DOCTORAL DISSERTATION SERIES
title

STUDIES !HPIQMNWIDN /N

mumcrn mam ml
mm

DAIIId MHLM
, SW£ CSlLom 1951
UNIVERSITY. 0CH

AUTHOR

DEGREE m

.

. PUBLICATION NO.
III

I

ML

a

y UNIVERSITY MICROFILMS
ANN ARBOR

• MICHIGAN

STUDIES IN PIGMENTATION IN MICROCOCCUS
PYOGENES VAR. AUREUS

by
David Kahler

A THESIS

ubmitted to the School of Graduate Studies of
Michigan State College of Agriculture and
Applied Science in p a r tia l fu lfillm e n t
of the Requirements fo r the degree of
Doctor of Philosophy

Department of Bacteriology and Public Health
1951

ACKNOW
LEDGMENT

The w riter wishes to express his sincere
appreciation to Dr, W
. L. Mallmann or the
Department of Bacteriology and Public Health
fo r his guidance and assistance in conducting
th is in v estig atio n and to the Chemistry
Department fo r the use of equipment.

TABLE OF CONTENTS
Page
INTRODUCTION - - - - - - - - - - - - - - - - - - -

1

LITERATURE REVIEW

3

- - - - - - - - - - - - - - - -

EXPERIMENTAL METHODS --------- - - - - - RESULTS

-

- - - - - - - - - - - - - - - - - - - - -

10
14

The Effect of Various Constituents of Chapman's
No. 110 Liquid Medium on Pigmentation - - - -

18

The Effect of the B Complex Vitamins Upon
Pigmentation - - - - - - - - - - - - - - - - -

21

The Role of the B Complex Vitamins in
Pigmentation Using a Semisynthetic Medium

24

- -

Relationship of Cocarboxylase to Thiamin in
Pigmentation of Staphylococci - - - - - - - -

32

Effect of S alts on the Pigmentation of
Staphylococci - - - - - - - - - - - - - - - -

33

The Role of Carbohydrates in Pigmentation

- - -

40

- - - -

46

Effect of Amino Acids upon Pigmentation
SUM
M
ARY

----- - - - - - - - - - - - - - - - - - - -

53

BIBLIOGRAPHY - - - - - - - - - - - - - - - - - - -

58

TABLES

INTRODUCTION
The fa c t th a t many microorganisms have exhibited some
type of coloration has aroused the c u rio sity of mankind since
i t s beginning.

Many attempts have been made to explain why

pigment production by a p a rtic u la r organism varies from time
to time under supposedly the same conditions.

As pigmentation

is usually accompanied by good growth, some workers have
stated th a t pigment is formed during c e ll p ro life ra tio n and
is the r e s u lt of active b a c te ria l metabolism.

Such a theory

discounts any idea th a t a p a rtic u la r metabolite d ire c tly
affects pigmentation.
Other workers have favored a theory of inheritance of
color and support th e ir theory with the evidence of Bunting's
work (3) with S e rra tla marcescens.

Using a synthetic medium

Bunting found th a t four types of colonies, namely:
bright pink, pale pink,
conditions,

and white,

dark red,

grown under controlled

a l l eventually came to an equilibrium with about

97.0 percent dark red and 3.0 percent bright pink colonies.
Most workers in th is f i e l d have f e l t th a t there are
p a rtic u la r growth fa c to rs or combinations of these facto rs
which cause an organism to produce pigment.

Evidence has

indicated th a t these facto rs are d iffe re n t f o r each species
of organism and may even vary within the species.

Supporters

of th is th ir d theory point out th a t such a hypothesis could

2

include the f i r s t theory as the organism forms products during
good growth which serve as metabolites fo r pigmentation.
With staphylococci many attempts have been made to study
the e ffe c t of various n u t r i l i t e s upon pigmentation.

However

these studies have been made upon media which consist of
components such as Cl) meat e x trac t,

(2) evaporated milk,

(3) ex tracts from or agglutinins prepared against staphylococci
or (4) alcohol p re cip itate d ex tracts of beef, brain, spleen,
heart and kidney.

The end r e s u lt has been th a t the information

has varied with the t e s t conditions and very l i t t l e
has resu lted .

agreement

Also methods of measuring pigmentation have

been f o r the most p a rt,

crude and therefore not too r e lia b le .

I t was f e l t th a t i f a method could be evolved by which
the amount of pigment could be measured with at le a s t a f a i r
degree of accuracy, then various media, both natural and
synthetic,

could be used to study the e ffec t of chemically

defined compounds upon pigmentation in S. aureus
(liicrococcus pyogenes var. aureus).
th is study was undertaken.

With th is object in mind

LITERATURE REVIEW
Pigmentation in microorganisms has at times been
instrumental in guiding the destiny of mankind since the time
before C hrist.

During the siege of Troy in 332 B.C. by

Alexander and his fellow Macedonians, the appearance of
blood-like droplets in bread was Interpreted to mean
destruction of those within the c ity

(27).

As a re s u lt,

the Macedonians continued th e ir assault and the c ity of Troy
fe ll.
Since the above event,

instances of "bloody bread"

have altered the destiny of man u n til 1819 when a s c ie n tif ic
explanation of the phenomenon was made by Bartolomea Bizio
(27X a professor a t the University at Padua.

Bizio promptly

recognized the fungus nature of the coloring matter and
proceeded to in fect other materials with the organism.
"Bloody spots" appeared within twenty-four hours.

He named

the causative organism S e rra tia marcescens. the genus in
honor of Serraflna S erra tia ,
on the Arno riv e r.
putrefying.

the f i r s t to operate a steamboat

The specific name means decaying or

In 1827 a complete report,

including the

chemical nature of the pigment, was made by Bizio.
The other carotenoid pigment producers have also had
in terestin g genealogies although most of these organisms were
investigated f o r th e ir pathogenic significance before

extensive work was done on th e ir chromogenic properties.
Robert Koch (35) f i r s t noted a micrococcus in pus in 1878,
and two years l a t e r Pasteur cu ltiv ated the organism in liquid
medium.

Rosenbaeh (34) made a thorough study of the organism

and the basis of i t s

pigment production,

and divided the genus

into two species — Staohvcoccus pyogenes aureus and
Staphylococcus pyogenes aibna.

Passet (35) added a yellow

v ariety , Staphvlococcus pyogenes c itr e u s .

Since th a t time

many attempts have been made to c la ssify these micrococci
using various c r i t e r i a such as fermentation reactions,
pathogenicity, hemolytic a c tiv ity ,

e tc .,

but the predominating

c h a ra c te ris tic has been the pigment as is evident by the
present nomenclature -- Micrococcus pyogenes aureus.
Zopf (38) studied the pigment content of eight
organisms and found lipoxanthins to be present in Bacterium
egrp.gium. Bacillus chrvsoloia and Staphylococcus aureus.
Gurd and Denis (15) reported th a t carotenoids were found in
Mycobacterium le p ra e .
technique,

Reader (26), using the chromatographic

id e n tifie d carotene in Sarclna aurantlaca and

Chargaff (7), working with the carotenoid pigments of Sarclnia
lu te a ,

Sarclna aurantiaca and Staphylococcus aureus. found

sarlnene, xanthophyll,

carotene, and zeaxanthine to be present.

Baumann et al (2 ) found th a t several species of Flavobacterium.
Corynebacterlum. Staphylococcus. and Mycobacterium synthesized

5

carotenes; however, attempts to transform the carotene to
vitamin A by the use of microorganisms failed .

However a

carotenoid-like structure was thought by Skinner and
Gunderson (31) to be the precusor of vitamin A in a species
of CorvnebpeterlumFrench (11) working with photosynthetic bacteria,
lib erated the c e ll contents by supersonic vibration.

A water

soluble protein which was attached to insoluble pigments,
bacteriochlorophyll and carotenoid, were lib erated .

Reimann

and Eckler (28), studying four variant types of Micrococcus
tetragenus. iso lated the following pigments:

yellow variant -

xanthophyll, pink variant - rhodaxanthin, brown variant carotin,

and mucoid pink variant - lycopene.

Sobin and

Stahly (30) extracted carotenoids with hot methanol from
Flavobaotgrium. Sarclna. Micrococcus. Erwlnla. Bafitp.pium.
Cp.llumonfls and Staphylococcus and found that some species
which are separated by biochemical reactions have identical
pigments.
Ingraham and Baumann (16), using a synthetic medium,
were able to demonstrate th at the amount of carotene produced
by a given organism varies with the medium, and th at the
content per gram of c e lls increases to a maximum a fte r which
i t decreases.

These workers suggested th at such a carotenoid

synthesis carried in terestin g biological implications since
the organisms do not contain chlorophyll.

6

Pinner and Voldrich (24) reported th at Staph. albus.
Staph* c itr e u s . and Staph. roseus are s p l i t off spontaneously
from Staph, aureus in broth containing aureus agglutinins
and th at albus can be reverted to aureus by using media with
a high concentration of anti-albus serum.

Eyatt, Jann and

Salle (4) obtained an extract from ruptured ce lls of a highly
chromogenic s tr a in of Staph, aureus and th is extract was used
to induce pigmentation in white stra in colonies.

Nutini and

Lynch (23) using an alcohol precipitated extract of beef
brain,

spleen, heart or kidney were able to convert the yellow

S form of Staph, aureus to the white R form with altered
biochemical c h a ra c te ristic s.
S till

The findings of Goldworthy and

(14) show th a t ox heart meat extract when added to

nutrient agar in h ib its pigment production in Bacillus
proaiglosus and S. marcescens but enhances pigment production
in Staph, aureus.
Two cultures of acid f a s t bacteria were found by Baker
(1) which produced pigment in the presence of lig h t and were
devoid of pigment in the dark.

Brief exposure to u ltra v io le t

and sunlight and longer exposure to e le c tric lig h t conferred
the a b ility to form pigment on fu lly developed unpigmented
cultures.

Kreitlow (19) exposed S. marcescens to a bright

red lig h t with the re su lt th a t a bright orange color was
produced a t 20 and 27 C while under a blue lig h t,

a highly

7

chromogenic organism l o s t i t s

pigmentation.

Cultures exposed

to white lig h t formed intermediates.
Sullivan (34) reported the yellow pigments of Bacillus
fucus. Micrococcus aurantlcus, M. c itr e u s . M. tetragenus
v ersat11i s . and Sarclna lu te a are formed very slowly in nonalbuminous media but are quickly formed in a peptone solution
plus s a lt s .

This worker found th a t magnesium s u lfa te and

dipotassium phosphate favored pigmentation.

Reid's (27)

exhaustive study of 76 cultures representing 24 genera of
chromogenic b acteria,

showed th at no pigment was produced when

the medium contained no nitrogen or inorganic s a lts ; however,
amino acids as the sole source of nitrogen or metals found in
pigments were not useful in stim ulating pigmentation.
Kharasch, Conway and Bloom (17) working with several cai'otenoid
producers showed th a t large concentrations of biologically
Important metals (manganese, copper,

iron) in h ib it organism

growth while smaller concentrations sometimes cause pigmentation
lo ss.

Turian's (36) studies with Mycobacterium phlei

demonstrated the opposite to be true in th a t more carotenoids
were produced when the medium contained traces of iron and
manganese.

Dewey and Poe (8) worked with minimum essen tials

fo r pigment production in S. marcescens and demonstrated
th at manganese,

s u lfs te ,

and phosphate were necessary.

8

Chapman (5) seeking a selectiv e medium fox* the iso la tio n
of staphylococci,

reported th a t a 7*5 percent sodium chloride

added to a Eacto phenol-red agar provided a good Isolation
medium and enhanced chromogenesis,

Fagracus (9) found that

the staphylococcus grew luxuriously with abundant pigmentation
when the sodium chloride concentration was carried as high as
10 percent.
Kharasch, Conway and Eloom (17) observed that the
development of pigment in S. marcescens does not require
glucose but depend upon substances with available aldehyde
or ketone groups.

Sevag and Green (29) investigated the role

of several breakdown products of glucose in the pigmentation
of Staph, aureus and concluded th a t there is some unknown
product of th e ir intermediate metabolism which is required.
The work of Kliger,

Grossowicz and Bergner (18) showed

th at both n ic o tin ic acid and thiamin must be present fo r the
f u l l u tiliz a tio n of available carbohydrates in staphylococci
but these workers did not mention the extent of pigmentation.
However the improved Chapman-Stone (6) medium the yeast
extract of which contains the above mentioned vitamins,
produces a deep pigment.

Gladstone (13) reported th at 25

stra in s of Staph, aureus grew well on a medium which included
16 amino acids with the following acids being e sse n tia l;

mmM

9

cystine, leucine,
phenylalanine,

valine, proline,

gylcine,

arginine and tryptophane.

aspartic acid,

Porter and Pelzer

(25) were unable to grow many stra in s of Staph, aureus in
Gladstone's basal medium and reported th a t bio tin ,
to n ic o tin ic acid,

in addition

and thiamin was necessary f o r growth.

The

findings of Moller, Wey, and Wacker (21) demonstrated th a t
about one-fourth of the staphylococci studied needed pantothenic
acid and about one-half required puriiies or u ra c il.

These l a s t

two groups of workers did not mention the e ffe c t of these
growth factors on pigmentation.

EXPERIMENTAL METHODS
The organisms which were selected Tor th is study were
three s tr a in s of Staphylococcus aureus CMicrococcus pyogenes
var.

aureus).

Two of the cultures No. 2 and 5 were iso la ted

from the th ro ats of students in the Department of Bacteriology
and Public h ealth a t Michigan State College.

The th ird

culture which w ill be designated 6 is 6538 from the American
Type Culture Collection.

Cultures 2 and 5 were selected

because of th e ir marked a b ility to form pigment on most so lid
media which supported th e ir growth.

Preliminary work was done

with culture 5 as of the three s tra in s used, th is

organism

was the best pigment producer.
The f i r s t attempts to study pigment formation in these
organisms were made on solid media.

Four media were compared,

namely (1) proteose peptone No. 3 agar plus 10 percent
evaporated milk,

(2) proteose lactose agar,

and (4) Chapman's No. 110 agar.

(3) dextrose agar

An attempt was made to measure

pigment production by visual observation at the end of a 10 day
incubation period.

However the amount of pigment could not

be measured by observing the growth on the agar p lates because
the degree of pigmentation in Isolated colonies was markedly
d iffe re n t from th a t in heavy confluent growth.
A method of measuring the amount of lig h t refle c te d
off the pigmented c e lls was tr ie d but l i t t l e

success resulted

11

because the differences of pigmentation on various media were
not s u f f ic ie n t fo r d iffe re n tia tio n .

Even when c e lls were

removed from the growing surface and placed on various back­
grounds, no marked improvement in d iffe re n tia tio n resu lted .
The method of Stahly e t a l (33), fo r measuring the
amount of pigment produced by c e lls grown on a solid medium
with a photoelectric colorimeter, met with more success than
the ones previously tr i e d .

The following formula was used

to determine the amount of

pigment:

Anount of pigment .
However i t was f e l t th a t a

more accurate method could

be

devised i f the pigment was

extracted from the c e lls with an

organic solvent.
The following method was devised:

200 ml portions of

the t e s t medium, prepared with double d i s t i l l e d water, were
placed in 500 ml Erlenmeyer fla s k s ,

the pH was adjusted to

7.00 and the medium was autoclaved f o r 15 minutes at 15 pounds
(121 C).

Three ml aliquots amounts of the medium were seeded

from a 24 hour n u trie n t agar s la n t.

After 12 hours incubation

at 30 C, transp lan ts were made to a second set of aliquots
which were also incubated 12 hours.

Four drops of these

12 hour cultures were added to the 200 ml portions of the
t e s t medium.

These flasks were shaken during incubation fo r

24 hours on a B urrell shaker at 30 C to promote maximum growth
by bringing oxygen and n u trien ts to the growing c e lls .

12

Following the shaking period the cells were separated
from the supernatant flu id by centrifugation and washed twice
with 0.85 percent saline.

Next 10 ml of 0.85 percent saline

plus glass beads were added to the c e ll mass and the suspension
was shaken fo r one-half hour in order to break up existing
clumps.

Further to insure a homogeneous suspension, 40 ml

of 0.85 percent saline was adaed to the c e ll suspension and
the resu ltin g suspension was passed through six layers of
cheese cloth*
The number of organisms was determined photometrically.
Population curves fo r the three te s t organisms were Trade by
plotting microscopic counts against lig h t transmission using
a Cenco Sheard Photometer S erial No* 2003 equipped with a blue
Vfratten F i l t e r No. 49.

(Figure 1)

This f i l t e r has a maximum

adsorption between 4400-4600 Angstrom Units.

As shown in

Figure 2 the maximum absorption of the methanol extracted
pigment was found to be at 4500 Angstrom Units; therefore,
little

erro r was introduced when a 1-10 d ilu tio n of the

suspension was adjusted to give a reading between 30 and 45
percent lig h t transmission.
Following the determination of the c e ll count, 35 ml
of the c e ll suspension was centrifuged, the clear supernatant
flu id was decanted and 15 ml of methanol was added to extract
the pigment from the c e lls .

To insure a complete extraction

the alcoholic c e ll mass was kept at 7 C fo r 24 hours.

The

Jfl

FIGURE

I

S. A U R E U S - 2 * .80- -

0.50--

20

40
PER CENT

60
T R A N S M IT T A N C E

80

13

extracted pigment was then separated from the c e lls by
centrifugation,

and passed through a Swinney f i l t e r i f the

extracted pigment solution was not absolutely clear*

The

amount of pigment was determined with a Beckman Spectophotometer Model B, S erial No* 20302, by p lottin g the
percent lig h t transmission against the dry weight of
pigment per ml of extracted pigment.

(Figure 3)

Readings

obtained from the Beckman were made at the point of maximum
adsorption which proved throughout the study to be 4500
Angstrom Units.
The amount of pigment per c e ll was calculated in the
following manner:

.
.
.
-it - Wt. of pigment in 15 ml methanol
Amount of pigment per c e ll - ----------No. of c e lls extracted

F IG U R E

l\)

350

450

550
WAVE

650
LENGTH

750
MILLIMICRONS

850

950

FIGURE

3

300- -

250- -

200 -

-

CD

UJ

100 -

-

so
40
P E R GENT

60
70
TRAN SM ITTA NCE

R3SULTS
Attempts to promote pigmentation in S. aureus have
resulted in the formulating of a wide variety of media.
recent years such media as dextrose agar,

In

proteose peptone

No. 3 agar plus 10 percent evaporated milk,

proteose lactose

agar, and Chapmen's No. 110 agar have been most frequently
used fo r studying pigmentation in staphylococci.

As workers

in th is f ie l d have not agreed th at any one of these media
are superior to the others in causing staphylococci to pigment,
i t was f e l t th at a comparison of these media would be p ro fitab le.
Also such preliminary work would offer some leads fo r fu rth er
investigation as to the specific factors and medium components
which are instrumental in causing pigmentation.
The constituents of the above media were as follows:
Dextrose Agar
-------------------------1000
Water
Beef e x t r a c t ---------------------------------- 3.0
T ry p to se--------------------------------------------10.0
D extrose--------------------------------------------10.0
Sodium c h lo r id e --------------------------5.0
A gar
15.0

ml
gm
gm
gm
gm
gm

Proteose Lactose Agar
Water —------------------------------------------- 1000
Proteose pepto ne
20.0
D extrose
0.5
L a c to s e ----------------------------------------------10.0
Disodium acid phosphate----------5.0
Sodium c h lo r id e --------------------------5.0
A gar
15.0

ml
gm
gm
gm
gm
gm
gm

15

Proteose Peptone No. 3 Agar
W ater------------------------------------------------------- 1000 ml
Proteose peptone No. 3 --------------------20.0 gm
D extrose-------------------------------------------------0.5gm
Sodium b h lo r id e --------------------------------5.0 gm
Disodium acid phosphate
5.0 gm
A g ar----------------------------------------------------------15.0 gm
Chapman's No. 110 Agar
W ater------------------------------------------------------- 1000 ml
Yeast e x t r a c t -------------------------------------- 2.5 gm
Tryptone------------------------------------------------10.0 gm
M annite
----------------------------------------------10.0 gm
L a c to s e -------------------------------------------------- 2.0 gm
Dipotassium acid phosphate
5.0 gm
Sodium c h lo r id e
----------------------------75.0 gm
G e la tin ---------------------------------------------------- 30.0 gm
A g ar--------------------------------------------------------15.0 gm
P etri plates were prepared of the above media and
quadruplicate seedings were made using washed ce lls of S. aureus
No. 5 taken from a n utrien t agar sla n t.

Two plates of each

medium were incubated at 20-22 C and the other two plates were
incubated at 37 C.

One plate at each incubating temperature

was sealed with parafilm tape in order to re ta in the moisture
of condensation so th a t the moist condition would be maintained
throughout the 10 aay growing period.

The other plate of

each medium was l e f t unsealed and as a re s u lt the agar and
cells lo s t considerable moisture a f te r 10 days incubation.
A comparison of the pigmentation produced under these conditions
was made.
The re s u lts of these te s ts are shown in Table 1.

The

data show th at none of the media which was kept in a moist

16

condition contained c e lls th at were pigmented.

Therefore i t

could be argued th a t dry conditions are necessary fo r pigmen­
ta tio n ; however, i t should be pointed out th a t,
parafilm tape i s porous m aterial,

although the

i t may have some influence

upon oxygen tension and thus affect pigment production.

It

is a well known fa c t th a t an adequate oxygen supply is
necessary fox* pigmentation.
As f a r as incubation temperatures were concerned, the
data show th a t,

a t the end of ten days,

there was very l i t t l e

difference in the amount of pigment produced.

A f a i r amount

of pigment was produced on dextrose agar but pigmentation on
proteose peptone No. 3 agar and proteose lacto se agar was
rath er inconsistent in th a t the former medium gave b e tte r
pigmentation a t 37 C and the l a t t e r at 20 C.

Chapman's

No. 110 agar medium gave the most consistent re s u lts in th a t
good pigmentation was obtained at both incubation temperatures.
The r e s u lts at the end of fiv e days were much the same as
those shown in Table 1 in th at the g reatest amount of pigment
and most consistently pigmented c e lls were obtained on
Chapman's No. 110 medium.

In the lig h t of the above re s u lts

Chapman's No. 110 medium merited fu rth e r Investigation.

Also

several of the chemical complexes in the medium could be
replaced by known chemical compounds, thus offering an
opportunity to detect the compounds instrumental in inducing
pigmentation.

17

In some recent work by Chapman (6),

i t was reported

th a t with medium No. 110, pathogenic staphylococci showed
greater pigmentation at 30 C than at 20 or 37 C.

An attempt

was made to confirm these findings using culture 5.

Thirty

p lates were seeded with one drop each of a saline suspension
from a 24 hour n u trie n t agar sla n t and 10 of these plates
were incubated at each temperature f o r 10 days.

The re su lts

were ra th e r inconsistent in th a t plates at each incubating
temperature showed greater pigmentation than other plates at
the two other incubating temperatures.

However one f a c t was

apparent, more plates showed good pigmentation at 30 C than
at the two other incubating temperatures thus confirming
Chapman's findings.
As these inconsistencies in pigmentation occurred to
some extent on a l l media and among colonies on the same p e tr i
dish,

an attempt was made to grow the c e lls in a liq u id medium.

One hundred ml portions of the modified liq u id medium of
Chapman were placed in 250 ml Erlenmeyer fla sk s.

These flasks

were seeded with two drops of a salin e suspension from a
24 hour n u trie n t agar sla n t.
each temperature,

Two flask s were incubated at

namely, 20, 30, and 37 C fo r a 10 day period.

Following incubation the c e lls were separated from the
supernatant f lu id by centrifugation.
in Table 2.

The re s u lts are presented

When visual observations of the c e ll masses were

18

made, i t

can be seen th a t the re s u lts at 30 and 37 C were much

the same in th a t a good pigmentation was obtained at both
temperatures.

However when the c e lls were extracted with

methanol according to the method or Stahly e t al (33), the
c e lls which were grown a t 30 C showed greater pigmentation
than those grown at 37 C.

As a re s u lt of these te s ts an

incubation temperature of 30 C was used in subsequent studies.
In order to accelerate growth by maintaining an adequate
supply of oxygen and n u trie n ts to the c e lls ,

the incubating

flask s in subsequent te s ts were shaken fo r 24 hours instead
of leaving them standing s t i l l fo r a 10 day growing period
as was done previously.

Approximately the same amount of

pigment was produced using these two periods of growth.
The Effect of Various Constituents of Chapman's No. 110 Liquid
Medium
The next step in the inv estig atio n of the facto rs which
are instrum ental in causing pigmentation was to study the
e ffe c t on pigmentation when one of the constituents was with­
held from Chapman's No. 110 liq u id medium.

For instance one

medium contained a l l the constituents with the exception of
mannite,

another a l l the components with the exception of

sodium chloride etc.

The amount of pigment produced was

determined by the method described in Experimental Methods.

19

The r e s u lts or these t e s t s are reported in Table 3.
In th is tab le the number

or

organisms per ml of medium, the

number of organisms extracted,
c e ll are given.

and the weight of pigment per

There are several in te re stin g observations

which can be noted from these data.

F ir s t th a t growth did

not occur when g e la tin was l e f t out of the medium despite
the presence of tryptone to serve as a source of nitrogen,
mannite and lacto se to serve as a source of carbohydrate,
and yeast ex tra c t to serve as a source of E complex vitamins.
Perhaps g e la tin could have served as a neu tralizin g agent to
some toxic end-product, however,
stra te d by subsequent data.

th is is unlikely as demon­

The most reasonable in te rp re ta tio n

is th a t one or several of the e sse n tia l amino acids were
(1) absent,

(2) present but in a very lim ited quantity,

or

(3) destroyed in the preparation of the tryptone with the
re s u lt th a t g e la tin served as the sole or main source of th is
e sse n tia l metabolite.

Therefore in the absence of g ela tin ,

the metabolite was not present in s u ffic ie n t amount to induce
growth.
Another fa c t to note is th at the number of c e lls per
Q
ml of medium varied from 1.1 to 2.6 x 10 while pigment
production varied over a much wider range,
0.95 to 5.37 x 10”10 mgm.

i t being from

In four out of the fiv e media the

amount of pigment did not show a d ire c t co rrelatio n with the

20

number of c e lls produced when one or the following were
lacking:

mannite, lacto se,

sodium chloride or tryptone.
Q
When mannite was lacking, 1.8 x 10 organisms per ml of
medium and 3*86 x 10”^° mgm of pigment was found while the
g

lack of tryptone resu lte d in 2.2 x 10 organisms and 1.79
-10
9
x 10
mgm of pigment.
The lowest b a c te ria l count, 1.1 x 10
organisms per ml of medium and the poorest pigment production,
0.95 x 10'*10 mgm, were found when yeast extract was absent
from the medium.

The complete medium showed a pigment

production of 4.70 x 10“^° mgm.
Some of the subsequent data show th a t a d ire c t co rrelatio n
occurs between the number of organisms and the amount of pigment.
The reason th a t such a co rrelatio n is not found in the data
given above is th a t such components as yeast ex tract,

g elatin ,

or tryptone have s u ffic ie n t amount of the supposedly lacking
constituent to contaminate the medium.

As a r e s u lt the

organisms have a l l the e sse n tia ls fo r growth and the removal
of one of the constituents simply a lte r s the amount which is
available.

I t is obvious th a t such a method gives only a

rough in dicatio n of the role of the individual constituents
to pigmentation.
these data,

However one fa c t does seem to stand out in

i t being th a t when yeast extract was lacking

from the medium, growth was poor and the amount of pigment
produced was considerably less than when the other components

21

were withheld.

As the most important components of the yeast

extract are the B complex vitamins,

the next lo g ical step was

to s u b s titu te such a complex fo r the yeast extract and to
observe the e ffe c t upon growth and pigmentation.
The Effect of the B Comply Vitamins Upon Pigmentation
The same medium as in the previous t e s t was used with
the exception th a t the B complex supplement v/as substituted
fo r the yeast e x tra c t.

The supplement contained the following

vitam ins:
(amount per ml of vitamin solution)
Thiamin-------------------------------------------------- 100 *
P y rid o x in e-------------------------------------------- 200 *
Calcium p e n to th e n a te ----------------------- 100*
R ib o fla v in --------------------------------------------100 *
N ia c in ----------------------------------------------------100 *
Para amino benzoica c i d --------------------100 *
I n o s i t o l ------------------------------------------------100 *
B i o t i n ----------------------------------------------------100 *
The vitamin supplement was added in the amount of 0.25 ml per
100 ml of medium, the concentration of each vitamin in the
finished medium being 0.25 per ml of medium with the exception
of pyridoxine.
the others.

This vitamin was added in twice the amount of

Also each of the vitamins was made up in separate

solution in the same concentration except fo r fciotln.

This

solution contained 0.5m*per ml as the b io tin requirement of
the t e s t organisms was considerably le s s than th at of any of
the other B complex vitamins.

22

The determination of the e ffe c t of these vitamins as a
complex and as separate u n its was made in the following manner:
the medium which contained the e n tire E complex was used as a
control.

Then a run was made in which one vitamin was l e f t out

of each medium and the number of c e lls and the amount of
pigmentation were determined.
I t is apparent th a t the data (Table 4) are much more
consistent in these te s ts than those shown in the previous
ta b le.

This would be expected as in th is case only one fa c to r,

the vitamin requirement, was being considered.

The amount of

pigment pi-oduced ranged from 0.48 to 4.31 x 10“^ mgm and the
q
number of organisms varied from 1.9 to 5.3 x 10 per ml of
medium.

The in te re s tin g observation under these circumstances

was th a t in the two cases where the amount of pigment was
small,
low.

the number of organisms produced was correspondingly
When the B complex was l e f t out of the medium, 0.48 x

1C“10 mgm of pigment was produced with a b a c te ria l population
9
of 1.9 x 10 organisms.
The absence of thiamin In the medium
resulted in 0.51 x 10”10 mgm of pigment and a b a c te ria l count
9
of 2.7 x 10 organisms.
When the remaining vitamins were
withheld from the medium, the following re s u lts were obtained:
pyridoxine lacking - 4.14 x 10~10 mgm of pigment with
5.1 x 109 organisms
rib o fla v in lacking - 4.06 x 10”10 mgm of pigment with
5.2 x 10 organisms

23

b io tin lacking - 4.31 x 10“^° mgm o f pigment with
5.5 x 109 organisms
n iac in lacking - 3.71 x 10"^° mgm with 4.3 x 109
organisms
para amino benzoic acid lacking - 3.77 x 10“^°
mgm or pigment with 4.4 x 109 organisms
calcium pentothenate lacking - 4.08 x 10“^° mgm
with 5.0 x 109 organisms
in o s ito l lacking - 3.68 x 10“^-° mgm or pigment
with 3.8 x 109 organisms.
The complete medium contained 3.81 x 10"10 mgm or pigment and
a b a c te ria l count o f 4.9 x 109 organisms.
A comparison or the re s u lts ,

when the B complex or

thiamin was lacking with the remaining data in the tab le,
shows th a t thiamin was e sse n tia l ro r maximum pigment production
and r o r good growth.

The data indicate th a t under these
Q
conditions the b a c te ria l growth must be over 3.0 x lCr7

organisms per ml or medium in order to a tta in good pigmentation.
The Tact th a t the amount or pigment produced by the complete
medium was le s s than when pyridoxine,

rlbo riav in , b io tin ,

and calcium pentothenate were leT t out or the medium is not
s ig n iric a n t as the dirrerences are well within the lim its or
error or the te stin g method.
B complex were su b stitu ted f o r

When the various vitamins or the
the yeast extract in Chapman's

modiried liq u id medium, good pigmentation did not occur unless
thiamin was included in the medium.

24

However as previously mentioned,

there were other

components in the medium such as g e la tin and tryptone which
may have appreciable amounts or B complex, and so a medium
of a more chemically defined nature was investigated*

This

was especially tru e in the lig h t of the work of Kliger,
Grossowicz,

and Bergner (18) who reported th a t both thiamin

and niacin were required f o r the f u l l growth of staphylococci.
T h e R o le o f t h e B C o m p le x V i t a m in s

in

P i g m e n t a t i o n U s in g a ^ S e m lr

The medium which was made fo r fu rth e r study was of a
semisynthetic nature and contained the following components:
W ater------------------------------------------- 1000
Vitamin-free casaminoacids5.0
------------------------------10.0
Tryptophane
C y s tin e
10.0
L a c to s e -------------------2.0
M annite
2.0
Dipotassium acid phosphate 5.0
Sodium c h l o r i d e
10.0
Magnesium su lfa te 7 HgO
20.0
Ferrous s u lfa te 7 HgO--------------10.0
Manganese s u lfa te
10.0

ml
gm
mgm
mgm
gm
gm
gm
mgm
mgm
mgm
mgm

The B complex vitamins were added to the above medium in the
same concentration (0.25 ml per 100 ml of medium) as used in
the previous modification of Chapman's No. 110 medium.

The

complete vitamin supplement was added except fo r the
p a rtic u la r vitamin under t e s t .

The t e s t medium was dispensed

in 200 ml amounts into 500 ml Erlenmeyer fla s k s ,

otherwise

the t e s t procedure was the same as th at used before.

25

The t e s t r e s u lts are presented in Table 5.

The most

s ig n ific a n t fa c t demonstrated was th a t the t e s t organism,
S. aureus 5, would not grow in a medium which did not contain
thiamin or n iacin and th a t growth was poor in the absence of
b io tin .

The data show th a t there was d e fin ite ly a niacin and

possibly a b io tin contamination from the g e la tin or tryptone
in the medium used in the previous t e s t .
thus,

confirm the work of Kliger e t a l.

These findings,
as f a r as the thiamin

and niacin requirements of staphylococci are concerned.
respect to b io tin ,

With

i t has been reported by several workers

th at various cultures of staphylococci d if f e r in th e ir need
fo r th is vitamin.

The fa c t th a t growth did occur demonstrates

one of three things,

namely;

Cl) the organisms can grow to a

very lim ited extent and thus produce a very small amount of
pigment in the absence of b io tin ,

(2) the organism can

synthesize a very lim ited amount of b.iotin which is required
fo r meager growth,

or (3) there is enough contaminating b io tin

in the so-called vitamin fre e casamino acid to su stain lim ited
growth.
The f i r s t p o s s ib ility is quite probable as the amount
of b io tin required fo r growth is very small as compared to
th a t of the other vitamins.

The second theory does not seem

to be sound fo r the following reason:
able to synthesize b io tin at a l l ,

i f the organisms were

they would be able to

26

synthesize a su ffic ie n t amount fo r good growth-

The th ird

hypothesis could also he a lo g ical explanation although
microbiological te s ts with the casamino acids have shown i t
to be b io tin fre e to the extent of 0.025/n/The f a c t th a t when b io tin was not added to the medium
poor growth and no pigmentation resulted,

and when i t was

incorporated into the medium good growth and good pigmentation
resulted, demonstrates th a t biotin is an essen tial vitamin.
This is tru e fo r the te s t organism S.. aureus 2 and 6, as well
as fo r 5.
The remaining vitamins, namely; pyridoxine, riboflavin,
para amino benzoic acid,

calcium pentothenate,

and in o sito l,

were e ith e r synthesized by the organism or were not required
fo r pigmentation as the amount of pigment varied from 3.40 to
4.20 x 10“^° mgm when any of the above vitamins were l e f t out
of the medium.

However i t is in terestin g to note th a t the low

figure of 3.40 x 10”^° mgm was paralleled by the comparatively
low b a c te ria l count of 2.7 x 109 organisms per ml of medium
and occurred when the medium lacked riboflavin.

The greatest

production of pigment, 4.20 x 10“10 mgm, was obtained when
the b a c te ria l count was comparatively high,
in o s ito l was withheld from the medium.
pyridoxine 3.55 x 10“^

and occurred when

In the absence of

mgm of pigment were found in the
Q
presence of a b a c te ria l population of 3.5 x 10 organisms.

27

When para amino benzoic acid or calcium pentothenate was
lacking,

the pigment production was approximately the same

(3.54 and 3.42 x 10

mgm resp ectiv ely ) with the same

b a c te ria l population (4 x 109 per ml of medium).

These data

show once again th a t the amount of pigment produced by the
organisms was closely a llie d with the b a c te ria l count and
th is relatio n sh ip was apparent even when the differences in
pigmentation were not g reat.
Although the amount of pigment which was produced when
each of the vitamins was withheld from the medium, was not
determined with cultures 2 and 6, px*ellminary te s ts showed
th a t thiamin and niacin were also required by these organisms.
In order to see i f the close co rrelatio n between the
amount of pigment and the number of organisms continued to
e x ist when the concentration of thiamin and niacin was
decreased,

a se rie s of t e s t s using 25.0, 12.5,

per 100 ml was tr ie d .

5.0,

and 2.5^

The three t e s t organisms were used.

The data fo r thiamin are presented in Table 6.

'With culture

2 the amount of pigment dropped from 2.32 to 2.20 x 10”^°
mgm and b a c te ria l counts from 1.66 to 1.20 x 10^ organisms
when the concentration of thiamin was decreased one h a lf.
With o n e -fifth the orig in al concentration of thiamin, 1.52
x 10”10 mgm of pigment and 520 x 106 organisms were found,
while one-tenth the o rig in al concentration of th is vitamin
resulted in 0.77 x 10“^° mgm of pigment and 350 x 106 organisms.

A

28

Culture 5 showed a greater drop in the amount of pigment
produced than did culture 2 over the same thiamin range.

With

th is culture the weight of pigment decreased from 4.42 to 4.32
x 10”10 mgm with the number of organisms dropping from 3.5 to
Q

3.3 x 10

with half the normal amount of thiamin present.

When

the concentration of thiamin was reduced to o n e-fifth the
original amount, 2.40 x 10”^° mgm of pigment was present with
a b a c te ria l population of 2.61 x 109 organisms.
d ilu tio n of thiamin,

one-tenth of the o rig in al,

The highest
gave 2.03 x 10“^c
Q

mgm of pigment with a b a cte rial count of 2.O x 10

organisms.

The decrease in the amount of pigment in culture 6 was
as follows:

the o rigin al concentration of thiamin gave 3.62

-10
9
x 10
mgm of pigment with 2.9 x 10 organisms,

one half of

the orig in al concentration gave 2.87 x 10“10 mgm of pigment
9
with 2.0 x 10 organisms, o ne-fifth of the original concentration
gave 0.78 x 10“^

mgm of pigment and 680 x 10^ organisms and

one-tenth of the original concentration gave 0.88 x 10~^° mgm
0
of pigment with 600 x 10 organisms.
The amount of pigment produced followed very closely
the b a c te ria l population except where 5.0 or 2.5* of thiamin was
used with culture 6.

Here slig h tly more pigment was produced

with the lower concentration of thiamin than with the higher,
although the b a c te ria l counts of the former were slig h tly
greater than those of the l a t t e r .

This difference i s best

29

explained by the Tact th a t there is some erro r when a small
amount of pigment was produced due to the inaccuracy of the
spectophotometer readings above 90.0 percent lig h t transmission.
In th is case the readings were 91.0 and 92.0 percent respectively
A much
be a d e fin ite

more s ig n ific a n t fa c t i s th a t there appears to
drop in the amount of pigment produced in a l l

three t e s t organisms when the thiamin concentration dropped,
below 12.5* .

In other words the amount of thiamin in the

t e s t medium could be halved without seriously affecting the
amount of pigment produced but below th is point pigment
production dropped sharply.

Again the weight of pigment

produced followed very closely the number of organisms,

the

b a c te ria l counts f e l l off considerably when 12.5/ was used.
This was also

well exemplified with culture 6 when 2 5.0^

were compared

with 1 2 .5 / •

Here the s lig h t drop in pigmentation

from 3.65 to 2.85 x 10“^° mgm was accompanied by a correspond­
ingly s lig h t drop in b a c te ria l count, 2.8 to 1.9 x 109
organisms.
The decrease in pigment production when the amount of
niacin was decreased from 25.0 to 2 .5 / per 100 ml of medium
is demonstrated in Table 7.

Again the concentrations tested

were the same as in the previous t e s t s ,
5 0, and 2 .5 / per 100 ml.

namely 25.0, 12.5,

30

Upon examination of the data using culture 2, i t was
demonstrated th a t pigment production dropped from 3.30 x 10“10
mgm with 25.0* of niacin to 3.11 x 10”^

mgm with 12.5* of

n iacin and f in a lly to 1.28 x 10“^° mgm of pigment with 5.0*
of n iacin .

No growth occurred when 2.5* of niacin was employed.
9
B acterial counts dropped from 2.31 to 2.20 x 10 organisms
when the concentration of niacin was o n e-fifth of the o rig in al.
When the concentration of niacin was reduced one-half,
using culture 5, the weight of pigment decreased from 3.32 to
3.28 x 10~10 mgm while the b a c te ria l population increased from
9
3.4 to 3.7 x 10 organisms.
Further reductions in the amount
of niacin in the medium resulted in the following:

o n e-fifth

of the o rig in al concentration gave 1.70 x 10"10 mgm of pigment
9

with 2.8 x 10

organisms, and one-tenth of the o rigin al
-10
9
concentration gave 0.90 x 10
mgm of pigment with 1.9 x 10

organisms.
Culture 6 showed a ranee of pigment production from
4.32 x 10”^

mgm with a niacin concentration of 25.0/ to

0.98 x 10~10 mgm with a niacin concentration of 2.5* •

The

intermediate concentrations of 12.5 and 5 .0 / of niacin showed
4.07 and 1.32 x 10~^° mgm of pigment respectively.

B acterial

counts ranged from 4.8 x 109 with the g reate st concentration
of niacin to 900 x 106 organisms with the lowest coneentx-ation
of niacin.

31

The re s u lts were comparable to those obtained with
thiamin in th a t reducing the concentration of niacin by oneh alf did not seem to decrease e ith e r the amount of pigment
formed or the number of organisms to any marked degree.
However when the concentration of niacin was reduced to
o n e -fifth or one-tenth of the o rig in a l concentration, the
response of the organisms was considerably d iffe re n t fo r
n iacin from what i t was f o r thiamin.

Pigment production

dropped below 1.0 x 10“^° mgm fo r a l l three organisms when
2 .5 y of thiamin was in the medium, while with niacin the
re s u lts were irre g u la r in th a t no growth occurred with culture
2, with cu ltu re 5, 1.33 x lO”'1
' 0 mgm of pigment was obtained
and 0.98 x 10“^° mgm of pigment was demonstrated with culture
6.

Such data would indicate th a t the c r i t i c a l concentration

of thiamin i s more constant among the species of staphylococci
than the c r i t i c a l concentration of niacin.
The p o s s ib ility of niacin being a precursor of the
carotenoid molecule is d i f f ic u lt to v isu alize although the
5 -carbon ring which makes up the n iacin molecule might serve
as a precursor* fo r the ionone ring of the carotenoid molecule.
The most probable role of niacin is th a t which i t

plays a

part with tryptophane in the formation of coenzyme I and
coenzyme I I .

These coenzymes are among the most Important

"hydrogen carriers'* within the b a c te ria l c e ll and are required

g

32

ro r many aerobic transformations.

Therefore niacin in th is

ro le is but in d ire c tly related to pigmentation by serving as
a precursor fo r enzymes which are necessary f o r the well­
being of the b a c te ria l c e ll.
R e la tio n s h ip

o f C o c a r b o x y l a s e t o T h ia m in i n
S ta p h y lo c o c c i.

Through phosphorylation thiamin is
coenzyme cocarboxylase.

P ig m e n ta tio n o f

converted to the

This coenzyme functions In the

metabolism of many organisms by permitting decarboxylation
of c e rta in ketoacids (e.g.

pyruvic acid).

Snell (32),

in

discussing the chemical aspects of b a c te ria l n u tritio n ,
reported th a t Streptococcus

ivarins

and Lactobacillus

ferment! grow when e ith e r thiamin or cocarboxylase is supplied
utiile S.

aureus grows i f e ith e r thiamin or i t s two moieties,

thiazole and pyrimidine, are present.

However nothing was

sta te d about the role of cocarboxylase In staphylococci.
With the above work In mind i t

was thought wise to see

i f cocarboxylase could be su b stitu ted fo r thiamin,

and i f i t

could, what e ffe c t i t would have on the pigmentation of the
three t e s t organisms.

The t e s t medium was the same as th at

used fo r the thiamin t i t r a t i o n with the exception th a t 2 5 .0 /
per 100 ml of medium of cocarboxylase was sub stituted fo r
thiamin.
The data which are presented in Table 8 show th a t there
was very l i t t l e

difference in the re s u lts when e ith e r thiamin

33

oi* cocarboxylase was present in the medium.

With culture 2,

2.33 x lCf10 mgm of pigment was produced with thiamin and
2. b8 x 10”10 mgm with cocarboxylase.

Culture 5 gave 3.88 x

10-10 mgm of pigment with both thiamin and cocarboxylase while
the re s u lts with culture 6 showed 4.27 x 10”10 mgm of pigment
with thiamin and 4.68 x 1CT10 mgm of pigment with cocarboxylase.
Bacterial populations were very sim ilar under the two t e s t
conditions.
Such data would indicate th a t one of the main functions
of thiamin is to serve as a precursor fo r cocarboxylase.

Some

work has also indicated th at thiamin is incorporated into the
carotenoid molecule during i t s formation.

From these two

fa c ts several theories of pigment formation can be postulated:
(1) thiamin may serve as a precursor fo r cocarboxylase and
enter into the formation of the carotenoid molecule at the
same time,

(2) thiamin may function during the logarithmic

stage of growth as a precursor fo r cocarboxylase and then when
th is requirement is s a tis fie d ,

thiamin enters into the formation

of the carotenoid or (3) both thiamin and cocarboxylase can
be incorporated into the pigment molecule.
Effect of Salts on the Pigmentation of Staphylococci
Reports have occurred in the lite r a tu r e which sta te that
various s a lts besides the phosphate buffers are required by
staphylococci when i t is grown in a semisynthetic medium.

34

The s a lts generally required Tor growth are sodium chloride,
ferrous s u lf a te ,

manganese s u lfa te ,

The e ffe c t of these s a lts

and magnesium s u lfa te .

on pigmentation in staphylococci

has never been reported although Turian (35) sta te d th a t
traces of iron and manganese are necessax-y fo r pigmentation
in Mycobacterium o h le i.

The s a lt s were addea in the following

concentrations per 100 ml of medium:
Sodium chloride
---------------------------- 1.0
Ferrous s u lfa te 7Hg0--------------------1.0
Manganese su lfa te if*#--------------------1.0
Magnesium s u lfa te 7HgO------------------2.0

mgm.
mgm.
mgm.
mgm.

The basal medium was the same as th a t used to determine the
vitamin requirements with the exception of the s a lts under
te s t.

Test procedure was the same as th a t used previously.
The weight of pigment produced by the three cultures

when the various s a lts were added individually to the medium
are presented in Table 9.

In the f i r s t

are the data fo r culture 2.

column of the tab le

When the four s a lts were added,

thus giving a complete medium, 3.93 x 10" ^
was produced but i f

mgm of pigment

the four s a lts were absent from the

medium, a sharp drop in pigment production re su lte d , the amount
being 1.29 x 10“10 mgm.

The addition of sodium chloride,

ferrous s u lf a te or magnesium su lfa te gave the following amount
of pigment:

1.26, 1.44, and 1.12 x 10~10 mgm respectively .

I t is apparent th a t with these s a lts in the medium, there is
no increase in pigmentation.

However upon the addition of

35

manganese s u lf a te ,
mgm.

pigment production increased to 2.32 x 10~^°
Q
The number of organisms varied from 1.5 x 10 when none

of the s a l t s was included to 2.2 x 109 organisms when sodium
chloride, ferro u s s u lfa te or manganese s u lf a te was added.
9
The complete medium showed a b a c te r ia l count of 1.8 x 10
organisms.
The r e s u lts

obtained with culture 5 are seen in the

second column of the ta b le .

Once again when the four s a l t s

were included in the medium, the maximum pigmentation was
obtained and when the four s a l t s were l e f t

out of th e medium

there was a decided decrease in the amount of pigment produced.
V/lth th is c u ltu re the former conditions gave 4.06 x 10"^° mgm.
of pigment and the l a t t e r conditions 1.52. x 10“^
pigment.

mgm of

When the four s a l t s were added separately to the

medium, the following amounts of pigment were obtained:

with

sodium chloride - 2.42 x 10”10 mgm, with ferrous s u lf a te 2.48 x 10”^° mgm, with magnesium s u lfa te - 2.26 x 10~^-° mgm,
and with manganese s u lfa te - 3.93 x 10 ^

mgm.

th a t with th i s c u ltu re a composite of the s a l t s

The data show
increased

pigment production considerably as compared to the medium
lacking these s a l t s .
a time,

Again when the s a l t s were added one at

the g re a te s t increase in amount of pigment produced

occurred when manganese s u lfa te was incorporated in the medium.

36

In a l l cases with cu ltu re 5 the b a c te r ia l populations were
g
s lig h tly over 4.0 x 10 organisms per ml or medium.
The th ir d column or the ta b le shows data obtained with
culture 6.

The in clu sio n or the lo u r s a l t s

a pigment production or 4.16 x 10”^

mgm.

in the medium gave
The medium without

the rour s a l t s showed a pigment production or 2.86 x 10”^

mgm.

For the th ir d time the data demonstrate th a t a g re a te r amount
or pigment was produced when the s a l t s were included in the
medium than when they were withheld.

However with t h i s

s tr a in

the add ition or the separate s a lt s did not r e s u lt in increased
pigmentation.

The addition or sodium chloride or rerrous

su ira te gave 2.27 x 10”10 mgm or pigment,

the add ition or

magnesium s u ir a te gave 2.45 x 10“^° mgm or pigment while the
addition or manganese s u ir a te gave 2.58 x 10-10 mgm or pigment.
The number or organisms per ml or medium with th is c u ltu re
9
was approximately 2.0 x 10 under a l l t e s t conditions.
From the above data i t

i s evident th a t the three organisms

varied in t h e i r pigment response to the rour s a l t s .

however the

one common r a c to r which they possess i s th a t a l l cu ltu res
responded with a g reate r output or pigment when a l l rour s a l t s
were present in the medium.

I t would be expected th a t the

b a c te r ia l population would be considerably g re a te r when pigment
production was higher;
were in the medium.

in other words,

when the rour s a lt s

This did not occur with c u ltu res 2 and 5.

37

In each case the b a c te ria l counts of the media with and
without the s a lts were approximately the same (culture 2 9
9
1.8 and 1.5 x 10 organisms, culture 5 - 4 . 6 and 4.0 x 10
organisms ).

The expected p attern is followed with culture 6

as there were approximately twice as
s a lts were included in

many organisms when the

the medium as when they were l e f t out.

From these data i t appeared as though one of the s a lts ,
magnesium s u lfa te ,

had

produced in cultures 2

namely

an e ffe c t upon the amount of pigment
and 5 but had very l i t t l e

the number of organisms produced.

e ffe c t upon

On the other hand, with

culture 6 the s a lts seemed to have an e ffe c t upon the number
of organisms as well as on the extent of pigmentation.
The data th a t show an increase in pigment production
upon the addition of manganese s u lfa te are very in te re stin g ,
especially in the lig h t of Turian's (35) findings with Mvco.
p h lei.

The ro le played by the manganese su lfate in the

metabolism,

and therefore in the pigmentation of the organism,

is not understood and c a lls fo r some speculation.
is not found in the carotenoid molecule,
of c a ta ly tic nature.

its

As manganese

probable role is

This has been found to be tru e in the

case of chlorophyll formation in plants and hemoglobin
formation in animals.

Manganese serves as an activ ato r fo r

the phosphotases, arginases, dipeptidases, end laccases and
therefore may be in demand in several instances in the formation
of the carotenoid pigments.

38

The ro le of the dipotassium phosphate in the growth
ana pigmentation of the three c u ltu res of staphylococci is
tre a te d separately due to i t s
e ffe c t of t h i s

many metabolic functions.

The

s a l t d ire c tly upon pigmentation has never been

reported; however, the need of the phosphate s a l t s fo r the
general well being of most organisms shows th a t at l e a s t
in d ire c tly i t

is

re la te d to pigment production.

One of th e

most important functions is the b uffer capacity.

Another

function is the p art played by phosphates in the sugar
metabolism of the c e l l s .

Five-tenths gm per 100 ml of

dipotassium acid phosphate was added to the t e s t medium.
This was s u f f ic ie n t to buffer the medium above pH 6.0 during
the 24 hour growing period.

The t e s t medium was the same

as th a t used in the previous t e s t with the exception of the
phosphate s a l t .
The contents of Table 10 compare the re s u lts obtained
with the th re e organisms when they were grown with and with­
out the phosphate s a l t .

As would be expected th e absence of

the phosphate re s u lte d in much poorer growth and much le s s
pigmentation than when th e s a l t was in the medium.

.Vith

cu ltu re 2 the d ifferen ces were not as great as with the other
two c u ltu re s ,
TO

x lO”-1
^

the weight of pigment f a ll in g from 2.33 to 1.72
^

mgm and the number of organisms dropping from 1.8 x 10

to 600 x 10^ per ml of medium.

With cu ltu re 5 the lo ss in

39

pigment production was from 3.88 to 1.90 x 10~10 mgm which is
no doubt the r e s u l t or the b a c te r ia l population decreasing
from 3.6 x 109 to 560 x 106 organisms per ml of medium.

The

g re a te s t lo s s in pigmentation i s seen with c u ltu re 6 where
the drop was from 4.97 to 1.36 x 10 ^

mgm.

With th is

organism

the number of b a c te r ia per ml of medium decreased from 2.95 x
109 to 700 x 10^ when the phosphate b u ffe r was l e f t

out of the

medium.
Also shown in the ta b le are the pH values of the
supernatant f l u i d

a f t e r cen trifu g in g .

The t e s t medium was

adjusted to a pH of 7.00 before seeding.

A fter growth the pH

of the t e s t medium containing the phosphate s a l t had dropped
approximately one u n it,

the pH values being 5.90 f o r cu ltu re 2,

6.30 fo r c u ltu re 5 and 6.18 f o r cu ltu re 6.
b u ffer was l e f t
u n its ,

.Vhen th e phosphate

out of the medium, the pH drop was over two

i t being 4.70 fo r c u ltu re No. 2,

4.75 f o r c u ltu re 5 and

4.97 fo r c u ltu re 6.
The lower pH which re s u lte d when the b u ffer was not
included in the medium was probably the p rin c ip a l reason fo r
the poor growth and low pigment production.
When the b a c te r ia l
g
populations were approximately 2.0 x 10 organisms f o r c u ltu re 2,
around 4 .0 x 109 organisms f o r cu ltu re 5 and 3.0 x 109 organisms
f o r c u ltu re 6,

pigmentation was good and when the number of

organisms f e l l

below these le v e ls ,

n o ticeab ly .

pigmentation decreased
j

40

The Role of Carbohydrates in Pigmentation
The sig n ifican ce of the carbohydrate content of a medium
in r e la tio n to the pigmentation of staphylococci has been
in v estig ated to some extent by Chapman (6).

He found th a t the

addition of evaporated milk and subsequently la c to s e ,
pigmentation markedly*

enhanced

In a medium which was reported in 1948,

Chapman (6) included mannite as well as la c to se ,
serving as a d if f e r e n tia tin g sugar.

the former

In the li g h t of Chapman's

experiences and as Sevag and Green (29) had reported in ­
conclusive r e s u lts as f a r as pigmentation was concerned when
carbohydrates or t h e i r interm ediate products were added to the
medium,

it

was f e l t th a t the e ffe c t upon pigmentation of the

most common laboratory sugars should be in v estig ated .
The sugars which were included in th is phase of the
study were la c to se ,

mannite,

when used sep arately ,
100 ml.
the

dextrose and sucrose.

Each sugar,

was added in the amount of 0.4 gm per

This is the t o t a l amount of sugar previously used in

media when 0.2 gm of la c to se and 0.2 gm of mannite were

added.

This was done to maintain a constant amount of sugar

in a l l media te s te d and thus to elim inate any possible chance
th a t g re a te r growth or pigmentation would be obtained in e ith e r
medium due to the one containing more carbohydrates than the
other.

i

41

In Table 11 the data are presented Tor th is experiment.
The pigment production varied from 2.79 to 4.09 x 10”"L0 mgm
while the b a c te r ia l populations were f a i r l y constant in th a t
Q
they ranged between 4.0 to 4.6 x 10 organisms per ml of medium.
The g re a te s t amount of pigment, 4.09 x 10“^-° mgm, was obtained
when both mannite and la c to se were the carbohydrate source,
followed by 3.60 x 10”10 mgm when sucrose alone was used.
With la c to se as the carbohydrate source in the medium,
3.50 x 10”10 mgm was obtained and with dextrose 2.79 x 10"10
mgm of pigment was found.
followsi

4.6 x 10

The b a c te r ia l populations were as

g

organisms when mannite or lactose was in
9
the medium, 4.4 x 10 organisms with sucrose as the source of
9
9
sugar, 4.1 x 10 organisms with dextrose and 4.0 x 10
organisms when la c to se and mannite were included.

Such

differences in b a c te ria l populations were not great enough
to be s ig n if ic a n t.
The differences in the amount of pigment produced when
the two sugars,
la c to se ,

lacto se and mannite or the single sugars,

mannite or sucrose were added to the medium were also

not gx*eat enough to be important.
as the source of carbohydrate,

however when dextrose served

the low fig u re of 2.79 x 10 ^

mgm of pigment merits some consideration.

As the b a c te ria l

populations were not consistent with the amount of pigment
produced; th a t i s ,

the t e s t s with the g re a te st number of

42

organisms did not give the g reatest amount of pigment,
in te rp re ta tio n is d i f f i c u l t .

However when lactose, mannite

or sucrose was added separately to the medium, the pigment
production and b a c te ria l populations were approximately the
same and a l l three showed more pigmentation and more organisms
than when dextrose was present in the medium.

Such findings

allow one to conclude th a t those sugars were superior to
dextrose in affecting pigmentation.

The possible explanation

of these findings might be found in the products which were
formed from these sugars during autoclaving.
According to Lewis (20) products deleterious to growth
of some organisms including staphylococci, were formed when
phosphates in a concentration above 0.3 percent and dextrose
in a concentration above 0.5 percent were autoclaved together.
On the other hand lactose had to be added in a slig h tly higher
concentration (0*6 percent) before these deleterious effects
were apparent.
growth,

As pigmentation is closely a llie d with good

i t is possible th a t Lewis * findings offer a p a r tia l

explanation of the differences in the amount of pigment
production with the various sugars.
Other workers have obtained varied re s u lts when growth
responses to autoclaved and f i l t e r e d sugars have been compared
Some data such as Lewis',

show th at i t is necessary to f i l t e r

sugars in order to get a tru e picture of the carbohydrate

43

metabolism.

Other work indicates th at th is is necessary for

only a few of the more susceptible sugars while s t i l l other
data, Fulmer e t al (12), show that a ll sugars can be auto­
claved as th is process produces intermediates which are
beneficial for growth.

There are innumerable reasons fo r

these discrepancies, such as the pH and amino acid content of
the medium, the various s a lts present in the medium, the time
and pressures used in autoclaving and the wide variety of
organisms which have been used fo r te stin g .
A search of the lite r a tu r e revealed th at nothing had
been reported on the effect of f il te r e d as compared to
autoclaved sugars upon pigmentation.

If there aflt some

detrimental or advantageous breakdown products derived from
sugars which are autoclaved and these products affect growth,
as some workers have reported,

there might also be some effect

upon pigmentation.
With the above in mind, a comparison between f ilte r e d
mannite and lactose and autoclaved mannite and lactose was
made.

A combination of these two sugars was used as i t had

been shown in preceding te s ts that these sugars gave maximum
pigmentation.

Tests were run using the three te s t organisms.

Mannite and lactose were added to the regular semisynthetic
medium in the concentration of 0.2 gm per 100 ml of medium*

44

The d ata from t h i s t e s t are presented in Table 12.

It

is apparent th a t with a l l th re e organisms th ere was d e f in ite ly
more pigment produced per c e l l when they were grown in a
medium using f i l t e r e d

sugars than when autoclaved sugars were

employed.

With c u ltu re 2 the medium containing the f i l t e r e d
-10
9
sugars gave 3.42 x 10
mgm of pigment with 3.4 x 10 organisms

while the medium with the autoclaved sugars gave 2.04 x 10
g

mgm of pigment with 2.0 x 10

organisms per ml of medium.

Using c u ltu re 5 the f i l t e r e d sugars showed 5.72 x 10“^-° mgm
of pigment and the autoclaved sugars 4.68 x 10”10 mgm of
9
pigment with b a c te r ia l counts of 4.1 and 4.7 x 10 organisms
re sp e c tiv e ly .

Culture 6 showed the g r e a te s t d ifferen ce in the

amount of pigment produced,

6.24 x 10“^^ mgm were found when

the medium contained the f i l t e r e d sugars and 4.39 x 10~10 mgm
when the medium included the autoclaved sugars.

With th is

c u ltu re the b a c te r ia l population under both t e s t conditions
q
was practica.-ly the same; 2.9 x 10 organisms with the
9
f i l t e r e d sugars and 2.8 x 10 organisms with the autoclaved
sugars.
These data o ffe r some in te r e s tin g
in te r p r e ta tio n of the r e s u lts
sugars were autoclaved,

information.

One

could be th a t when these two

some of the interm ediates which were

formed were detrim ental to the form ation of the carotenoids
in staphylococci.

Another in te r p r e ta tio n of th e r e s u lts

is

45

th at with the breakdown of some of the sugar during autoclaving, there was not as much of the original sugar l e f t
in the medium as when the f il te r e d sugars were used.

As

th is sugar combination seems to give good pigmentation,

an

alte rin g of these sugars in any way such as autoclaving,
re su lts in le s s pigment being produced.

On the other hand

i t does seem quite plausible th at with some other sugar or
sugar combination, products more favorable fox* pigmentation
would be formed from the autoclaved sugar than from the
unaltered, f il te r e d sugar.
The relatio n sh ip of the amount of pigment to the
number of c e lls produced c a lls fo r some discussion.

In

much of the previous data which have been given there was
a direct relatio n ship between the amount of pigment and the
number of ce lls produced.

When counts were high, pigmentation

was good and when the counts were low, the amount of pigment
dropped off considerably.
s a lts ,

However with the alterin g of the

and now with the f ilte r e d and autoclaved sugars,

does not seem to be the case.

this

In both instances pigmentation

seemed to become a facto r independent from growth.

Of course

th is was tnxe only i f a p arti culai- lev el of growth was
obtained.

Such evidence supports the theory th at pigmentation

can be altered by some grov/th factors and is not simply a
phenomenon which occurs when the b a c te ria l population atta in s
a p a rtic u la r lev e l.

46

I t is true th at there was some discrepancy in the
b a c te ria l counts of culture 5 in th at there were slig h tly
greater numbers of c e lls in the autoclaved sugar medium
than in the f i l t e r e d sugar medium.

However, th is difference

only serve fu rth e r to support the argument th a t there are
growth fa c to rs which d ire c tly affect pigmentation end have
put a minor e ffe c t upon growth.
Effect of Amino Acids upon Pigmentation
No investigation of the e ffe c t of various metabolites
upon pigmentation would be complete without some work being
done on alterin g the amino acid composition of the media.
Reid (26) reported th a t nitrogen as well as inorganic s a lts
were necessary fo r pigmentation.

Gladstone (13) developed

a synthetic medium of 16 amino acids and found that 11 of
these acids were necessary fo r the growth of 25 strain s of
sttphylococci.

This worker was interested simply in growth

of the organism and did not mention the extent of pigmentation
in these cultures.
For such a study, i t would be necessary to use a
chemicaliy defined medium sim ilar to Gladstone's in order
th at i t

could be determined i f any p a rtic u la r amino acid

had an e ffe c t upon pigmentation.

When a synthetic medium

is used, additional problems are presented which are not
apparent when a peptone or even casamino acids are used for

47

the nitrogen source.

The i n i t i a l problem is to devise a

medium in which the organism w ill grow.

I f th is can be

accomplished, the next d iffic u lty to overcome is to obtain
s u ffic ie n t growth for pigment extraction.
The medium which was trie d contained the following
co n stitu en ts:
Basal Medium
W ater-------------------------------------------------------------M annite---------------------------------------------------------Lactose
-----------------------------------------------------Dipotassium acid phosphate------------------Sodium c h l o r i d e
Magnesium su lfate 7HgO
Ferrous s u lfa te 7Hg0 "
Manganese su lfa te H«*

1000 ml.
2.0 gm.
2.0 gm.
5.0
0.1
0.2
0.1
0.1

gm.
gm.
gm.
gm.
gm.

Thiamin ---------------------------------------------------------- 250.0 y
Niacin ------------------------------------------------------------ 250.0 y
B i o t i n -------------------0.12 y
Valine ------------------------------------------------------------ 175.0
Leucine ---------------------------------------------------------- 150.0
G ly cin e---------------------------------------------------------- 40.0
1 P r o l i n e ------------------------------------------------------ 60.0
M ethionine---------------------------------------------------- 60.0
Phenylalanine---------------------------------------------- 60. 0
Aspartic a c i d ------------------------------------------------150.0
A rg in in e-------------------------------------------------------- 40. 0
H i s t i d i n e ------------------------------------------------------ 40.0
Cystine ---------------------------------10.0
Tryptophane-------------------------------------------------- 10.0

mgm.
mgm.
mgm.
mgm.
mgm.
mgm.
mgm.
mgm.
mgm.
mgm.
mgm.

In order to determine i f the above medium contained
su ffic ie n t n u trien ts fo r growth of the three t e s t organisms,
the following procedure was trie d .

The medium was dispensed

in three ml amounts and seeded from a 24 hour nutrient agar
sla n t.

Two tra n s fe rs through the broth were necessary before

growth was considered successful.

The re su lts of these t r i a l s
part of Table 13.
6 did not.

are observed in the fix*st

Cultures 2 and 5 grew in the medium while

The l a t t e r cultux*e was reseeded into the above

broth and shaken during incubation at 30 C and s t i l l no growth
occurred.

Attempts were also made to grow th is organism at

37 C without success.
The following constituents were added to a previously
tr ie d medium in the combinations given below.
Vitamins (nonessential)
Pyrodoxine ------------------------------------------ 500.0 V
250.0 *
Calcium pentothenate --------------------250.0 V
Riboflavin ----------------------------------------250. 0 V
Para-amino benzoic acid --------------I n o s i t o l --------------------------------------------250.0 i
2.0 y
Folic acid ----------------------------------------Amino acids (nonessential)
Alanine ------------------------------------------------ 100.0 mgm.
75.0 mgm.
Glutamic acid ----------------------------------27.0 mgm.
Tyrosine ------------------------------------------75.0 mgm.
Lysine -------------------------------------------------Purine bases
Adenine--------------------------------------------------0.5mgm.
Guanine--------------------------------------------------0.5mgm.
U r a c i l ----------------------------------------------------0.5mgm.
X anthine------------------------------------------------0.5mgm.
(1) Basal

medium -

calcium pentothenate.

(2) Basal

medium -

calcium pentothenate - purine bases.

(3) Basal

medium -

5 nonessential B complexvitamins.

(4) Basal

medium -

5 nonessential B complexvitamins - purine
bases.

(5) Basal medium - 5 nonessential B complex vitamins - purine
bases - f o lic acid
(6; Basal medium - 4 nonessential amino acids.

49

Calcium pentothenate, purine bases and fo lic acid were
included in the various media as the work of Moller et al (20)
had shown that about one-fourth of the races of staphylococci
studied,

required pantothenic acid,

one-half needed purine

bases and a few would not grow without fo lic acid.
The three cultures were seeded into these media in the
same manner th a t had been used before.
in the remaining portion of Table 2 2 .

The resu lts are shown
Cultures 2 and 5 grew

in a ll the combinations of medium which were trie d while
culture 6 fa ile d to grow.

A fin a l t r i a l was made in which

the nonessential amino acids, namely, alanine, glutamic acid,
tyrosine and lysine were added individually; however s t i l l
no growth was obtained.

Due to these re s u lts the investigation

of the extent of pigmentation in a purely synthetic medium was
confined to cultures 2 and 5.
The medium which was used fo r pigmentation te s ts was
the basal medium which contained the sugars, s a lts ,

essential

B complex vitamins and the 11 required amino acids plus
alanine,

glutamic acid, tyrosine and lysine.

The.

la tte r

acids were added separately to lo ts of the above medium to
determine the significance of each in pigment production.
I t was necessary to shake the incubating cultures for 48 hours
rather than 24 hours in order to obtain su fficien t cells fo r
methanol extraction.

Two hundred ml of the medium in flasks

was seeded and treated in the usual manner.

50

In Table 14 are shown the d ata using c u ltu re 5.

The

amount or pigment produced v aried from 4.10 to 4.77 x 10“^
mgm.

The l e a s t amount or pigment was obtained when the medium

consisted or the required amino acids plus glutamic acid
(4.10 x 10”^*® mgm), and the g r e a te s t amount when the medium
contained the 11 required amino acids alone or with alanine
(4.77 x 10-10 mgm).

Vlihen ly s in e or ty ro sin e was used in the

medium with the required amino acids,
mgm

or

4.41 and 4.28 x 10”^°

pigment re sp e c tiv e ly were produced.
From the standpoint or th e amount

or

pigment produced,

the d iffe re n c e s do not seem to be g reat enough to be
s ig n if ic a n t.

However when th e d a ta are examined in the lig h t

of the number of c e l ls produced,
apparent.

some in te r e s tin g r e s u lts

The g re a te s t number of c e l l s ,

are

790 x 10 , were

found when the le a s t amount of pigment was produced; th a t

is

with the medium which contained th e 11 required amino acids
6
plus glutamic acid.
The lowest b a c te r ia l count, 425 x 10 ,
occurred when the amount of pigment was the g re a te s t,

th a t is

with the required amino acids alone or with alan ine.

The

other two media containing the required amino acids plus
ly sin e or ty ro s in e showed b a c te r ia l populations of 510 x 10
organisms per ml of medium which were in versely proportional
to th e amount of pigment produced.

I

51

The r e s u lts , when c u ltu re 2 was t r i e d ,
Table 15.

are shown in

The data are e r r a c tic due to in s u f f ic ie n t c e lls

fo r e x tra c tio n and to the vai’iable r a ti o
c e lls and pigment production.

of the number of

For example when the medium

contained the 11 required amino acids plus alanine,
s u f f ic ie n t c e lls were obtained.

Pigment production and t o t a l

c e ll v a ria tio n s were shown as follow s:
acids alone gave 4.03 x 10
c e lls ,

—

10

in ­

the 11 required amino

mgm of pigment and 358 x 10

6

with ty ro sin e - 2.72 x 10“10 mgm and 576 x 106 c e lls ,
■j

with glutamic acid - 2.96 x 10“

c

mgm and 520 x 10

c e lls ,

and with lysine - 3.43 x 10-10 mgm and 394 x 10^ c e l ls .
Upon examination of the data obtained with cu ltu re 5,
the i n i t i a l conclusion th a t could be drawn is th a t pigmentation
was not affecte d by the addition of any of the au x iliary amino
acids which were te s te d .

A lev e l of pigmentation of approximate]

4.40 x 10“10 mgm was reached when any of the above media was
tr i e d .

On the other hand the growth of the organism was

d e f in ite ly

affected by the addition of the amino acids.

Therefore under these t e s t conditions pigmentation was not
d ir e c tly proportional to growth.
Such a high lev el of pigmentetion in con trast with
b a c te r ia l counts which were r e la tiv e ly low requires some
explanation.

One possible answer may be the length of time

th a t the c e lls were grown,

i t being twice as long as when

52

the semisynthetic medium was under t e s t .

As mentioned before

a 48 hour growing period was necessary to obtain s u ffic ie n t
c e lls fo r methanol e x tra c tio n .

Another p o s s ib ility i s th a t

considerable e rro r was introduced in the pigment production
determinations when i t was necessary to use values a t the
lower end of the curve denoting weight of pigment.
lig h t transm ission covered a range of 78.0 to 83.0.

The percent
The f a c t

th a t the most accurate readings on any type of a lig h t tr a n s ­
mission instrument are obtained between 25.0 and 75.0 and
tn a t below and above these values,
magnitude,
r e s u lts .

the e rro r increases in

causes some doubt as to the v a lid ity of these
This is

also tru e when the number of organisms was

determined as the percent l i g h t transmission obtained with
these c e ll suspensions was above 77.0 percent.
The in co n sisten t re s u lts obtained with culture 2 are
also best explained by the inaccuracies which enter the
calcu latio n s when percent lig h t transmission values are high.
With t h i s

organism much g reater erro r would be expected,

in the determination of pigment weight,

as

the spectophotometer

readings were a l l above 90.0 percent although the b a c te ria l
counts were above 66.0 percent.

SUM
M
ARY
The r e s u lts obtained In th is study have been discussed
f o r the most p a rt in th a t po rtion of the th e s is where the
data were given.

Therefore, th i s

sectio n w ill be confined

to some concluding or summarizing remarks of the observations
which have been made in the study of S.

aureus.

The method which was devised fo r determining the amount
of pigment has proved s a tis fa c to ry fo r the majority of the
te s ts

made.

Close agreement has been obtained with the method

of Stahly e t al (32) as shown in Table 16.

With a small amount

of pigment (A) 0.0190 u n its were obtained with Stahly*s method
and 1.91 x IQ*10 mgm of pigment 7/ith the method used in these
te s ts .
close,

With g re a te r amounts of pigment agreement was also
(B) and (C) showing 0.0243 and 0.0266 u n its respectively

of pigment with S ta h ly 's method and 3.88 and 4.32 x 10“^° mgm
resp ectiv ely of pigment with the extracted pigment method.
Enough samples were not te s te d to determine th e su p erio rity
of one method over the other but the agreement of both show
th a t the method used in t h i s study was s c ie n tif ic a lly

sound.

Attempts to a ttr ib u te pigmentation to p a r tic u la r
chemical compounds have been made by many workers with a
v ariety of organisms.

Some have gone as f a r as to s ta te th r t

organisms can be made to produce pigment simply by the
addition of a p a r tic u la r s a l t ,

sugar, vitamin or amino acid or

can completely suppress pigmentation by withholding any such
compound from the medium.
organic s a lts

Sullivan (1905) reported th a t the

- malate, t a r t r a t e and oxalate allow growth but

do not allow pigmentation while la c ta te ,
give good pigmentation.
used in th is study.

c itra te

or succinate

Several species of micx*ococci were

From the observations made in th is study

th is was not the case with staphylococci.

*i/ith th is organism

the degree of pigmentation can be a lte re d by the addition of
various chemical compounds but i t

cannot be induced or

completely suppressed in the presence or absence of such
components.

Perhaps the methods of measuring the extent of

pigmentation have been responsible fo r some of the re s u lts
which have been obtained in the p ast.
In some instances changes in the amount of pigmentation
were closely a llie d with the b a c te ria l population.

A good

yield of pigment was obtained when the b a c te ria l counts were
re la tiv e ly high and low pigment formation re su lte d when
re la tiv e ly low b a c te ria l counts occurred.

This was well

exemplified whexi the vitamin supplement was su b stitu ted fo r
the yeast ex tract in the liq u id modification of Chapman's
medium and when decreasing amounts of thiamin and niacin were
used in the semisynthetic medium.

In both cases a lte rin g

the amount of vitamin re su lte d in changes in the b a c te ria l
populations which in turn caused a proportional change in the
amount of pigment produced.

55

Although a d ire c t c o rre la tio n was also very apparent
with a l l th ree t e s t organisms when the amount

or

pigment was

compared in the presence and absence of the phosphate s a lt .
The data obtained with the s a lts and sugars for the most p art
presented a d iffe re n t p ictu re.

Variations in the amount of

pigment produced were not accompanied by sim ilar changes in
the b a c te r ia l populations.

The stu d ies made with the various

s a lts demonstrated th a t the addition of manganese s u lfa te
increased pigmentation by one th ir d of th a t obtained with the
other s a lts

(ferrous s u lfa te ,

magnesium and sodium chloride)

th a t were tr ie d .
Likewise the comparison of the various sugars using
culture 5 showed th a t differences in the amount of pigment
were not co rrelated d ire c tly with c e ll numbers.

There was

le s s pigment when dextrose served as the carbohydrate source
than with any of the other sugars but b a c te ria l populations
were about the same throughout.
The comparison of the f i l t e r e d

and autoclaved mannite

and lactose solutions presented a sim ilar picture in th a t,
g reater pigmentation was obtained with the f i l t e r e d carbo­
hydrates than was obtained with the autoclaved carbohydrates
although the number of organisms per ml of medium remained
approximately the same.

56

The re s u lts

obtained when the amino acid content or

the medium was a lte re d would ind icate th a t alanine, ty ro sin e,
glutamic acid and ly sin e had no e ffe c t upon pigmentation
although the v a lid ity of these r e s u lts may be open to question
due to the lim ited number of organisms which grew in a purely
synthetic medium.
To summarize,

i t may be sta te d th a t sp ecific s a lts

and

sugars had a d ire c t e ffe c t upon pigmentation and very l i t t l e
or no e ffe c t upon c e ll p ro life ra tio n a f te r the growth require­
ments of the organism were apparently completely s a tis f ie d .

BIBLIOGRAPHY
1.

Baker J . A. ,1938 Light as a fa c to r in the production of
pigment by c e rta in b a c te ria ,
J . Eact. 35.:
625.

2.

Baumann C.L., Steenbock H., Ingraham M. & Fred E. 1933
Microorganisms and the synthesis of carotene and vitamin
A. J . Biol. Chem. 103: 339.

3.

Bunting M. J . t 1940 The production of sta b le populations
of color v a ria n ts of S e rra tia marcescens #224 in
rapid ly growing c u ltu re s. J . Bact.
69•

4.

Byatt P.A., Jann G.L. and S alle A.J. 1948 V ariation in
pigment production in Staoh. aureus. J . Bact. 55:
787.

5.

Chapman G. H. , 1943 Determination of the chromogenic
property of staphylococci, J . Bact. £5u 405.

6.

Chapman G.H., 1948 An improved Stone medium fo r the
Is o la tio n and te s tin g of food poisoning staphylococci.
Food Research JL£: 100.

7.

Chargaff E ., 1934 Etude des pigments carotenoides de
quelques b a c te rie s . Annales I n s t. Past. 5g.: 415.

8.

Dewey B.T. and Poe C .F ., 1943 A simple a r t i f i c i a l medium
fo r pigment production by members of the genus S e rra tia .
J . Bact. 45: 495.

9.

Fagraeus A strid, 1948 The influence of sodium chloride
on growth of staphylococci and some other b acteria.
Acta. Path, and Microbiol. Scand. £6.: 655.
Cited
from Biol. Abst. 24: 18980 (1950).

10.

F ildes P., Richardson G., Knight B. and Gladstone G. P .,
1936 A n u trie n t mixture s u ita b le f o r the growth of
Staphylococcus aureus. B rit. J . Exp. Path. 17: 481.

11.

French C. S., 1940 The pigment-protein compound in photo­
sy nth etic b a c te ria . J. Gen. Physiol. 23: 469, 483.

12. Fulmer E .I ., Williams A.L. and Werkman C.H. 1931.
The
e ffe c t of s t e r i l i z a t i o n of media upon theix* growth
promoting p ro p erties toward b a c te ria . J. Bact. 21:
299.
13.

Gladstone G .P.,1937 N u tritio n of Staphylococcus aureus.
B rit. Jour. Exp. Path. 1.8: 322.

58

14.

Goldsworthy N.E. and S t i l l J . I . , 1936 The e ffe c t of meat
e x tra c t and other substances upon pigment production.
J . Path. Bact. 43: 555

15.

Gurd F. B. and Denis W., 1911 Thebiochemistry
le p ra e . J . Exp. Med. 14: 606.

16.

Ingraham M.A. and Baumann C.A. , 1934 Ther e la tio n
microorgamisms to carotenoids and vitamin. A.
J . Bact. 28: 31.

17.

Kharasch M.S., Conway E.A. and Bloom W.L. , 1956 Some
chemical fa c to rs influencing growth and pigmentation
of c e rta in microorganisms. J . Eact. 22.: 533.

18.

K liger I . J . , Grossowicz K. and Bergner S ., 1943 Studies
on th e ro le of n iacin and thiamin in the metabolism of
glucose by Staoh. aureus. J . Bact. 46: 399.

19.

Kreitlow K.L., 1941 Effects of ra d ia tio n of S e rra tia
marcescens J . Bact. 42: 127.

20.

Lewis 1 .11., 1930 The in h ib itio n of Phvtomonas
in cu ltu re media containing sugars. J . Bact. 19,: 432.

21 .

E o ller E.F., Weygand F. and Wacker A., 1949 N u tritio n of
staphylococci. Zeitschr. Naturforch. 4§.:

22.

Niven C.F. and Sherman J.M. , 1944 N u tritio n of the
enterococci. J . Bact. 4Z: 335.

23.

N utini L.G. and Lynch E .k ., 1946 Further studies on the
e ffe c ts of tis s u e e x tra c ts on Staphylococci aureus.
J . Exp. Med. 48: 247.

24.

Pinner M. and Voldrich M., 1933 Derivation of Staphylococcus
aIbus c ltre u s and roseus from Staphylococcus aureus.
J . In f. Dis. 50: 185.

25.

P orter J.R. and Pelczer M.L., 1941 The n u tr itio n of
Staphylococci aureus. J . Bact. 41: 173.

26.

Reader V., 1925 The carotenes in Sarcina lu te a . Biochem.
Jour. 19: 1049.

27.

Reid R.D., 1936 Studies on b a c te r ia l pigments. J .
31: 1936.

of Bacillus
of

Bact.

I

59

28.

Reimann H.A. and Eckler C. , 1941 The pigments from
v a ria n ts of ^lcrococcus tetra g en u s. J . Bact. 42: 435.

29.

Sevag M. G. and Green M.N., 1944 The ro le oi* carbohydrates
in the development of pigment by Staoh. aureus.
J . Bact. 43: 496.

30.

Sobin B. and Stahly G.L., 1942 Is o la tio n of B acterial
carotenoid pigments. J . Bact. 44: 265.

31.

Skinner C.E. and Gunderson, 1932 Production of vitamin A
by species of Cornebacterium. 97: 53.

32.

Snell E.E., 194y N u tritio n of microorganisms. Ann. Rev.
Micro. Biol. 3: 97.

33.

Stahly G .I., Sesler C.L. and Brode W.L., 1942 A method
fo r measuring b a c te ria l pigments by use of th e spectro­
photometer and p h o to electric coloi*imeter. J . Bact. £2.: 14

34.

Sullivan M.X., 1905 Synthetic cu ltu re media and the bio­
chemistry of b a c te ria l pigments. J . Med. Res. JJfe: 109.

35.

Topley W.W.C., Wilson G.S. and Miles A.A.,1946 P rinciples
of bacteriology and Immlnity 3d ed. Williams and Wilkens
Co., Baltimore, Med.

36.

Tux*iam G. , 1950 The biosynthesis of carotenoids by
paratuberculosis b a c te ria . H elvetica Chim. Acta. 33: 13.
Cited from Biol. Abs. 2£: 16248 (1950)

37.

Werkman C.W. and Wilson P.W. , 1951 E a c te ria l Physiology,
Academic Press I n c ., New York.

3o.

Zopf W., 1889 Pigments found in several species of
b a c te ria . Ztschr. f . wiss. Mikrobiol. £: 172.

Table 1*

A Comparison o f Pigment; Production by
aureus
on Solid Media a t Two Incubation Temperatures
with P e tri P lates Sealed and Unsealed
Incubating Temperatures

Media

21 C

37 C

Dextrose
Agar

Sealed
Unsealed

N P
F P

N P
F P

Proteose
Peptone
#3 Agar

Sealed
Unsealed

N P
F P

N P
GP

Proteose
Lactose
Agar

Sealed
Unsealed

N P
GP

N P
F P

Chapmans
#110
Agar

Sealed
Unsealed

N P
GP

N P
GP

N P - - - No v is ib le Pigment
F P ----- F a ir Pigmentation
G P ----- Good Pigmentation

Table 2.

A Comparison of Pigmentation by S. aureus
5 a t three Incubating Temperatures Using
10 Days Growth

Incubating
Temperatures

Pigmentation
Observed in
Cells

20 C

F P

30 C

GP

37 C

GP

F P ----- F air Pigmentation
GP
Good Pigmentation

Pigment at 1on
by Methanol
Extraction
Orange
Very Dark
Orange
Dark
Orange

T a b le 3 .

T he E f f e c t o f O m ittin g V a r io u s C o n s t i t u e n t s o f
C hap m ans # 1 1 0 M edium o n G r o w th a n d P i g m e n t a t i o n
of
aureus 5

Omitted
Constituent

No.of Organisms
/ml Media x 10

No. of Organisms
Extracted x 10

Wt. of Pigment/
Cell x 10-10

Complete
Medium

* —_

314

4.70

Without
Marini te

1.8

314

3.56

Without
Lactose

2.6

314

2.54

Without
Sodium
Chloride

2.1

314

5.37

Without
Yeast
Extract

1.1

314

0.95

Without
Tryptone

2.2

307

1.79

Without
Gelatin

N.G.

----------

----------

T a b le 4 .

T h e E f f e c t o f O m i t t i n g V a r i o u s B C o m p le x V i t a m i n s
o n P i g m e n t a t i o n When S u b s t i t u t e d f o r Y e a s t E x t r a c t
i n C hap m ans # 1 1 0 M edium

Omitted
Constituents

No* of Organisms
No. of Organisms Wt • of Plgmer
/ml of MediumCx 109 ) ExtractedCx 109 ) Cell (x 10~10

Complete
Medium

4.9

350

3.81

Without
B Complex

1.9

350

0.48

Without
Thiamin

2.7

350

0.51

Without
Pyridoxine

5.1

350

4.14

Without
Riboflavin

5.2

357

4.06

Without
B iotln

5.3

350

4.31

Without
Niacin

4.3

350

3.71

Without
P. A. B. A.

4.3

350

3.77

Without
Ca Pento
thenate

5.0

350

4.08

'Without
In o s ito l

3. 8

350

3.68

T a b le 5 .

T h e E f f e c t o f O m i t t i n g V a r i o u s B C o m p le x V i t a m i n s
f r o m a S e m i - s y n t h e t i c M edium o n P i g m e n t a t i o n b y
S . a u reu s.

Omitted
Constituents

No. of Organisms Wt• of Pigmen

/ml of MediumCx 10^) ExtractedCx 109 ) Cell (x 10~lc

o
t

Complete
Mealum

No. of Organisms

281

4.09

—

«

Without
Thiamin

N.G.

Without
Niacin

N. G.

Without
B iotin

0.7

350

0.57

’Without
Pyricoxine

3.5

326

3.55

Without
Riboflavin

2.7

350

3.40

Without
P. A.B. A.

4.0

350

3.54

Without
Ca Pento­
thenate

4.0

350

3.42

Without
In o s ito l

4.7

350

4.20

~

~

~

w

_

T a b le

6.

T he E f f e c t o f V a r io u s C o n c e n tr a tio n s
P ig m e n ta tio n o f T h ree C u ltu r e s o f £ •

Organism

of Thiamin /
25.0

12.5

o f T h ia m in o n
aureus

100 ml of Medium
5.0

2.5

2
Wt. of Pigment
x 10*"10

2.32

2.20

1.52

0.77

No. of Organisms
x 109/ ml of
Medium

1.7

1.2

0.52

0.35

Wt. of Pigment
x 10-*10

4.42

4.42

2.40

0.78

No. of Organisms
x 109/ ml of
Medium

3.5

3.3

2.7

2.0

Wt. of Pigment
x 10"10

3.62

2.85

0.78

0.88

No. of Organisms
x 109/ ml of
Medium

2.9

2.0

0.68

0.60

5

6

T a b le 7 .

T he E f f e c t o f V a r io u s C o n c e n tr a tio n s
P ig m e n ta tio n o f T h ree C u ltu r e s o f § .

Organism

of Niacin /
25.0

o f N ia c in on
aureus

100 ml of Medium

12.5

5.0

2.5

2
Wt. of Pigment
x 10**10

3.30

3.11

1.26

No. of Organisms
x 109/ ml of
Medium

2.3

2.2

0.66

N.G.

Wt. of Pigment
x 10”10

3.32

2.28

1.70

0.90

No. of Organisms
x 109 / ml of
Medium

3.4

3.7

2.8

1.9

Wt• of Pigment
x 10-10

4.32

4.00

3.39

0.98

No. of Organisms
x 109 / ml of
Medium

4.9

4.1

1.3

0.90

5

6

Table 8.

A Comparison of the Amount Pigment Producted When
Cocarboxylase Is S u b stitu ted Tor Thiamin in a
Semisynthetic Medium Using Three Cultures or
aureus

Wt. of Pigment (x 10~10) /

It

Organism

Cocarboyxlase

Cell

Thiamin

2

2.58

2.33

5

3.88

3.88

6

4.68

4.27

T able 9 .

The E f f e c t o f Sodium C h lo rid e , F e rro u s S u l f a t e , Manganese S u l f a t e and
Magnesium S u lf a te on Pigm ent P ro d u c tio n and Growth i n T hree C u ltu re s
o f S. a u r e u s *

Chemical
Constituents

Wt. of Pig.
x 1CT10

No. of
Organisms
x 109

Culture 6

Culture 5

Culture 2

Wt. of Pig.
x 10“10

No. of
Organisms
xlO 9

Wt. of Pig.
x 10"10

No. of
Organisms

Four Salts
Added

3.93

1.8

4.06

4.0

4.16

2.0

No Salts
Added

1.29

1.5

1.52

4.6

2.86

1.0 x 109

Sodium
Chloride
Added

1.26

2.2

2.42

4.4

2.27

1.0

Ferrous
Sulfate
Added

1.44

2.2

2.48

4.0

2.27

900 x 106

Manganese
Sulfate
Added

2.32

2.2

3.93

4.4

2.58

1.4

X

109

Magnesium
Sulfate
Added

1.12

1.7

2.26

4.6

2.45

900

X

106

X

X

109

109

Table 10.

A Comparison or the Number or Organisms, Weight
or Pigment and pH Obtained with and without d i­
potassium Acid Phosphate Using Three Cultures
or
aureus

No. or Organisms
x 10^/ml or Medium

Organi c r:

2

With
KgHP04

1.8

2.33

5.90

0.58

1.72

4.70

3.7

3.88

6.30

Without
KgHP04

0.56

1.90

4.75

With
KgHP04

2.95

4.27

6.18

Without
KgHP04

0.70

1.36

4.97

Without
KgHP04
5

6

pH or Super­
Wt • or Pigment natant Fluid
After Growth
x 10
/C e ll

With
KgHP04

T a b le 11#

T he E f f e c t o f V a r io u s C a r b o h y d r a te s i n
s y n t h e t i c M edium o n P i g m e n t a t i o n b y

Carbohydrate
Lactose &
Mannite

No* of Organisms
(x 109 )/ml of Medium

a S e m iaureus

Wt. of Pigment
Cx 10“10)/C ell

4.0

4.09

Lactose

4.6

3.43

Mannite

4.6

3.50

Dextrose

4.1

2.79

Sucrose

4.4

3.60

Table 12.

A Comparison of Pigment Production by Three Cultures of
aureus
When Grown on Media Containing Filtered or Autoclaved Lactose and
Mannite.
No. of Organisms
(x 109 )/ ml Medium

Organism

Filtered
Sugars

Autoclaved
Sugars

Wt. of Pigment
(x 10'10)/ Cell
Filtered
Sugars

Autoclaved
Sugars

2

2.2

2.2

3.42

2.04

5

4.1

4.7

5.72

4.68

6

2.9

2.8

6.24

4.39

Table 13.

The Growth Response of Three Test Organisms
to Various Synthetic Media

Growth of Organism
Media

Culture 2

Culture 5

Culture 6

B.M.

4

4

-

B. M. 4 C.P.

4

-4

-

B.M. 4 C. P.4
P.B.

4

4

-

B.M.4 None sse n tia l
Vitamins

4

4

B.M. 4- None sse n tia l
Vit .4 P. B.

4

4

B.M.4- None sse n tia l
Vit.4 P.B.4
Folic Acid

4

4

B.M.4 Four
Amino Acids

4

4

B.M.
C.P.
P. B.

basal medium
calcium pentothenate
purine bases

T a b le 14*

T h e E f f e c t o f V a r io u s Am ino A c id s
o f S . aureus 5 .

Amino Acids

No* of Organisms
(x 106)/ ml Medium

on P i g m e n t a t i o n

Wt. of Pigment
Cx 10~10)/ Cell

11 Required Amino
Acids

435

4.77

11 Required Amino
Acids +
■ Tyrosine

510

4.38

11 Required Amino
Acids + Alanine

435

4.77

11 Required Amino
Acids «
►
» Glutamic Ac*

790

4.10

11 Required Amino
Acids + Lysine

510

4.41

T a b le 1 5 .

T h e E f f e c t o f V a r i o u s Am ino A c id s o f P i g m e n t a t i o n
o f S. au reu s 2 .

Amino Acids
Added

No. of Organisms

Wt • of Pigment
(x 1 0 '10)/ Cell

(x 10^)/ ml Medium

11 Required Amino
Acids

368

4.06

11 Required Amino
Acids + Tyrosine

576

2.72

11 Required Amino
Acids + Alanine

------■
K

11 Required Amino
Acids *
►Glutamic Ac<

520

11 Required Amino
Acids +- Lysine

394

-------

2.96

1

* In su ffic ie n t Cells fo r Extraction

3.48

T a b le 16*

A C o m p a r is o n o r S t a h l y ' s M e th o d w i t h t h e E x t r a c t e d
P ig m e n t M eth o d T o r D e t e r m i n i n g A m ount o r P ig m e n t
P roduced.

Medium

Stahly *s
Method

Extracted
Pigment Method
(x 10"10)

Semisynthet ic
Medium A

0.0190

1.91

Semisynthetic
Medium B

0.0248

3.88

Semisynthet ic
Medium C

0.0266

4.32