolysing power*

The data as presented perhaps indicates that sodium asparaginate glycerol agar lacks some constituent essential for the maintenance of high" proteolytic a c t i v i t y .

An examination of the data with respect to the influence of iron

i n the i n i t i a l experiment indicates an even greater v a r i a b i l i t y "between

the cultures than was apparent with milk i t s e l f , the presence of iron

stimulating such species as A - l , A-5 and A-9 to a marked degree, and

showing no effect on the rate of protein breakdown i n the case of A-2,

A-3 and A-10. The tendency of the species to decrease i n proteolytic

a c t i v i t y as they are carried on the a r t i f i c i a l medium, while not as con­

sistent as with milk alone, i s even more marked. While the average

percentage increase of the cultures when calculated on the basis of the

control drops from 146 to 49 per cent i n milk, i t drops from 241 per cent

to 91.5 i n the presence of iro n , a decrease 1.5 times as great. Hence

the iron has apparently accentuated the variation i n proteolytic a c t i v i t y

which was recorded within a given species i n milk alone.

The species do not give a consistent response to iron as determined

over the eighteen months* period, but, considering the average percentage

stimulation for the different cultures, i t w i l l be seen that A-4 and A-l2

show the strongest stimulatory effect, as compared with A - l , A-5, A-10 and

A - l l , which responded to iron i n Experiment 1. The response to iron by

A-8 as determined by experiments not reported herein i s perhaps more

marked than this data would indicate, and hence i t i s regarded, with A-4

and A-12, as the species showing the greatest stimulatory effect upon

the addition of iron to the basic milk medium.

- 14 -The extreme v a r i a b i l i t y of species of Actinomyces has also been

reported by Waksman (53), who found str i k i n g quantitative variation when

studying amino nitrogen production by various species. In view of the

data presented i n Table I i t would seem possible that the microorganisms

once removed from their natural habitat-the soil-and carried on a r t i f i c i a l

media for an extended period of time, change to unstable physiological

forms, which change may result i n quantitative variation. I t was therefore

decided to carry the cultures i n s t e r i l e s o i l i n an attempt to restore any

lost measure of a c t i v i t y and to maintain them at a constant physiological

l e v e l .

S t a b i l i t y of Actinomycetes on a S o i l Medium

Quantities of fine sandy loam were placed i n large test tubes and

s t e r i l i z e d for three hours at 15 pounds pressure on three successive days.

After testing the s o i l for s t e r i l i t y , tubes were inoculated with a water

suspension of the twelve cultures and incubated at 28° C, s t e r i l e water

being added as required to maintain the moisture content of the s o i l .

The a c t i v i t y of representative species was determined at intervals over

a f i f t e e n months' period. The inoculum i n each case was prepared by

adding a small portion of the s o i l culture to a 3-ml. melted agar medium

tube, sloping, and allowing to harden. After a week's incubation, cultures

prepared i n t h i s way exhibited some surface growth which was transferred

to a fresh agar slope to be used for inoculation of the milk cultures

after a further seven-day incubation period. Table I I presents a summary

of the data ootained with representative cultures after they had been

carried i n s o i l for periods of three months, twelve months, and f i f t e e n

months.

Table I I

Effect of Maintaining Stock Cultures on S t e r i l e Soil-as measured "by Response to Iron*

Species Fo. .

3 months on S o i l

Milk Milk + Fe Per Gent Increase

12

'• Milk

months on S o i l Per Cent

Milk * ;Fe Increase

15

Milk

months on S o i l Per Gent

Milk -t- Fe Increase

A - l 12.2 11,7 13.0 12.9

A-E 13* 3 8.1 ISe 5 54.3

A-3 17.6 17.9

A-4 12.2 18.3 50.0 22.4 33.6 50.0 19.0 31.0 63.

A-5 24.5 30.8 25.7 11.6 14.4 24.1

A-6 14.2 14.4 18.2 19,8 8.7 17.0 20.2 18.8

A-7 '. .• • 9.3 9.1 7.8 7.9 8.1 11.0 36.7

A-8. 15.6 31.5 101» 22.9 42.0 86. 20.3 39.5 94.5

A-9

A-10 18.2 24.6 35.1 17.6 22.2 26.1

A - l l 8.6 8.6 10.8 11.6 7.4 9.1 11.8 29.6

A-12 8.4 16. 3 94. 11.6 25.3 118.0 8.8 16.6 88.0

C ** 6.0 . 6.0 5.9 5.9 6.0 6.0 ** Control, Uninoculated. * A l l figures represent non-protein nitrogen as

per cent of t o t a l nitrogen.

From Table I I i t i s apparent that the differences i n a b i l i t y to

degrade milk protein which were observed with regard to the various c u l ­

tures i n the i n i t i a l experiment reported i n Table I, and which tended

to disappear as the cultures were carried on the laboratory medium, have

begun to reappear. While some of the strains, notably A-l and A-2, have

not reached the level of proteolytic a c t i v i t y f i r s t reported, cultures

A-3, A-4, A-5 and A-8 far exceed i t , and cultures A-6 and A-IO approximate

the o r i g i n a l figures closely. A-7, A - l l and A-12 are remarkaole i n that

l i t t l e change i n the rate of degradation of milk protein can be recorded.

In the presence of iron, cultures A-4, A-8 and A-12 show a strong and con­

sistent stimulation, contrasting with the v a r i a b i l i t y of their response

as recorded in the previous table. The other cultures studied show

l i t t l e or no stimulatory effect. With the exception of A-5, the figures

denoting nonprotein nitrogen as percentage of t o t a l nitrogen agree

closely for the sets completed at twelve and at f i f t e e n months, both i n

the absence and i n the presence of iron. That this i s an important con­

sideration i s apparent when the di v e r s i t y of the data i n the preceding

table i s recalled. The extreme v a r i a b i l i t y of the actinomycetes, which

has been reported Dy so. many investigations, has been largely overcome,

and i t i s suggested that by maintaining isolations of these microorganisms

on s t e r i l e s o i l , the great d i f f i c u l t i e s encountered i n any study of th i s

group may be at least to some extent eliminated.

The tendency on the part of the cultures to show decreased a c t i v i t y

after numerous transfers on an a r t i f i c i a l medium, and to regain their

l o s t a c t i v i t y after prolonged contact with s o i l has also been observed

with regard to gelatin liquefaction. Giant colonies of the actinomycetes

were seeded on plates of 15 per cent gelatin and incubated for a period

of ten days, after which time the width of the liquefied c i r c l e on the

plate was measured. This technique was f i r s t carried out i n July, 1938,

using the cultures after they had "been carried, i n the stock medium for

six months, and repeated i n February, 1941, using the same cultures which

had been carried on the laboratory medium during the intervening time,

and also the corresponding cultures freshly isolated from the s o i l medium

after having been i n contact with i t for a period of f i f t e e n months.

The data are presented i n Table .•III.

Table I I I

Comparison of the Rate of G-elatin Liquefaction of Species of Actinomyces carried on an A r t i f i c i a l Medium and i n S t e r i l e S o i l

Species A r t i f i c i a l Medium A r t i f i c d a l Medium S o i l Medium No. 8 months' cultures 26 months' Cultures 26 months' cultures

Width of li q u e f i e d Width of Liquefied Width of Liquefied zone i n Cm. _ zone i n Cm. zone i n Cm.

: A-l ' • ' liS-A-2 .6 .7 1.7 A-3 ' * " • .'.4.; .7 ' A-4 • 2.4 1.9 2.4 , A-5 .5 .5 1.5 A-6 .9 1.0 1.0 A-7 2.0 1.0 1.0 A-8 1.2 3.1 A-9 .5 .5 .5;. A-10 1.0 .6 1.0 A - l l 1.3 .4. .8 A-12 .7 .7 1.0

An examination of the data reveals that the behavior of the cultures

closely p a r a l l e l s that recorded i n Tables I and I I . After contact with

the a r t i f i c i a l medium for some time, the cultures are seen to decrease

i n a c t i v i t y or to show no change, but the cultures freshly isolated from

the s o i l medium are s i g n i f i c a n t l y more active, with the exception of

cultures A-6, A-7, A-9 and A-12. Of these, A^7, A-9 and A-12 showed l i t t l e

appreciable change i n the preceding data, while A-6 decreased s l i g h t l y

and then regained i t s former proteolytic a c t i v i t y after maintenance on

s o i l .

The relative rates of starch hydrolysis, while not correlating com­

ple t e l y with proteolytic a c t i v i t y , indicate that the same general tendency

may apply to metabolic functions other than those concerned with protein

degradation. I t would thus seem probable that a higher le v e l of bi o l o g i c a l

a c t i v i t y results when the actinomycetes are maintained under natural con­

ditio n s , and that the v a r i a b i l i t y which has been reported with regard to

quantitative studies of these microorganisms may be largely eliminated

through the use of s o i l as the stock medium.

Effect of Soil, Ash ' "/

Since the b i o l o g i c a l a c t i v i t y of cultures of actinomycetes was con­

siderably increased through contact with the s o i l medium, and since iron,

a material of almost universal di s t r i b u t i o n i n s o i l s , exhibited a stimu­

latory effect on certain of the cultures, i t was decided to investigate

the possible influence of other metallic constituents on the proteolytic

a b i l i t y of representative cultures. Since the p o s s i b i l i t y that the

increased a c t i v i t y of cultures i n contact with the s o i l medium was due to

substances of an organic nature was considered, i t was decided to remove

s o i l organic matter by i g n i t i o n , and to test the stimulatory effect of

the resultant ash. Two-gram samples of s o i l of the same sample as that

on which the cultures had been maintained were ignited i n porcelain

crucibles u n t i l t h e i r weight remained constant after continued heating.

The ash was transferred to 500 ml, of milk i n l i t e r flasks and autoclaved

with the milk to f a c i l i t a t e solution of at least part of the oxides

formed on ig n i t i o n . The flasks were then inoculated and incubated i n

the" usual manner. Cultures A-4, A-8 and A-12 from the laboratory medium

were tested i n t h i s way. The data are presented i n Table IT.

Table IV .

Response of Species A-4, A-8 and A-12 to the Presence of S o i l Ash

Species No.

Wt. of Ash i n grams.

Non-protein Nitrogen as Per Cent Total Nitrogen

Per Cent Increase

Species No.

Wt. of Ash i n grams. Milk... . . Milk +. Ash

Per Cent Increase

A-4 11.0 21.7 97.2 A-8 1.5 10.3 ••"• 14.7 42.7 A-12 1.5 9.7 11.3 16.5 0* 6.3 6.3 '

* Control, uninoculated.

A comparison of the response of the species to s o i l ash, and to

f e r r i c n i t r a t e as reported i n Table I, reveals that they d i f f e r consid­

erably. The response of A-4, with an average stimulation of 44.1 per

cent from f e r r i c nitrate,increases to 97.2 per cent when i n contact with

s o i l ash. This i s higher than i t s response to iron after contact with the

s o i l medium, when the percentage stimulation was 50 to 63 per cent. The

response of A-8 to s o i l ash i s only s l i g h t l y greater with an increase

from 36.4 per cent to 42.7 per cent for the more varied enrichment, but

this figure i s less than half the percentage stimulation recorded for

iron when the stock cultures were carried on a s o i l medium. A-12, on the

- 20 -

other hand, shows only 16.5 per cent stimulation from the s o i l ash as

compared to an average stimulation of 78.1 per cent as reported i n

Table I, and of 100 per cent when kept i n contact with s o i l . I t should

be noted here that A-12 was among those species exhibiting no increase

in proteolytic a c t i v i t y after extended contact with the s o i l medium,

while both A-4 and A-8 showed substantial increases.

Effect'of Trace Elements

Since i t seemed possible that some other constituent of the ash was

exerting a stimulatory effect, a qualitative analysis of t h i s s o i l was

carried,out. Twenty grams of the s o i l were digested with hydrochloric

acid according to the method of Van Bemmelen and Hissink(49), the organic

matter was removed from the f i l t r a t e by evaporation with n i t r i c acid, and

the extract tested by the qualitative procedures of IToyes (37) supplemented

by floyes and Bray's (38) procedures for the rare elements 0 Since a hy­

drochloric acid digestion had been used i t was not possible to test for

the metals of Group I. The amounts of the various elements were roughly

estimated, and are reported as "absent", "trace" or "heavy" i n Table V.

It i s f u l l y realized that the procedures used are not as sensitive

as some of the micro-analytical methods which have been evolved, but

they may be carried out with ra p i d i t y and by using a large sample and

concentrating the f i l t r a t e , any trace elements which would be available

and present i n the s o i l solution should be recovered. Very heavy pre­

ci p i t a t e s of manganese, iron, and aluminum were obtained, and the possi­

b i l i t y that trace elements were occluded and carried down with these

materials should not be overlooked.

Table .?

Summary of Metallic Elements found i n Upland Glac i a l S o i l , using Qualitative

Analytical Procedures

Element Occurrence Element Occurrence

Lead absent Manganese heavy

B i smuth absent Iron heavy

Copper trace Nickel trace

Cadmium trace ^'Cobalt trace

Ar senic absent Vanadium absent

Antimony trace Zirconium absent

Tin trace Titanium heavy

Selenium absent Strontium absent

Tellurium absent Barium absent

Aluminum heavy Calcium heavy

Chromium absent Magne slum heavy .•

Zinc trace

- 22 -

The effect of these metals on the proteolytic a c t i v i t y of

representative species of actinomycetes was determined, using the con­

centration and source of the cation as outlined i n Table 71.

Table VI

Source of Metallic Constituents and Concentrations used to Determine thei r Accumulative Effect on

Actinomyces i n Milk

Metal Salt Used Concentration of Cation

per ml. of milk

iron Fe{SF03)5.9H2Q 9.90 gammas

manganese MnS04.2H20 6.22 "

copper CuSp4.5H20 3.31 "

cobalt Co(F0 3) 2.6H 20 3.31 "

aluminum A1G13.6H2G. 3.31 "

zinc 2nCl 2 , 3;31 " •

t i n , SnCl2.2H2G • 3.31 »

nickel M(F0 3) 2.6H 20 3.31 "

cadmium Gd (F0 3] 2.4H 20 3.31 "

Y/hen the accumulative effect of these metallic ions as reported i n

Table VII i s compared with that of 47.6 gammas of iron, i t ' w i l l be seen

that certain species respond more strongly to the varied enrichment.

A-4 shows a response of 209.5 per cent i n the l a t t e r case, while the aver­

age stimulation was 44.1 per cent i n Table I and 50 to 63 per cent after

the cultures had been returned to the s o i l . The a c t i v i t y of A-8 i s

- 23 -

Table VII

Response of Species of Actinomyces to traces of Certain Metals

Culture Non-Protein Nitrogen as Per cent Total Nitrogen Per Cent

No. Milk Milk + Metals Increase A-4 9 & 5 29.4 209.5

A-5 8.2 9.4 11.6 A-6 9.6 11.8

, A-7 7.2 11.5 59.7

A-8 10.3 23.6 129.1

A-10 9.0 9.8 8.0

A - l l 6.9 7.7 11.6

A-12 9.9 9.8 -c* 6.1 6.1

* Control, uninoculatecL

stepped up from an average of 36.4 per cent over an eighteen months5

period to 129.1 per cent, an increase over the a c t i v i t y of the s o i l c u l ­

tures, which responded from 86 to 101 per cent. A-7, which did not show any

appreciable stimulation from iron alone, shows a significant increase of

59.7 per cent from the varied enrichment. On the other hand A-12, which

did not respond to any extent to the addition of s o i l ash to the basic

medium, showed no increase from the various metals added above. The

a c t i v i t y of the other species tested (A-5, A-6, A-10 and A - l l ) was i n ­

fluenced only s l i g h t l y by either the varied enrichment or by iron alone.

In the case of species A-4 and A-8, the concentration of iron added was too low to account for the large increases i n non-protein nitrogen recorded i n Table VII.

The immediately preceding experiments have indicated the influence

of other metals i n the physiology of actinomycetes. Apparently some

other metal or metals are important i n the metabolism of A-7, v/hich ex­

hi b i t s l i t t l e or no stimulation from iron alone. Cultures A-4 and A-8,

which have shown a consistent response to iro n , exhibit an additional

effect from the presence of other cations. A-12, on the other hand,is

not stimulated by the metals added at these concentrations, although show­

ing large increases i n non-protein nitrogen i n the presence of 47.6 gammas

of f e r r i c n i t r a t e . The requirements of the other species studied

apparently do not include the materials outlined i n Table YJ, although the

slight increases reported may suggest a response at a higher concentration*

While no conclusions can be drawn as regards the necessity of particular

metals for a given species, such requirements are suggested. At the same

time, the increase i n proteolytic a c t i v i t y reported i n Table I I appears

to be due, not to the organic f r a c t i o n , but to the wide variety of

metallic constituents normally present i n the s o i l .

'Discussion and Summary...- Part .A

The proteolytic a c t i v i t y of cultures of actinomycetes and their

response.to the addition of traces of iro n i n soluble form were measured

by determining the non-protein nitrogen produced i n milk cultures. In

preliminary experiments, the cultures were shown to respond i n the

greatest degree to f e r r i c nitrate added at the rate of 47.6 gammas of

cation per ml. of milk, although no toxic effect was ooserved at higher

concentrations. Cultures incubated at 28° C. for seven days exhibited

the maximum response to iron, the percentage stimulation increasing up

to this time, then gradually decreasing. The quantitative variation en­

countered i n the non-protein nitrogen production of the cultures made

i t apparent that a l l experimental procedures and methods must he closely

checked before comparable data could he obtained. The source of inoculum

and method of inoculation affected the rate of proteolysis and the con­

sistency of the cultures, and the results were found to be most s a t i s f a c ­

tory when 0.5 ml. of a suspension of mycelial growth i n water was used.

Although the standard procedure was followed with extreme care, and

the duplicate determinations of non-protein nitrogen checked satisfactor­

i l y , variations too significant to be overlooked i n a quantitative study

were recorded when the proteolytic a c t i v i t y of the species was determined

at different times. A series of experiments over an eighteen months*

period indicated that, i n general, the protein degrading power of the

species tended to decrease as stock cultures were carried on the labora­

tory medium. Thus the average non-protein nitrogen production of the

twelve cultures i n milk dropped from 146 per cent to 49 per cent during

the eighteen months' period.

Considerable differences i n proteolytic a c t i v i t y between the species

were observed i n i t i a l l y ; A-3, A-7 arid A - l l exhibited only s l i g h t a c t i v i t y ,

whereas A - l , A-2, A-6 and A-10 were very active i n th i s regard; but as

they were carried on the a r t i f i c i a l substrate, their comparative acti¥ity

appeared to become equalized and minimized. Thus the range recorded for

the different species decreased from 37.7 - 7i6 per cent to 10.7 - 6.9

per cent during the course of the study.

The addition of iron to the milk medium accentuated the differences

between the species as a result of stimulation on the part of certain

cultures,, Considering the effect of the iron over the eighteen months*

period, the same tendency for decreased a c t i v i t y , v/hile not as regular

as i n milk i t s e l f , was observed, the average percentage stimulation on

the basis of the control dropping from 241.1 per cent to 91.5 per cent.

Whereas the response of the individual cultures was extremely variable,

the average percentage stimulation indicated that A - l , A-4, A-8 and A-12

responded most strongly to iron® Thus i t became apparent that, speaking

generally, the a c t i v i t y of the species was progressively decreased i n

accord with the length of time the stock cultures were i n contact with

the a r t i f i c i a l medium u n t i l they reached a more or less constant l e v e l .

A l l cultures had been carried i n stock on the a r t i f i c i a l medium for

eight months prior to the commencement of -the work. In t h i s time i t

i s possible that cultures A-5, A-7, A - l l and A-12 may have reached a

constant le v e l of a c t i v i t y and hence varied only s l i g h t l y during the course

of the study.

Since i t i s of the utmost importance to maintain the s t a b i l i t y of

the species, they were, returned i n pure culture to their natural habitat,

the s o i l . The proteolytic a c t i v i t y of isolations made at the end of a

year was then determined and i n nearly a l l cases was shown to have increased

considerably; and moreover, with but one exception, the non-protein n i ­

trogen determinations checked s a t i s f a c t o r i l y when cultures were compared

at different times. Evidently contact with this widely diverse material

had, i n the course of twelve months, raised the l e v e l of b i o l o g i c a l a c t i v ­

i t y and maintained i t so that fresh isolations showed high and consistent

results. That such a conclusion i s important i s obvious when the

numerous reports of variation and inconsistency occurring i n the l i t e r a ­

ture concerning t h i s group are recalled. A-4, A-8 and A-12 responded

strongly to the addition of iron, i n contrast to their inconsistent

reaction when carried on the a r t i f i c i a l medium.

The influence of s o i l ash on the proteolytic a b i l i t y of the three

cultures showing the strongest response to f e r r i c n i t r a t e suggested

that other metals might have an influence i n maintaining certain cultures

i n a high state of a c t i v i t y , since A-4 responded more strongly to the

presence of s o i l ash than to iron alone. By qualitative analysis t h i s

s o i l was found to- contain a' wide variety of metals which, when added i n

trace amounts to milk, indicated that at least certain cultures responded

to metallic substances other than iron. Cultures A-4, A-6, and A-8 were

stimulated to a greater degree by the more' varied enrichment, but A-12,

despite the fact that i t responded to iron alone, was not stimulated

appreciably by s o i l ash or by the various trace elements added. This

species did not exhibit any increase i n proteolytic a c t i v i t y after con­

tact with the s o i l medium and apparently reacts only to iron at a higher

concentration than was dissolved from the s o i l ash or was added with the

varied enrichment.

I t has thus been shown that the cultures of actinomycetes under

study vary markedly i n their a b i l i t y to degrade milk protein; that iron

stimulates the proteolytic a c t i v i t y of certain of the cultures; that, i n

general, continuous c u l t i v a t i o n on an a r t i f i c i a l medium impairs their

hydrolytic a b i l i t y ; and that the maintenance of the stock cultures i n

s t e r i l e s o i l restores to a large degree their lost a c t i v i t y . Certain

other metals appear to show a stimulatory effect, but since the study

i s mainly concerned with the influence of iron on proteolysis, a study

of other metallic substances has not been undertaken.

PART B - .STUDIES. -ON:OXYGEN" REQUIREMENTS

Preliminary experiments involving the use of shake agar cultures

demonstrated differences i n the oxygen requirements of the actinomycetes

under study. In view of the fact that Brown and Baldwin (5) and Allyn

and Baldwin (l) reported the influence of oxidizing and reducing sub­

stances on the growth levels of various b a c t e r i a l species, and demonstrated

the opposite effects of ferrous and f e r r i c iron i n the oxidation-reduction

potential of a medium, i t was believed that the differences rioted i n the

oxygen requirements of the cultures might afford a possible explanation

for the stimulatory effect of f e r r i c nitrate as n o t e d i n P a r t A«

Methylene Blue Studies

The influence of methylene blue and f e r r i c n i t r a t e on the growth

levels of the various species i n sodium asparaginate glycerol agar sim­

p l i f i e d by omitting dextrose, were studied. Water solutions of methylene

blue and f e r r i c nitrate were s t e r i l i z e d separately and added aseptically,

to tubed 10 ml. quantities of the medium immediately prior to inoculation

with a water suspension of the required culture. The series were i n ­

cubated at 28° C for a period of ten days.

An examination of the data as presented i n Table VIII reveals

specific differences between the cultures. A l l the "control" cultures

with the exception of A-2, A-7 and A - l l produced only surface growth,

while the named cultures showed sub-surface growth, and hence a greater

tendency towards anaerobiosis. The presence of methylene blue i n a l l

Table T i l l

The Influence of Methylene Blue and Ferric t i t r a t e on the Growth Levels'of Species of 'Actinomyces i n Shake Agar Cultures

Culture Mo* Control

Methylene Blue .0001$

Methylene Blue oQQZfo

Fe 10 y per ml. A-l Surface sub-surface* sub-surface 5 mm. sub-surface* A-2 sub-surface* sub-surface'- a few

colonies through tube

sub-surface* -colonies through tube

surface and sub-surface a few colonies through tube :'

A-3 heavy: surface surface and sub surface

sub-surface* heavy surface. - a few colonies through tube

A-5 heavy surface surface and sub­surface

sub-surface* - a few colonies through tube

heavy surface

' A-6 ' heavy surface surface and sub^ surface - a few colonies through tube

sub'-surface 5 mm. many colonies through tube

heavy surface - a few small colonies through tube

A-7 surface and sub­surface, many colonies through tube

sub-surface* sub-surface* many small c o l ­onies through tube

surface and sub­surface

A-6 heavy surface numerous small colonies through tube

f a i r l y heavy through tube

surface - small c o l ­onies through tube

A-9 heavy surface surface - a few c o l ­onies through tube

large colonies through tube .

heavy surface

A-10 heavy surface surface and sub­surface

numerous colonies• . . • through tube

heavy surface - a few large colonies through tube

A - l l

A-12

surface and sub-surface many colonies through tube heavy surface

sub-surface - a few colonies through tube surface and sub­surface

sub-surface* - many co 1 onie s t hro.ugh tub e sub-surface* - many small colonies through tube

surface and sub-surface - a few colonies through tube surface - small c o l ­onies through tuoe

* Denotes a heavy growth ring l-2mm.. below the surface of the medium.

cases allov/ed growth to occur deeper i n the medium, the higher concentra­

tion being more effective i n this regard. The addition of iron at a

concentration of 10 garnnas per ml. had no effect on the growth levels of

A-5 and A-9, but tended to bring growth to the surface i n the case of

A-2, A-7 arid A - l l . In a l l other cases, the effect of iron i s similar

to that of methylene bluej i n other words, i t too tended to lower the

level of growth i n the shake agar cultures.

It would appear that methylene blue, by functioning as a hydrogen

acceptor, f a c i l i t a t e s the energy exchange i n this simple synthetic

medium, thereby permitting a l l cultures to show discernible growth at a

lower l e v e l than i n the controls. In the presence of iron, the growth

lev e l i s lowered i n the case of the more aerobic cultures, while with

the so-called anaerobic cultures, A-2, A-7 and A - l l , the growth i s

brought to the surface. Ho explanation of this observation can be offered

at the present time.

Attempts to define, by the use of a suitable range of oxidation-

reduction indicator dyes, as recommended by Clark (6) and discussed by

Hewitt (26),. the actual potential produced by the various species, proved

unsuccessful, due to abundant pigment formation by the organisms when

milk constituted the medium and to high t o x i c i t y of the dyes when a synthe­

t i c medium was used. At the same time1, a non-toxic dye concentration was

too low to allow observation of the color changes. Methylene blue,

however, proved to be highly satisfactory for this purpose?due to the

fact that the color change i s sharply defined i n milk, and the potential

of the medium several days after inoculation l i e s more or less i n the

v i c i n i t y of the change i n color of this indicator. I t has therefore been

- 31 -employed to determine the reducing power of the various species and to

study the effect of iron on the potential attained during the growth of

the microorganisms. Preliminary experiments demonstrated that a l l the

strains exerted a f a i r l y strong reducing action, as measured by methylene

blue at a concentration of 1:100,000. The a c t i v i t y of the cultures was

then determined at higher concentrations: Methylene blue was added to

milk to give concentrations of 1:50,000, 1:10,000 and 1:1,000, and

s t e r i l i z e d i n 10-ml. quantities. Table IK records the data at the end of

a five-week incubation period. Considering their tolerance to higher

concentrations of methylene blue, the species under study are observed

to divide themselves into three groups: (a) those withstanding a con­

centration of 1:1,000 - A-2, A-7, A-10 and A - l l ; (b) those withstand­

ing a concentration of 1:10,000 - A - l , A-3, A-5, A-9; and (c) those

withstanding a concentration of 1:50,000 -A-4, A--6, A-8 > A-12.

A study of the influences of iron on methylene blue reduction by

the various species was undertaken. To this end.methylene blue was

added to milk at a concentration of 1:100,000 and the milk was divided

into three parts. One-third was tubed i n 10-ml. quantities to serve as

a control; to a second portion was added a water solution of f e r r i c

n i t r a t e to give a concentration of 53 gammas per ml. of milk; the remain­

ing one-third was tuoed,and to each tube was added a l i t t l e reduced iron,

as recommended by Scott (43) and Hastings and McCoy (25) for the main­

tenance of a reduced potential i n a medium. This material after s t e r i l i z ­

ation with the milk maintained the indicator i n a reduced condition as the

leuko compound except for a narrow surface band which re-oxidized to

methylene blue on cooling. Table Z records the data as obtained with this series after a f i v e day incubation period.

Table IX

The Tolerance of Species of Actinomyces to Varying Concentrations of Methylene Blue

i n Milk

Species l o . 1:50,000 1:10.000 1:1,000

A-l

A-2

A-3

A-4

A-5

A-6 A-7

A-8

A^9

A-10

A - l l

A-12

Heavy surface ring. Complete reduction below pigment zone.

White surface ring. Heduction complete at bottom.

Black surface growth. Se­duction complete below pigment. Whole tube digested. Serum greenish. Surface ring and growth down sides. Reduction complete i n lower half of tube. Reduction complete at bottom. Blue surface ri n g . Reduction complete at bottom.

Whole tube digested. Serum yellowish. Black surface growth. Se­duction complete below pigment.

Brown surface growth. Re­duction complete below pigment.

Blue-green surface r i n g . Re­duction complete i n lower half of tube. Yellow surface growth.,. Whole tube digested.

Surface growth and colonies down sides of tube. Complete reduc­ti o n at bottom of tube, White surface ring. Reduction complete at bottom.

Yellow colonies down sides. 75$ reduction at bottom.

Growth down sides. Slight reduction at bottom.

Slight surface ring. Slight reduction at bottom..

Small blue colonies down sides. Reditction complete at bottom; p a r t i a l through rest of tube. Slight growth down sides. Reduction complete at bottom; p a r t i a l through lower half of tube. Small colonies down -g- of tube. Reduction complete at bottom.

Yellow colonf ies down sides of tube. Reduc­tion complete at bottom.

Small blue colonies down sides. Mo re­duction.

Small blue colonies near bottom of tube. Fo reduction.

Small colon­ies near bot­tom of tube. 50% reduction at bottom, i

Table X

The Influence of Fe r r i c Nitrate and Reduced Iron i n the Reduction of Methylene Blue i n Milk by Species of

Actinomyces

Culture No»

Methylene Blue ; Methylene Blue + F.e(JT0R).„

Methylene Blue + Reduced Iron..

. A - l complete reduction complete reduction at bottom - 75$ reduction above

no re-oxidation

A-2 > .'• no reduction no reduction no re-oxidation A-3 complete reduction 50$ reduction through­

out tube sl i g h t re-oxidation

A-4 no reduction ' no reduction s l i g h t re-oxidation A-5 sli g h t reduction

at bottom no reduction p a r t i a l re-oxidation

A-6 no reduction no reduction complete re-oxidation

A-? ; sli g h t reduction at bottom

sl i g h t reduction at bottom

no re-oxidation

A-8 no reduction sli g h t reduction at bottom

complete re-oxidation

A-9 no reduction no reduction no re-oxidation

A-10 ... 90$' of tube reduced

80$ of tube reduced no re-oxidation

A - l l complete reduction at bottom

complete reduction at bottom

blue surface band 50$ reduced

A-12 no reduction no reduction complete re-oxidation

- 34 -

It w i l l De observed that, considering the a b i l i t y to reduce or to

discharge the color from methylene blue, or to re-oxidize methylene white,

the species f a l l into three groups.* (l) Those exerting a strong reduc­

ing action i n the medium,- Species A - l , A-7, A-10 and A - l l ; these effected

at least p a r t i a l reduction of methylene blue and showed no a b i l i t y to

oxidize the reduced iron tubes. (2) Those exerting no reducing action

i n the medium - Species A-2, A-5 and A-9; these did not reduce methylene

blue i n the given time and showed no a b i l i t y to oxidize the reduced iron

tubes. A-3 i s also placed i n th i s group since i t effected both reduction

and a slight re-oxidation. (5) Those exerting an oxidizing action -

Species A-4, A-6, A*-8, A-12j with these cultures there i s no reduction of

methylene blue but a more or less complete re-oxidation of methylene white.

This c l a s s i f i c a t i o n of the species agrees with the i r tolerance to

methylene blue as ascertained i n Table IX, with the exception of A-l and

A-2. On the basis of tolerance to methylene blue A-2 appeared more

anaerooic than A - l ; but i n the above data the order i s reversed. Since

A-2 showed sub-surface growth i n shake agar cultures (Table T i l l ) , i t was

decided that i t should be placed with A-7, A-10 and A - l l as species showing

the strongest tendency towards anaerobiosis.

The presence of f e r r i c n i t r a t e i n many cases tended to prevent as

complete reduction of methylene blue as i n the "control" cultures, i n d i ­

cating that the oxidation-reduction potential of the medium had been raised.

The three cultures showing the greatest response to f e r r i c n i t r a t e i n

the degradation of milk protein are among the most aerobic. This per­

haps suggests that the presence of iron raises the potential of the medium

so that the microorganisms can grow down to a greater depth i n the

medium, the resu l t being greater proteolytic a c t i v i t y .

Influence, of,Iron xmder Anaerobic Conditions

As a result of these studies i t was decided to determine the effect

of iron on the a c t i v i t y of A-8, chosen as an aerobic species, and A - l l ,

chosen as an anaerobic species., when placed under anaerobic conditions.

Heavy mineral o i l , added to the surface of the milk flasks p r i o r to ster­

i l i z a t i o n to form a layer about a half an inch thick, was used as a means

of preventing the absorption of atmospheric oxygen by the milk after

s t e r i l i z a t i o n . While i t i s possible that complete exclusion of a i r cannot

be obtained i n this manner, at least a considerably reduced oxygen tension

results. 1 Table XI

A Comparison of the Response of Species A-8 and A - l l to the Addition of Ferric If i t rate to Milk under Aerobic

and Anaerobic Conditions

species No.

A-8

A-8

A-8

A-8

A - l l

A - l l

A - l l

A - l l

Iron Added •r.

47.68

47.68

47.68

47.68

C - uninocu-lated

L _

O i l

150 ml,

150 ml.

150 ml.

150 ml.

Non-protein Nitrogen as per cent of Total Nitrogen

9.6

21.7

607

8.3

7.8

8.0

7.8

9x8

6.1

Per cent Increase

26.0

23.8

2.5

25.6

The data as presented i n Table XI show cl e a r l y that the a c t i v i t y

of A-8 i s materially lessened under anaerobic conditions, when there

was l i t t l e stimulation from iron. Whereas the percentage stimulation

i n aerobic culture was 126 per cent, i t dropped to 23.8 per cent upon

the exclusion of a i r . The anaerobic species, A - l l , on the other hand,

showed l i t t l e or no stimulation from iron under aerobic conditions, but

was stimulated by 25.6 per cent i n the absence of atmospheric oxygen.

This increase i s believed significant,since A - l l was noteworthy i n that

i t did not respond to iron at this period of the study. Apparently the

presence of iron i n anaerobic culture f a c i l i t a t e s the exchange of

oxygen in the case of A - l l but has l i t t l e effect on the aerobic A-8 when

atmospheric oxygen lias been excluded. I f such i s the case, a moderate

supply of a i r should increase the a c t i v i t y of the iron i n the case of A-8

and should decrease i t s effectiveness when A - l l i s used. Experiments

were set up to prove or disprove t h i s p o s s i b i l i t y .

Flasks were prepared as above; i n addition to adding the o i l p r i o r

to autoclaving, 150-ml. quantities of s t e r i l e o i l were added to two of

a series of flasks at definite intervals during the cooling of the milk,

i n an endeavor to l i m i t the amount of a i r absorbed by the milk during

cooling. These were then inoculated by placing 0.5 ml. of a water sus­

pension of the required seven-day culture below the surface of the o i l , ,

and incuoated at 28° C for one week. The data reported i n Table XII

indicate that, when species A-8 i s used as the source of inoculum, the

response to iron becomes greater as atmospheric oxygen i s absorbed by

the culture medium. Whereas no response was noted when more or less

complete exclusion of a i r was effected, an 18.7 per cent increase was

recorded when the milk had been allowed to stand for twenty hours p r i o r

Table XII

The Influence of Aerobic to Anaerobic Conditions on the A c t i v i t y of A-8

Treatment with O i l Non-protein Nit

per cent of .. .Nitrog

rogen as Total en • Per Cent

Milk Alone Milk + Iron Increase Ko o i l 9.2 16.1 72.7 Added before s t e r i l i z a t i o n 6*3 6.2 «.

Added 4 hours after s t e r i l i z a t i o n 7.2 7.9 9.7 Added 8 hours after s t e r i l i z a t i o n 8.0 8„3 Added 20 hours after s t e r i l i z a t i o n 8.0 9 © 5 18.7 Control - uninoculated 6.0 6.0

to the addition of the o i l . While this figure i s much lower than the

72.7 per cent increase obtained i n the cultures exposed to the atmosphere

during the incubation period, the increase would suggest that oxygen i s

essential for the highest proteolytic efficiency of A-8, and that iron

gives i t s maximum stimulatory effect i n the presence of a i r .

From Table XIII,the response of A - l l under identical conditions

shows the opposite trend. This species exhibits i t s maximum response to

iron under most nearly anaerobic conditions, while the presence of a i r

depresses i t s stimulatory effect. Thus, when oxygen was excluded, an

increase of 35.3 per cent was recorded, whereas no stimulation or only

negligible response resulted when even small amounts of a i r were absorbed

by the medium. While this increase does not approach that recorded for

A-8 under optimum conditions, i t i s believed s i g n i f i c a n t , since A - l l

showed l i t t l e response to ir o n i n other experiments performed at t h i s

Table XIII

The Influence of Aerobic to Anaerobic Conditions on the A c t i v i t y of A - l l

Treatment with O i l lion-protein Nitrogen as

per cent of Total Nitrogen Per Cent

Increase

Treatment with O i l

Milk Alone Milk + Iron Per Cent Increase

No o i l 6.7 6.8 1.5 Added before s t e r i l i z a t i o n •6.5 8.8 55.5 Added 4 hours after s t e r i l i z a t i o n 5.5 Added 8 hours after s t e r i l i z a t i o n 6.7 6»7 -Added 20 hours after s t e r i l i z a t i o n 6.5 6.7 3.0 Control - uninoculated 6.0 6.0

time. Thus species A-8 and A - l l are d i r e c t l y opposite i n their reaction

to these conditions.

On the basis of the data presented i n t h i s section, i t would appear

that response to iron i s largely based on oxygen requirement. The aerouio

species are stimulated by the addition of iron to the medium, whereas

those species i n which a stronger reducing a c t i v i t y was demonstrated,

exhibit l i t t l e or no response to iron under aerobic conditions, but when

ai r i s excluded from the culture, significant stimulation occurs* The

difference between the oxygen requirements of the species would appear

to be one of degree only, since a l l the cultures w i l l grow i n the presence

of a i r , but iron does not appear to have i t s maximum stimulatory effect

on the more anaerobic species except i n the absence of oxygen.

Discussion and SummaryPart B A study of the stimulatory effect of iron on proteolytic a c t i v i t y as

- 39 -

determined i n Part A indicated certain differences between the various

species. When the stock cultures had been stabilized through contact

with s o i l , A-4, A-8 and A-12 responded markedly to the presence of iron,

while the other species were inconsistent or showed l i t t l e stimulatory

effect. This appeared to suggest some fundamental difference between

the species.

Preliminary experiments i n shake agar cultures coupled with th e i r

general cultural characteristics suggested certain differences i n oxygen

requirements, and these differences were believed to afford an explanation,

for the varying response to iron. In shake agar cultures three species,

A-2, A-7 and A - l l , exhibited sub-surface growth and hence the greatest

tendency toward anaerobiosis. The presence of methylene blue allowed

growth i n the case of a l l species to grow deeper i n the medium, whereas

the presence of f e r r i c n i t r a t e allowed the more anaerobic species to

grow at the surface, and forced the species which i n the controls exhib­

ited surface growth, to develop below the surface of the agar. This

data strengthened the conviction that iron played some fundamental part

i n the respiratory complex, and that the oxygen requirements of the

species determined the conditions under which the enrichment would be

most effective.

Accordingly, a study of the oxidation-reduction requirements of the species was undertaken, using methylene blue, the indicator which proved most satisfactory for the purpose. On the basis of tolerance to methylene blue and their reduction and re-oxidation of the dye, the spe­cies were divided into three groups: those exerting a strong reducing action - species A-2, A-7, A-10 and A - l l ; those exerting no reducing action - species A-4, A-6, A-8 and A-12; and those with an intermediate

reducing power - species A - l , A-3, A-5 and A-9.

The presence of f e r r i c nitrate at the concentration used i n milk

throughout the study retarded i n many cases the rate of reduction of the

methylene blue, suggesting that i t had some effect i n r a i s i n g the potential

of the medium. This would account for the lower growth level observed i n

shake agar cultures with species A - l , A-3, A-4, A-5, A-6, A-8, A-9, A-10

and A-12, but affords no reason for the surface growth with A-2, A-7 and -

A - l l .

In an effort to find some explanation for t h i s observation, species

A-8 and A - l l were chosen for study under anaerobic conditions as secured •

through providing a layer of mineral o i l on the surface of the culture,,

A-8, an aerobic species showing strong response to iron i n the presence

of a i r , was not stimulated by iron under anaerobic conditions, but showed

increased stimulation with the amount of a i r absorbed by the freshly ster­

i l i z e d milk. A - l l , on the other hand, responded to iron under conditions

of anaerobic culture, while showing no significant response when oxygen

was present. This species, i t i s true, had shown response to f e r r i c n i ­

trate i n the data presented i n Table I, but i t s reaction was inconsistent,

and in the control cultures run at the time of these experiments not more

than 3*0 per cent stimulation was recorded, whereas under anaerobic con­

ditions an increase of 25.6 - 35.3 per cent was noted and i s believed

s i g n i f i c a n t .

I t i s d i f f i c u l t to state at the present time as to whether or not

Warburg's theory that, respiration or the u t i l i z a t i o n of molecular oxygen

by l i v i n g c e l l s i s a process resulting from catalysis by iron, i s a p p l i ­

cable i n the case of the data recorded. However, the importance of iron

. . . . . . 41 - • for the respiratory enzymes as reviewed by the University of Wisconsin

Biochemists (48), and the opposite reactions of the aerobic and the more

or less anaerobic species, would suggest a fundamental difference i n the

respiratory enzyme complexes of A-8 and A - l l .

In view of the data outlined above there appear to be certain rather

s t r i k i n g differences and s i m i l a r i t i e s among the twelve species under study,

but detailed explanations cannot be set forth at the present time regard­

ing the actual function of the iron enrichment with respect to the oxida­

tion-reduction requirements of the individual species.

PART C - MZY1E STUDIES

While i t has been demonstrated i n the preceding section that iron i s

effective in. stimulating proteolysis through i t s influence on the oxidation-

reduction potential of the medium, i t was s t i l l thought possible that i t

might play a part i n influencing the production of the proteolytic enzyme

complex.

Waksman (51) takes as part of his system of c l a s s i f i c a t i o n the action

of the species i n milk, dividing them into f i v e major classes, based on

the i r a b i l i t y to bring about the c l o t t i n g of milk and to effect protein

breakdown. I t i s noteworthy that no species isolated i n t h i s laboratory

over a three-year period has had the a b i l i t y to form a clot i n milk. In

certain instances, however, species have been observed to c l o t milk i n the

presence of a greater amount of iron than i s optimum for the stimulation

of proteolysis.

He stated that, while the optimum temperature for growth l i e s be­

tween 25 and 28° 0 for the saprophytic groups, the maximum rate of pro­

teolysis takes place at 37° G when hydrolysis of protein i s measured by

- 42 -

ammonia production using brom cresol purple as the indicators The

species used i n the present study, however, i n every case showed maximum

ammonia production at 28° 0, and three cultures, A-5, A-9, and A-12,

refused to grow at 37° C.

In the same paper Waksman described methods for iso l a t i n g a rennet­

l i k e enzyme from a well hydrolyzed milk culture by preci p i t a t i o n with

alcohol, and for separation of a crude enzyme complex capable of proteo­

l y t i c action by treating vegetative growth oy the "acetone-dauerhefe"

method. He reported a f a i r l y complete separation of the two enzyme sys­

tems by these procedures.but no confirmation of t h i s has been obtained

with the species studied herein. The material prepared by either method

clotted s t e r i l e milk occasionally^but no consistent behavior could be

discovered- on the other hand, both preparations brought about peptoniza­

tion of the milk protein, suggesting that no complete separation of the

two enzymes had been effected.

Since the preparation of an alcohol precipitate provided an active

proteolytic complex, t h i s method was used to determine the effect of

iron on i t s a c t i v i t y . Two-months* old cultures of A-8 and A - l l i n milk

and i n milk containing 47.6 gammas of iron, i n which the casein was

almost completely hydrolyzed, was f i l t e r e d , the f i l t r a t e precipitated with

95 per cent alcohol, and the precipitate so obtained f i l t e r e d o f f ,

washed with alcohol and ether, and dried over sulphuric acid. Weighed

portions of the dried precipitate were added to 10-ml. quantities of

s t e r i l e milk and s t e r i l e milk containing 47.6 gammas of iron as f e r r i c

n i t r a t e . 1-ml. of toluene was added to prevent bacterial growth and

the tubes were incubated at 28° C for a period of four days, after which

time non-protein nitrogen was determined. Since only small amounts of

the f i l t r a t e s were available for the t o t a l nitrogen determinations, the

micro-Kjeldahl method was used after i t had been found to give almost

identical results with the macro-method. The data are presented i n

Table XIV.

Table XI?

.Response of. Various Proteolytic Enzyme Preparations to the Presence of Iron

Source of Enzyme

gms.'of Enzyme added

Son-protein Kitrogen as per cent of Total

Nitrogen Milk M l k + Fe

A-8 .05 13.1 15.7 a X 24.9 28.4

A-8 Fe* • .05 50.1 43.9 - » X 55.7 50.5

A - l l . * X 18.0 15.4 .2 23o o 24.9

A - l l Fe* .1 25.5 24.4 .2 38.2 35.7

* Cultures A-8 and A - l l grown i n milk containing 47.6 gammas of iron were the enzyme sources.

A study of Table XIT reveals that iron does not appear to stimulate

the proteolytic enzyme complex per se, since no significant stimulation

was found with, any of the preparations studied. The material from the

parent iron cultures, on the other hand, i s more active than a corres­

ponding amount of enzyme from a culture to which no iron had been added,

suggesting that the iron stimulates production of the proteolytic enzyme

complex. While i t must he taken into consideration that the alcohol

precipitate from the cultures containing iron was smaller, and hence the

enzyme complex was more concentrated, t h i s large increase i n a c t i v i t y

- 44 -

appears to be s i g n i f i c a n t , at least i n the case of species A-8. Thus

i t may be concluded that f e r r i c n i t r a t e brings about greater production

of the proteolytic enzyme complex, and that t h i s stimulatory effect i s

responsible,in part at least^for tfee large increases i n non-protein n i ­

trogen recorded for certain species e a r l i e r i n the study.

The p o s s i b i l i t y that iron was stimulating not only the enzyme com­

plex, but also the amount of growth, was considered. Since the physical

nature of milk does not allow the separation of vegetative growth from

the medium, the species were cultured on sodium asparaginate glycerol

f l u i d medium both i n the presence and i n the absence of f e r r i c n i t r a t e .

The nature of the growth of actinomycetes i n f l u i d media made i t convenient

to f i l t e r off the colony mat and determine i t s weight i n the manner used

for study of the fungi. Accordingly, 100 ml. quantities of sodium

asparaginate glycerol f l u i d medium of the' same constitution as the s o l i d

medium used for carrying the stock cultures (with, of course, the omission

of the agar) were placed i n 500 ml. fla s k s . A water solution of f e r r i c

n i t r a t e was added to certain of the flasks to give a concentration of

5.68 gammas of iron per ml. of medium, and after s t e r i l i z a t i o n , they were

inoculated with representative species. After a ten-day incubation period

at 28° C, the medium was f i l t e r e d through a weighed paper and the weight

of vegetative c e l l s determined. The data i s presented i n Table XT.

The presence of i r o n apparently does not influence the amount of

vegetative growth produced by the cultures to any appreciable extent,

although A-8 does show some increase. However, the increase reported i n

Table XT with regard to A-8 has not been confirmed i n duplicate experi­

ments, and hence i t i s probably correct to conclude that iron exerts no

- 45 -

Table XT

Influence of Iron on the Amount of Vegetative Growth Produced by Species of Actinomycetes

Culture . No.

Weight of Tegetative Growth i n mgms. . Sasic medium Basic medium + Fe

Per Cent Increase

A-4 26.2 25.2 -A-5 24.5 • 9.8

. A-6 34.5 31.0 A-7 25,8 26.2 1.6 A-8 21.7 26.7 23.0

: V; A - l l ' 23.4 26.3 14.0 A-12 25.7 22.7 -

stimulatory effect upon the amount of growth produced by the various

species.

Discussion and Summary - Part. C /

Since increased proteolytic a c t i v i t y had been reported i n the case

of certain species i n the presence of iron, i t was decided to determine

i t s stimulatory effect on the proteolytic enzyme complex. Species A-8

arid A - l l were chosen for t h i s phase of the investigation. A-8, when

carried on s o i l , responded markedly to f e r r i c n i t r a t e and was quite active

p r o t e o l y t i c a l l y . A - l l , on the other hand, was not stimulated appreciably

by iron, and did not hydrolize milk protein to any great extent. Milk

cultures of the species with and without iron were used as the source

of the enzyme material, which was prepared by precipitation with alcohol.

The data, while inconclusive i n some respects, demonstrated that

f e r r i c n i t r a t e , at the concentration to which species 1-8 i t s e l f responded,

had l i t t l e or no effect on the a c t i v i t y of the enzyme preparation. At

the same time, the actual production of the enzyme"complex was stimulated

•markedly, i n view of the fact that preparations from parent cultures

with and without iron differed widely i n a b i l i t y to bring about non-protein

nitrogen formation. While i t i s recognized that the enzyme material from

the parent iron culture was more concentrated than that without i r o n ,

at the same time, the large increases reported with A-8 i n the presence

of iron would lead one to conclude that the amount of enzyme produced i n

the presence of iron was considerably greater than that formed i n i t s

absence. A similar study of A - l l supports the observations made as

regards A-8, but since t h i s species was not as active p r o t e o l y t i c a l l y and

did not show large responses to iron, the data i s perhaps not as clear-cut.

A comparison of the actual weight of,vegetative growth from the

various species i n the presence and i n the absence of iron prompted the

conclusion that t h i s metal had no stimulatory effect on the amount of

colony mat produced.

It would appear that, while iron has no effect on the amount of ve­

getative growth produced, and does not stimulate the proteolytic enzyme

complex per se, i t does perform a function i n increasing the amount of

enzyme complex formed i n a given milk culture. Thus, iron apparently

stimulates certain species under aerobic conditions both by increasing

the amount of proteolytic enzyme complex produced, and, as was pointed

out i n Part 3, through i t s function with regard to the oxidation-reduction

requirements of the species.

- 47 -

IT. GENERAL STffiffflRY

The influence of iron on the metabolism of Actinomyces was i n v e s t i ­

gated, using non-protein nitrogen production i n milk as the basis of study.

Twelve cultures representative of species normally found i n the upland

g l a c i a l s o i l of coast regions of B r i t i s h Columbia were selected for the

study.

Significant variations recorded between experiments performed at

different times were shown to follow definite trends. Speaking generally,

the proteolytic a c t i v i t y of the species decreased i n accordance with the

length of time the stock cultures had been maintained on an a r t i f i c i a l

medium. The large differences recorded between species i n the i n i t i a l

experiments tended to become equalized and minimized, u n t i l , after an

eighteen months* period, l i t t l e difference i n non-protein nitrogen pro­

duction was observed. The addition of f e r r i c nitrate to the milk medium

resulted i n a larger spread of a c t i v i t y between the species at the com­

mencement of the work, due to the stimulatory effect of iron i n certain

instances. But this effect was inconsistent, and a c t i v i t y decreased

u n t i l a minimum l e v e l was reached. On the average, a slight stimulatory

effect from iron was s t i l l apparent.

The data suggested the u n s u i t a b i l i t y of the laboratory medium for

maintaining the a c t i v i t y and s t a b i l i t y of the cultures over a period of

time. In an effort to overcome the d i f f i c u l t y , the species were returned

to their native habitat - the s o i l - i n pure culture. Isolations at the

end of a year displayed, i n most instances, a greatly increased hydrol-

y t i c a c t i v i t y , and three species, A-4, A-8 and A-12, responded markedly to

iron. Apparently contact with this complex natural medium had largely

overcome the i n s t a b i l i t y of the species, raising them to a higher l e v e l

of biological a c t i v i t y when the extreme v a r i a b i l i t y of the cultures was

reduced to the minimum. By providing s t e r i l e s o i l as the medium for the

maintenance of the stock cultures, and by using fresh isolations for

quantitative studies, v a r i a b i l i t y , which has heretofor been considered an

inherent p e c u l i a r i t y of the actinomycetes, may be largely overcome.

Since s o i l ash was shown to have a stimulatory effect on certain of

the species, their response to a wide variety of trace elements was

studied. The accumulative effect of the metals added indicated that,

while A-12 responded only to iron, A-7 was stimulated by some other cation,

and A-4 and A-8 showed an increased response when other metals were

added with iron. The efficiency of s o i l i n maintaining the species at a

high l e v e l of biological a c t i v i t y thus appeared to be due to the variety

of metallic elements i t contained.

The p o s s i b i l i t y that iron might bring about i t s stimulatory effect

through i t s influence on the varying oxidation-red-uction requirements

of the species was considered. Accordingly, i t was shown that, while an

aerobic species responded to iron i n the presence of oxygen, a less

aerobic species was stimulated only under anaerobic conditions. Certain

fundamental differences i n the respiratory mechanism of the contrasting

species were thus suggested.

No stimulatory effect of iron on crude proteolytic enzyme pre­

parations could be demonstrated, but the presence of the metal appeared

to stimulate proteolytic enzyme production, at least i n the case of those

cultures exhibiting strong hydrolytic a c t i v i t y .

Thus, when the v a r i a b i l i t y encountered i n any study of the actino­

mycetes has been reduced to a minimum by maintaining the stock cultures

- 49 -

i n s t e r i l e s o i l , a strong and consistent response to f e r r i c nitrate was

observed to be due to an increased energy exchange resulting i n a

greater production of the proteolytic enzyme complex.

- 50 -

V. BIBLIOGRAPHY

(1) Allyn, W.P., and Baldwin, I. L., 1932. The Oxidation-Reduction Po­t e n t i a l i n Relation to the Growth of Anaerobic forms of Bacteria. J. of Bact. 23s 369 - 398.

(2.) Associates of Rogers, L. A. 1935. Fundamentals of Dairy Science. Second Edition. Reinhold Pub. Oomp., Hew York.

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