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ANN ARBOR • MICHIGAN

THE NUTRITION A N D FACTORS
A F F E C T I N G THE GROWTH OF
BACTERIA IN SOLUBLE OILS

by
H I L L I A R D PIVNICK

A THESIS
Submitted to tne Graduate School of M i c h i g a n
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
Department

cf Bacteriology

1952

AC KN OWLE DGEMENTS
Scientic matters are readily expressed in easily under­
stood terms which have very exact meanings but there are no
words which can possibly express the depths of a m a n ’s thoughts.
It is with this in mind that I humbly offer the following
ac k n o w l e d g e m e n t s .
To Dr. F. W. Fabian for his wise and experienced guidance
in the pursuit of these studies and for personal
in the daily struggle,

I am most grateful.

counselling

I am likewise

greatly indebted to the many men in industry without whose
help this project

could not have been carried out:

as Dr. Edwin Place,

Industrial Physician,

Lubrication Engineer,

and Mr. L. Garlieb,

of the Oldsmobile Division,

.Motors Corporation, Lansing,

General

and Dr. E. W. Adams and Mr* A.

Lindert of the Standard Oil Company
emulsion samples and advice;

such men

(of Indiana)

;V.

for materials,

to the great number of chemical

and pharmaceutical

companies for generously supplying materials

for investigation;

to my friends

Health,

in the Ontario Department of

C. Christie, A. Miller and M. Magis,

samples and cultures;

to the "media room"

for emulsion

in the bacteriology

department for expertly filling the many orders for media.
It is an especial
encouragement

pleasure to acknowledge the cheerful

of my wif e — a silent but stimulating partner

in these many research projects.

TABLE OF CONTENTS

Pa ge
1

INTRODUCTION
FART I

PART II

PART III

PART IV

PART V

M E T H O D U S E D IN DETERMINING- NUMBERS OF
EACTERIA IN SOLUBLE OILS
R e v i e w of L i t e r a t u r e

5

E xperimental

7

Results and Discussion

8

THE GROWTH OF EACTERIA IN SOLUBLE OIL
EM U L S I O N S
R e view of Literature

13

Expe rime ntal

15

Results and Discussion

20

COLIFORM BACTERIA IN SOLUBLE OIL
EMULS IONS
R e v i e w of L i t e rature

23

Experimental

24

Resul ts and D i s c ussi on

27

D E R M A T I T I S A N D CUTTING OILS
R e v i e w of L i t e ra ture

30

Experimental and Results

37

Discuss ion

42

D I S I N F E C T I O N OF SOLUBLE OIL EMULSION S
Review of Literature

43

Exper imental

46

Results and Discussion

51

Pag©

PART VI

FART VII

NUTRITIONAL STUDIES OF THE BACTERIA
IN SOLUBLE OIL EMULSIONS
R e v i e w of Literature

56

Experimental

59

Results and Discussion

62

BACTERIA IN SOLUBLE OIL EMULSIONS
Review of Literature

68

Experimental

69

Results and Discussion

72

Culture Descriptions

76

LI T E R A T U R E CITED

109

Introduction

The ability of bacteria to utilise hydrocarbons has been studied by
microbiologists for over a half of a century* but it is only recently
that the subject has received the attention which it deserves*

The work

of Zobeli and others has emphasised the role that microorganisms play in
the breakdown and* to a smaller degree* in the synthesis of petroleum —
an urgent study in a mechanised world*
As a basis for orientation towards the work which follows* some of
the fundamental aspects of the petroleum industry will be discussed after
which a description of cutting oils* especially soluble oils* will be
given*
Refining of Petroleum
Crude petroleum exists in subterranean deposits usually in the free
or liquid state but also often in close association with sands or shale*
When a well is drilled* the petroleum is forced to the surface by gas
stored under high pressure in association with the subterranean oil pools*
If

the gas pressure is not adequate or becomes depleted with time* then

other methods mist be used to bring the oil to the surface*
The crude oil is often in the form of an emulsion with brines which
are

chemically

similar to sea water; and before they are refined* the

emulsion oust be broken to separate the petroleum from the water*
The refining procedure may be divided into two phases* (a) the dis­
tillation and fractionation of the petroleum to break the coaplex mixture
into its desired components and (b) the treatment of these cooponents to

-2

remove undesirable materials*

The distillation depends on the heating

of the crude oil with fractionation to produce the "various *cuts" • Vacuum
may be used*

The number of fractions depends on the manufacturer's

quipment and market demands*

e—

However* the fractions may be roughly

grouped as gasoline, kerosene* diesel oils* fuel oils* lubricating oils*
asphalt and road oils*

Treatment of the oils following distillation Is

usually necessary to remove undesirable confounds which are unstable or
malodorous*

The most comaon method of treatment is to

the fraction

with concentrated sulfuric a d d to form a sludge of petroleum sulfates
with the unsaturated hydrocarbons and also to precipitate organic bases*
phenols* organic sulfur compounds and other undesirable substances* After
the sludge has been removed* the residual acid is neutralised with sodium
hydroxide and washed with water*

A few other methods of treatment In­

clude extraction of undesirable coiqpounds with various solvents such as
furfural, acrolein and nitrobenzene and treatment with materials such as
sodium plumblte or cupric chloride to convert foul-emailing mercaptans
to non-smelling disulfides*
Chemistry of Petroleum
The chemistry of petroleum Is so complex that even today* after many
decades of Intensive research* there are huge gaps In our knowledge con­
cerning the nature of many of its components*

The coaqpositlon of crude

oil varies in different oil fields and even in wells in the same fields*
Indeed* a single well may produce crude oil of varying composition at
different times*

- 3-

Genfifally crude oils are classified according to their coop ononis
into paraffin base* asphaltic (naphthene) base and mixed base*

The par—

affin base crudes consist predominately of aliphatic hydrocarbons with
some cyclic conpounds while the asphaltic base consists mainly of naphthenic or saturated ring structures with some aliphatice*
crude oils contain a more equal mixture of the two*

The mixed base

If wax is present*

the crude oil is called "wax-bearing** *
The aliphatic hydrocarbons are mixtures of saturated
straight and branched chains*

unsaturated*

The carbocydics or ringed conpounds are

of two general classes; the aromatics (unsaturated compounds) and the
cycloparaffins or naphthenes (saturated compounds)*

Both classes of

carbocydics may or may not have aliphatic side chains of varying num­
ber* size and conplexity*
Cutting Fluids
Cutting fluids are lubricating and cooling compounds used to facil­
itate metal-cutting operations*

The best lubricating action is furnished

by 100 per cent fatty oil such as lard oil or rape seed oil* while the
best cooling action results from 100 per cent water*

Petroleum oils lack

the lubricating properties of fatty oils and the cooling properties of
water*

However, they are the most common component of cutting fluids

because of their cheapness* rust-preventing characteristics* relative
inertness and ability to be synthesised into compounds with good lubri­
cating properties*

-feCutting fluids have been classified by mangr workers, bat they may
be conveniently divided into three groups:
(a)

The alkaline solutions which may consist of a soap solution*

with or without added base; or they may be solutions of such substances
as borax* sodium carbonate or trisodiua phosphate*

The use of such cut­

ting fluids is limited principally to cooling and laying dust in grinding
operations*
(b)

The soluble or eimil s1 fiable oils*

These oils consist of min­

eral oils* with or without fatty oils* that have been compounded with
emulsifying agents to make them miscible with water*

K few emulsifying

agents are saponified fatty acids* able tic acid* petroleum sulfonates*
phenols and naphthenates*

Mixed with water these soluble oils are used

in cutting and grinding operations where cooling requirements predomi­
nate, but where lubricating and anti-corrosive activity are also required*
(c)

The straight oils*

or without fatty oils*

These oils consist of mineral oils* with

They are used where greater lubrication than that

afforded by soluble oil emulsions is required*
All oils used* whether mineral or fatty* may or may not be treated
chemically or contain additives to improve their lubricity*

Such treat­

ment is enployed to Increase the film strength of lubricants which are
used in high pressure work and also to give anti weld properties to the
oils*

The commonest chemical treatment is with sulfur or chlorine*

-5 -

AddLtives ihlch #re used nay contain arsenic or phosphorous (

5

)*

These compounds , under extremes of temperature and pressure * form iron
oxides which serve as a lubricating film when the organic lubricant
breaks down and thus prevent welding of chips to the tools*
Bacteria are not known to grow in straight oils as water is neces­
sary for their existence*

However, they do grow in the soluble or emol—

sifiable oils ( 62, 36, 34, 25
water*

) when these have been nixed with

Growth in this medium is thought to be responsible for several

undesirable occurrences such as obnoxious odors, discoloration of the
emulsion and an increase in acidity with resultant breaking of the emul­
sion*

There is also considerable opinion, but no proof, that these bac­

teria are responsible for dermatitis of workers in contact with the oils*
For these reasons studies of the flora of soluble oil emulsions and fac­
tors which affect their growth have been undertaken*
PART I*

METHOD USED IN DETERMINING NUMBERS
GF BACTERIA IN SOLUBLE OILS
Review of Literature

It is true, but nonetheless unfortunate, that mich quantitative
bacteriology is carried on with little regard for suitability of the
diluent and media employed*
well established methods ( 3

Workers in water and milk bacteriology have
) ( 4

) based on considerable research,

but in many other fields there are no authoritative works to guide the
researcher*

It has been observed that some laboratories use distilled

water, tap water, 5>0— 5>0 mixtures of the two or physiological saline

with apparently little consideration for the pH or osmotic pressure of
the material being investigated*

Supposedly, -the Ideal diluent would be

some of the material being Investigated*

But since such diluents are

rarely feasible, it is desirable to use a diluent which will have the
least effect on the bacteria being Investigated*
Zobeli ( 64 ), searching for a diluent which would be suitable for
marine bacteria, found that the bacteria in marine mud perished rapidly
In tap water, distilled water, 0*85 per cent saline and a solution form­
ulated upon the average composition of river water*

Using the counts

obtained when antodaved sea water was used as a diluent as 100 per cent,
he found that when sea water and distilled water were mixed In varying
ratios, there was the least survival In those diluents having the great*
est proportion of distilled water*

The mixture giving the greatest sur­

vival at 60 min contained 75 per cent of sea water and 25 per cent of
distilled water*

In work with petroleum products, there has been a var­

iety of methods alloyed for growing and enumerating the bacteria con­
cerned*

Stone, Fenske and White ( 54) used nutrient agar as a plating

medium because this medium gave slightly higher counts than a mineralsalt oil agar and because all organisms ^llch they transferred from the
latter medium grew when transferred to nutrient agar*
tion the diluent which they used*

They did not men­

Bushnell and Haas ( 15 ) used a min—

eral-ealt agar containing hydrocarbons but did not mention the diluent*
In Investigations of soluble oil emulsions Lee and Chandler ( 34 ) used
distilled water as a diluent and nutrient agar as a plating medium while

Liberthson ( 36 ) mentions neither the medium nor the diluent*

Buffett*

Gold and Weirich ( 25 ) used the standard agar plate method for obtaining
counts*
Experimental
The experiments reported here embraced a study of materials and
methods for obtaining plate counts of bacteria in soluble oils*
the following procedure was common to all this work*

Howerer*

Media were held at

U5*C before pouring; unless otherwise stated* duplicate plates were pour—
ed; decimal dilutions were so made that one ml of suspension was added
to each petri dish; dilution bottles were shaken 25 times by hand; plates
were Incubated at ^0mC for 1*8 hours and'ciauxte&^on1 a@Quebec coloxy count­
er*
Media tested were nutrient agar (Difco) and nutrient agar to which
1 per cent of a soluble oil had been added*
water*

The diluent was distilled

Nine samples of soluble oil emulsions from industrial sources

were plated and duplicate plates poured with each of the

Three

samples were plated with a medium composed of 1*5 per cent agar and 1
per oent of a eoluble oil*
Diluents tested were those frequently encountered in bacteriological
work; distilled water* 0*85 per cent saline* M/20 phosphate buffers and
M/20 phosphate buffers containing 0*85 per cent saline*

Buffers ranged

from pH 7*0 to pH 8*0 in 0*2 pH unit increments while buffered salines
were used at pH 7*0* 7*6 and 8*0*

The inoculum used for investigation of diluents was usually a mix­
ture of 20 different soluble oil emulsions obtained from industrial
sources*

Occasionally* as noted* individual samples were used*

All

samples were refrigerated at *>0*F upon arrival and held at this tenper—
ature*
The following procedure was used to minimise the amount of work and
equipment necessary for the experiments with diluents*

Plate counts

using nutrient agar* with distilled water as a diluent* were made on
frigerated samples of emulsions*

re­

When the bacterial count was obtained*

a portion of the saaple was removed from the refrigerator and so diluted
that a one ml portion would give plates ideal for counting*

Plate counts

in quadruplicate* of the final suspension were made at one minute and at
intervals thereafter*

The average of the four plates was used to calcu­

late the percentage change ttiich occurred after one minute*
Results and Discussion
Table 1 shows the effect of adding one per cent of soluble oil to
nutrient agar*

Six of the nine samples had higher counts cm the nutrient

agar without oil* and five of the six counts were significantly higher*
Since the soluble oil produced a very opaque medium when added to nutri­
ent agar* probably the decreased count in the presence of the oil was
due to masking of colonies rather than inhibition of growth by the oil*
Several colonies growing on the nutrient agar with oil grew well when
fished to nutrient agar without oil*
Plating media composed only of distilled water* 1*5 per cent agar
and one per cent soluble oil showed no growth after two days at

-9 -

Table 1
A conparative count of bacteria in soluble oil emulsions using nu­
trient agar and nutrient agar containing one per cent soluble oil as
plating media*
Bacteria per ml
Sanple

Nutrient agar

Nutrient agar-*- oil

1

760,000

510,000

2

2,200,000

1,01*0,000

3

2,710,000

1,620,000

h

2,065,000

805,000

5

2UU,350,000

295,600,000

6

il*,35o

13,300

7

78,1*00

82,200

8

16,500

8,250

9

75o

1,U50

Table 2 shows the effect of distilled water, as a diluent, on or­
ganisms in soluble oil emulsions*

There was a definite decrease in bac­

terial papulation of Sample 6 and Mixture A with increased time*

However,

the changes in Sasple 1 and Mixtures B and C are within experimental
error ( 43 )•

Table 3 *-a representative of two experiments identical in

all respects except t h a V ^ e experiment not shown counts wdre/a *a3%eFac*e
15 mins*

Both experiments showed identical trends*

was decidely toxic*

Physiological saline

Buffer at pH 7*0 gave the most consistent results

-10*

Table 2
in n um be r

Per cent change/of bacteria from soluble oil emulsions
suspended in distilled water

Enilsion

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Per cent change_ _ _ _ _ _ _ _
60 min
5 min
30 min
15 min
90 ndn
120 min

Sm*)le 1

- 8*1

-2.8

2,5

-25.5

-

1.7

-1*6

Sanple 6

-12,5

-7.0

-18*6

-22.9

-

-53*1

-52.2

Mixture A

-

-

-U.5

-23*0

m

-

-

Mixture B

-

-7.8

-5.1

- 5.3

-9.7

m

-

Mixture C

-

•

1%7

5.0

1.6

m

m

- a no e x p e r i m e n t s made

2U0 min

-11-

Table 3
in n um b e r

Per cent change/of bacteria from mixture of soluble oil emulsions
suspended in various diluents_ _ _ _ _ _ _ _ _ _ _ _
Bact. per nl
at 1 kin

Diluent
1 Distilled niter
2 0*85

per cent saline

Per cent change
30 nin
60 min
90 nin

160

15.7

5.0

171

•33*9

-85.U

1.6

-96a

3 Buffer

pH 7.0

165

3.0

1.2

2.1

kBuffer

pH 7.2

165

- 1.2

•4.2,1

2.1;

5 Buffer

pH 7*k

211

-0*9

6.2

3.8

Buffer

pH 7.6

172

-lfc.5

- 8.1

1*6

7 Buffer

pH 7.8

IS?

5.7

19a

9.5

Buffer

pH 8*0

CO
rH

5.2

0,6

-17.7

9 Buffered saline

pH 7.0

77

118.2

106,6

62.U

10 Buffered saline

pH 7.6

51

2?3.1i

263.0

202,0

U Buffered saline

pH 6.0

89

28J.

7U.2

76^

6

8

-1 2 -

with a mean deviation for the two experiments of 2*6 per cent from the
count at one minute*

Buffers at the other pH* s gave deviations which were

generally within experimental error*

However, buffered saline gave re­

sults totally unlike those given by saline alone or buffer alone*

Al­

though all diluents received identical inocula, the mean count at one
minute in diluents 1 to 8 inclusive was 170 per nl; while in diluents 9
to 11 inclusive, buffered salines, the mean count was 72 per ml*

This

indicated that buffered salines caused a rapid decrease in bacterial
population as determined by the plate count*

The decrease was not due

to a killing effect because at JO minutes and thereafter the counts
were considerably greater than at one minute*

This would definitely

indicate that the buffered saline caused an immediate clunping, but
"that on standing, the cliuyts of bacteria broke apart*

The buffer had

a sparing activity towards the toxic effect of sodium chloride because
in one experiment there were six bacteria per ml in diluent 2 (0*85 per
cent saline) at 90 minutes while diluents 9, 10 and U

(buffered salines)

had a mean count of liyg bacteria per ml at the same time*

The other ex­

periment showed similar results*
A rather extraordinary phenomenon involving diluents is included in
these studies as an example of some of the difficulties encountered*

An

emulsion containing 0*05 per cent of parachlorometacresol, inoculated
with a mixed inoculum, when plated after 68 days, yielded about 6,000
colonies

if 0*1 ml of emulsion were plated directly but if dilutions

of It 100 were plated no colonies appeared*

It was thougit that dilution

of a nutritional factor was involved and the following were added to the
nutrient agar without benefit:

parachlorometacresol in eight concentra­

tions , soluble oil , heat killed bacterial suspensions , vitamin mixtures,
yeast extract and incubation in a carbon dioxide-air mixture* It was
that
suggest©d/the diluent might be at faalt and experimentation showed that
if soluble oil emulsion, or phoqphate buffer at pH 9*0 was. used as
diluents the count remained stable for one hour: but buffer at pH 7*0
caused a decrease of 81* per cent in one minute and a decrease of 99.6
per cent in 30 minutes*

In this particular case the toxic effect of the

diluent was traced directly to hydrogen ion concentration*
It can be concluded that nutrient agar is suitable for enumeration
of aerobes in soluble oil emulsions*

However, one must be constantly on

guard concerning the diluent used for plating work*
saline are definitely useless:
should be used with caution*

Saline and buffered

distilled water and M/20 phosphate buffers
Soluble oil evulsions are not usable because

they make the medium too cloudy for counting if one ml is added to a petri
dish*

A wise precaution would be the direct plating of 0*1 ml of an emul­

sion as well as plating dilutions, of the emulsion*
PART II*

THE GROWTH OF BACTERIA IN SQLUHUE OIL EMULSIONS
Review of Literature

It has been known for many years that bacteria were present in cut­
ting fluids*

However, early workers were mainly concerned with health

hazards, especially the role that cutting fluids played in causing and
spreading dermatitis*

-lllCantenporary workers are chiefly Interested In catting fluids as a
cause of dermatitis, but there are a few who are Interested In the detri­
mental effects that organisms have on the soluble oil emulsions*

Lee and

Chandler ( 34 ) Investigated samples from various departments of one com­
pany for five months and found that bacterial counts were usually around
25 million per ml, seldom lower than 15 million and sometimes greater
than 50 million*

They found practically a pure culture of the same or­

ganism in all the departments of this plant and in another plant some
distance away*

Chandler ( 1 7 ) in a personal communication stated that

this organism was still present in practically pure culture in this same
plant several years later*

They ( 34 ) found that replacing the emulsion

or sterilising it by beat was useless because circulation of the emulsion
through the machines recontaminated it*

Fresh emulsion in use for two

hours contained lli,U80,000 bacteria per ml and in four days they had in­
creased to 37 >800,000.

The organism, a Pseudomonas * grew equally well

in emulsions made with mineral oils alone or mineral and lard oil c<aabinations*

Duffott, Gold and Weirlch ( 25 ) conducted a very extensive

survey of the bacteriology of soluble oil emulsions but unfortunately
only a short note was published and further information was unobtainable
( 60 )•

They found that the majority of 63U samples taken from J2S dif­

ferent factories in northeastern United States contained over one million
bacteria per ml*

The higiest count was

million*

Most of the work reported does not state the source of contamination
of the emulsions*

Westveer ( 62 ) suspected the source of contamination

to be river water used to make the emulsion, aerial contamination, con—

taminated metal parts and the workers' hands*

He also suspected that

refuse thrown into the emulsion by workers might serve as a source of
contamination*

( 29 )

Stone, Fenske and White (54 ) and Gray and Thornton

showed that some soil organisms were capable of fermenting hydro­

carbons similar to those found in petroleum, but they did not work with
soluble oil emulsions*
Experimental
Experiments were designed to grow mixed cultures in emulsions of
several soluble oils and also pure cultures in a single soluble oil*

The

ability of organisms from several suspected sources of contamination to
grow in soluble oil emulsions was investigated*

Also the increase in

bacterial population was followed in fresh emulsions in a local machine
shop*

Counts on several emulsions obtained from industrial sources were

mads*
The growth curves of a mixture of four emulsions obtained from mach­
ine shops were obtained in fresh emulsions of 13 soluble oils*
oils ware mineral oils emulsified with petroleum sulfonates*
contained saponified abietic acid as an emulsifier*

All 13

One also

Fifty ml of a four

per cent emulsion of each oil were sterilised in 15>0 ml Erlenmeyer flasks
at 15 pounds steam pressure for 15 minutes and two gm of heat-sterilised
iron chips was added to each flask*

Each flask was then inoculated with

0*1 ml of the mixture and incubated at room tenperature (23 to 27*C)*
Plate counts were made immediately and at Intervals up to 21 days*

r«r

£

j-

Lg« 1

Diagraeatic sketch of single tinit for growing bacteria in soluble oils.

-0l6—

The above work was repeated using only U
nique*

oils and a different tech­

When the work was repeated, the apparatus was designed to eliminate

seme of the variables such as aeration and shaking which were encountered
with the use of the Erlenmeyer flask*

The apparatus Shown in Fig* l

was

constructed as follows*
A and B were one-gallon jugs each fitted with a three-holed rubber stopper
which was wired securely*

B stood on a shelf above A*

for coopressed air to jugs A and B respectively.

C and D were inlets

E was a 2$0 ml separatory

funnel containing JO ga of iron chips resting on glass wool located at
point F.

G and H were 7 m

imately 2 man at J*

glass tubes with H being constricted to approx­

The operation of the apparatus was simple.

Compressed

air was used to force the emulsion from A to B and also to start a siphon­
ing action which returned the emulsion from B to A*

With the atop-cock of

funnel E closed, conpressed air was introduced at C and this forced the
emulsion through tube G into flask B until flask A was emptied*

To eapty

flask B , conpressed air was introduced at D and this started a siphoning
action.

As soon as the siphon was started, the stop—cock of funnel E was

opened allowing the emulsion to return through the iron chips F into flask
A*

The apparatus shown in Fig* 1

was set up in groups of three, each

member of the group being interconnected by air lines*
which is shown in Fig* 2

The thole apparatus

consisted of 12 units set up in four groups*

Aseptic precautions were observed in sterilising, assembling and main­
taining the system*

The apparatus was sterilized by* filling for 2U hours

with a It 1250 solution of Roccal (high molecular alkyl dimethyl benzyl—

ammonium chlorides) and was flushed out several times with tap water im­
mediately before filling with soluble oil emulsion*

The soluble oil emul­

sion was a four per cent emulsion in tap water prepared in gallon Jugs
which had been sterilized with a 200 p*p*m* chlorine solution*

The air

supply was filtered through sterile cotton, a method which was found, by
occasional checks, to remove all microorganisms*
After filling the apparatus with the emulsion, the emulsion was inl­
inediately inoculated with two ml of an emulsion which consisted of a
mixture of 20 samples obtained from industry*
the beginning and at intervals thereafter*

Plate counts were made at

The emulsions were circulated

twice daily but occasionally a day was missed*

However, the emulsions

were always circulated twice immediately prior to removing samples from
E for plate counts, and all were treated exactly the same to minimise
variables*
Thirteen pure cultures were isolated from three smaples of emulsion*
They were inoculated into flasks containing *>0 ml of sterile emulsion
made with a single oil and incubated at room temperature*

Plate counts

were made imediataly after inoculating and at intervals thereafter*
The possible sources of contamination of soluble oil emulsions were
investigated by introducing probable contaminants into soluble oil emul­
sions*

The contaminants were river water, sputum, human feces, unfiltered

air, floor sweepings and sludge from an oil pit in a local factory*

Wash­

ing the hands in the emulsion was not tried as a source of inoculum be­
cause most of the above materials would probably be on the hands at some
time*

The oil emulsions were circulated in a system shcmm in Fig# 3
was designed to simulate industrial conditions#

which

This entailed aeration

of the emulsion by means of compressed air and circulation of the emulsion
through iron chips#

A single unit of the apparatus is shown in Fig# 3

and a photograph of the whole apparatus is shown in Fig#

4

#

Details of

the apparatus are as follows:
A
D
C
D
3
F

—
—
—
-

G
H
I
J

—
—

Air inlet#
Screw clasp for adjustment of air
flow#
Meted, screw cep#
Oil emulsion flow tube#
Gooch funnel set in screwcap C,plugged with cotton#
Rubber band encircling the funnel E to keep it from
slipping through the hole#
Three grams of iron chips ($0 chips per gm)#
Stainless steel wire screen#
Gallon bottle containing ^600 ml of two per cent oil emulsion#
Water manometer#

Aseptic precautions were observed in setting up the many units of
the system#

The gallon bottles were

with 200 p#p#m# of chlor­

ine for U8 hours and washed thoroughly with hot t«p water immediately be­
fore use#

The iron chips and the screens were sterilized in a hot air

oven and all of the other apparatus was sterilized in the autoclave#

The

conpressed air sapply was filtered through a sterilised adsorption column
packed with cotton#

It was then bubbled through sterile distilled water

to decrease moisture loss in the emulsion caused by the aeration#

Ai \

holes in the cap C were sealed with melted paraffin after the necessary
glass tubing and funnel had been installed#

The Gooch funnels* C * were

plugged with cotton except for flask No# U which was exposed to aerial
contamination#
and emulsion#

Flask $ served as a control on the sterility of equipment

V'*

^.... .

Fig. J

Fig.

i• •

Dl.graa.tie ak.teh or . i n * . unit for growing bacteria m

k

TOlubl. oil#.

Photograph or imltipl. unit. oT th. aingl. unit *own in Pig. 5.

-1 9

The emulsions, containing two per cent of a soluble oil* were made
with tap water except in flask No* 1 where river water was used*

The in­

oculum was placed in funnel £ and the air was adjusted to circulate the
emulsions in the various systems at approximately the same rate*

Circu­

lation was carried on for hS minutes to distribute the organisms before
the initial samples were taken for a plate count*
Plate counts were made at 0, 9, 19, UJ, 86, 161, J53 and 780 hours
using a beef extract-peptone agar containing two per cent agar instead
of the customary 1*5 per cent agar*

This modification was alloyed in

order to inhibit the spreading which frequently occurs when soil organ­
isms are grown on agar plates*
scribed phosphate buffer*

Diluent eaployed was the previously de­

Plates were incubated for 72 hours at ^0*C*

The ability of bacteria to grow in emulsions in machine shops was
investigated in two different types of distributing systems in a local
automobile factory*
"B**

These systems toill be designated as system "A" and

The fresh emulsions in these systems were not inoculated but were

seeded by bacteria left when the pits were cleaned out*

System A was

filled with a two per cent emulsion of a petroleum oil emulsified with
petroleum sulfonate, and system B contained a translucent solution, poss­
ibly a soap-type coolant*

A sample of amulsion from System A, taken when

it was being cleaned out, contained 16,^00,000 bacteria per ml, and a
sample of sludge in the bottom of the pit contained 710,000 bacteria per
gm*

Samples were plated using methods described in Part I*

-20-

Samples from eight machine shops In different localities were ob­
tained and plated to make a survey of the bacterial populations in emul­
sions used in industry*

The oils used to make the emulsions had been

manufactured by several different companies and the emulsions had been
in use for various lengths of time before plating*

Thirty-six samples

■were plated by the methods previously described in Part I*
Results and Discussion
The growth curves of mixed cultures in -the 13 soluble oils run in
the Erlenmeyer flasks were very similar to the growth curves obtained in
the more conplicated apparatus*

Moreover, there was very little differ­

ence in the growth curves obtained in the emulsions of the 13 soluble
oils irrespective of the oil used*

For this reason only three curves,

representative of the group, are shown in Fig*

5

* Although

1 three

emulsions contained approximately the same number of bacteria after two
days, emulsions A and B showed greater numbers at three days and emulsion
A was able to sustain a far greater bacterial population than the other
two*
The pure cultures grew well in the soluble oil emulsion*
them did not Increase to at least one million per ml*

The other 12 organ­

isma reached their wayimum populations at various times*

Growth curves of

three cultures, representative of the group, are shown in Fig*
Figs*

5

multiply —

and 6

Only one of

6

•

show the extreme rapidity with which these organisms

almost a hundred fold Increase within 2h hours*

Both

LOGARITHM OF BACTERIA PER ML

•

-4

»

LOGARITHM OF BACTERIA PER ML

K*

ft

•jaid

I
:

I

*p«ipi4 s

O
►

fO
P

*

tt

K

w

§
a
?
«

The sources of contamination of soluble oils are shown very well
in Fig*

7

*

The results for sputum* aerial contamination

and control are not graphed because the flask containing the sputum was
sterile at nine hours* the control remained sterile throughout the exper­
iment and the flask exposed to aerial contamination contained no bacteria
up to l6l hours but had 2*600,000 per ml at 353 hours and 395*000 at 780
hours*

Fig* 7

requires little discussion*

sources of contamination are numerous*

It is obvious that the

The greater numbers in the flask

inoculated with feces may be due to the nature of the organisms or the
relatively large amount of organic matter Introduced with the inoculum*
The decline in population with time is characteristic of these or­
ganisms in a closed system*

Under industrial conditions either the emul­

sions are replaced every few weeks or additional emulsion is added each
day to compensate for evaporation of the water and loss of oil which forms
a film on the finished work*

One large system investigated contained

35*000 gallons of emulsion and this required a daily addition of about
1*000 gallons*

It serviced several dozen machines*

The lubricating en­

gineer in charge said that the main reservoir had not been enptied in
more than a year*

Under such conditions* where the emulsion is renewed

continuously the bacterial population tends to remain static*
The growth of bacteria in soluble oil emulsions under industrial con­
ditions is shown in Table
caused

4

*

The translucent coolant in system B

a sligit lag phase and it was not able to support as much growth

as the emulsion in system A*

It was inpossible to obtain a sanple from

system A at the time it was being filled*

L O G A R IT H M

SLUOGE
RIWER WATER
S O IL
FECES
SW EEPINGS

O ---- — -o
# --- —
□ --- —
■ --- —
A— -—

•
a
■

.

4

i >-

IOO

1

200

I

I

I

I

L

300

400

300

600

700

TIME » HOURS

Tig* 7

""

Growth earns of possible contssdnantg in soluble oils*

-22-

Table

4

Increase In bacterial populations
of 2 systems in a machine shop
Bacteria per ml
Days in Pee

0
1

I
2

5
6
7

8
9

10

91,000,000

,

9 000,000

The levels of bacterial populations in J/S industrial samples of
soluble oil emulsions are well demonstrated in the histogram shown in
Fig*

8 * Thirteen of the samples contained between one and 1C million

bacteria per nil and 11 contained from 10 to 100 million*
It may be concluded that bacteria, whether normal to soluble oil
emulsions or from such sources as soil, water and feces, grow with con­
siderable rapidity when introduced into fresh emulsions*

Some soluble

oils maintain a higher level of bacterial population than others, and
there are indications that pure cultures of bacteria, or even mixed in—
ccula from different sources, may attain different levels of population
in the same emulsion*

LO G A R ITH M OF B A C TE R IA P E R M L
Frequency distribution of bacterial populations in 36 samples
of soluble oil evulsions obtained from Industrial sources*

-25PART III.

COLIFORM BACTERIA IN SOLUBLE OIL EMULSIONS
Review of Literature

Coliform bacteria are ubiquitous in nature, and it is, therefore,
not surprising that they have been found in soluble oil emulsions*

Page

and Bushnell ( 41 ) found Bacillus aero genes and Bacillus coli communis
in cutting conpound*

Duffet, Gold and Weirich ( 25 ) found Aerobacter

aerogenea and Escherichia coli in soluble oil enulsions and Weirich ( 59 )
has stated that B* coli is the second or third most frequently found or­
ganism in cutting oils*
Antagonists of coliform bacteria Which are particularly active are
two members of the genus Pseudomonas, Ps* fluorescene and Ps* aeruginosa*
Of these two organisms only the letter has been reported in soluble oil
emulsions ( 2 5 ).
Pa* aeruginosa produces two substances which are antagonistic to
5* coli*

They are an enzyme-like substance called pyocyanase and a pig­

ment, pyocyanin*
The antagonism of Pa* aeruginosa toward E* coli is very pronounced
as the two following examples will demonstrate*

Havens and Dehlers ( 30 )

found that Ps* aeruginosa was always present in the gut of a minnow, Gambusia affinis, and that when this minnow was introduced into polluted
water, the coliform r«g>idly disappeared*
of the Ps* aeruginosa

One loopful of a pure culture

isolated from the minnow, when introduced into a

2U hr culture of E* coli, conpletely destroyed that organism within 72 hrs*

-2l*Rochalx and Vieux ( 44 ) found that If B* pyocyaneua and E* coli were
mixed in equal quantities in nutrient broth, and the mixture serially
subcultured daily they could still isolate both organisms after 13 djys,
but if the tiro organisms nere allowed to remain in the same tube for 15
days, then only Ps* aeruginosa could be recovered*
There are many

other members of the genus Pseudomonas reportedto

be present in soluble oil emulsions but their ability to act antagonist­
ically has not been reported*

However, their ability to so act must be

suspected from a consideration of the previously discussed

workofLee

and Chandler ( 34) and Chandler ( 17 )•
Experimental
These experiments were designed to find out whether the nnormal"
flora of soluble oil emulsions from industrial sources were capable of
acting antagonistically toward coliform organisms*

The inocula were sol­

uble oil emulsions in which either coliform or "normal” organisms had
grown for long periods of time*
(designated No* 3)

As a source of coliforms, an enulsion

used which had been inoculated UO days previously

with about two gm of feces* (See Part II)* It had a total count of
bacteria
6,850,OOCy per ml of which 75 per cent were capable of fermenting lactose
in Endo medium*
from two sources*

Flora "normal" to soluble oil suspensions was obtained
One was from an emulsion (designated No* 7) which had

been inoculated UO days previously with sludge taken from a machine shop
pit*

It contained 270,000 bacteria per ml*

The other was a mixture (des­

ignated M) of soluble oil emulsions obtained from several machine shops*
It contained 81*000,000 bacteria per ml*
The ratio of coliform to "normal" organisms was varied*

In three

experiments the inocula were introduced into fresh* two-per cent soluble
oil emulsions to give approximately the following ratios of coliform to
non-colifonm

Flask A* l:lj Flask B* 10:1; Flask C* 1:10*

In the fourth

experiment the coliforms and non-coliforms were not introduced into fresh
emulsion but 1800 ml of emulsion No* 3 in which coliforms had grown for
UO days was mixed with 1800 ml of emulsion No* 7 in which "normal" bac­
teria had grown for the same length of time*

The initial inoculum of

bacteria per ml of emulsion and the per cent of coliforms are shown in
Table 5

for all four experiments*
Table 5

Initial inoculum in experiments designed to show the
antagonism of bacteria "normal" to soluble oil emulsions
toward coliform bacteria*
Flask

Coliform
per ml

Non-coliform
per ml

Source of
Coliform

Source of
Non-Coliform

Per cent
Coliform

A

2,250

1,950

No. 3

Mixture M

55*6

B

27,500

5,000

No. 3

Mixture M

814*6

C

3 ,1*00

27,100

No. 3

Mixture M

10*8

D

2,600,000

750,000

No. 3

No. 7

77.7

-26-

Total counts of bacteria were made at 0* 7, 17, 26 and 70 days by
the previously described methods, aid coliform organisms were determined
by plating appropriate dilutions of the emulsions with Endo medium*

At

26 days, the most probable number (UPH) of ooliforas was determined In
lauryl sulfate tryptoae broth ( 37 ) to compare the method of coliform
determination with that of the Endo plate method (See Table 6

)? and at

70 days, only the UPN was used because as little as 0*1 ml of the two per
cent emulsion rendered the Endo plates useless for detection of coliform
organisms*
As a check on the Endo plate method 17 colonies which were thought
to be lactose fermenters and 17 which were thougit to be non-fermenters
were fished from the 26th day plating to brilliant green bile lactose
broth fermentation tubes*

Sixteen of the supposed lactose fermenters

produced gas and one did not*

Conversely, of the 17 supposed non—fer—

mentors, 16 did not produce gas in brilliant green bile lactose broth,
but one did*

As a check on the UPN, all positive tubes of lauryl sulfate

tryptose broth from the 70 day determination were inoculated into bril­
liant green bile lactose broth and streaked on easin methylene blue agar*
All were confirmed on these media*
The presence of coliforms in soluble oil emulsions in machine shops
was also investigated*

The 1CPN of coliform bacteria was determined in

22 samples obtained from one plant and the presence of coliforms, but not
the nuober, was determined in eight samples from another machine shop*

-27-

Results and Discussion
The ability of the flora "normal" to soluble oil emulsions to act
antagonistically toward coliform bacteria is shown beyond doubt in Fig*
9

• In all instances the relative numbers of coliforms was very «w>»n

after 26 days*

At 70 days, Flasks A and D contained *00J and *000U per

cent of the total number of bacteria as coliforms while Flasks B and C
contained no colifetnas*
When the coliform and "normal" organisms were inoculated into fresh
emulsion (Flasks A, B and C) both types of bacteria grew well*

However,

even though the absolute number of coliforms per ml increased in «ii
three flasks, the per cent coliform increased only in Flask C and in
this flask only up to 17 days*

In Flasks A and B there were very rapid

decreases in the relative numbers of coliforms*
The antagonism appears to be a one-eided antagonism in the sense of
Wakeman ( 58 )•

Regardless of the ratio of oolifarm to "normal" organ­

isms at the beginning, the organisms Which were considered "normal" to
soluble oil emulsions were able to suppress the coliforms*
A mixture of equal volumes of emulsion No* 3 an^ No* 7 was contained
in Flask D*

Emulsion No* 3 had been inoculated UO days previously with

feces, and emulsion No* 7 had been seeded with bacteria from a machine
shop source for the sane time*

As can be seen from Fig*

9

, Flask D,

there was very little increase in total count and none of this was due
to coliforms*

On the contrary the coliforms decreased rapidly*

7f

90

PC

f

TO

4
SO
40

4

I

TO

I

COt/FORM

90

so
40
30

9

PERCFAE

AMR Ml

FJLASM

LOtARMM

OF BACTFR/R

9

10

£0

r/M£,DAYS

I«

4

70
60

VJ

S

SO

K.

4

90

9

I

40
30

ai

to

to

t

COLIFORM

r

90

9

PERCENT

LOfAR/FFM

OF BACTFRIA PfR Ml

^

to

AO

to

90

40

60

r /M £ , D A Y S

L06A A /T N A S O Y TO TA L M m a C *
LO G A A tTM K O f C Q U fT Q e#

O -

P£A C£*r COJAOAM

C r-

ig* 9 Antagonism ot bacteria "normal" to soluble oils towards coliform bacteria*

-28
A comparison of the MPN method and the Endo agar plate
method for determining coliform organisms in soluble oil
emulsions is shown in Table 6

*

Table 6
Flask

Bacteria per ml
Endo

U.P.N*

A

1,900

UJO

B

70,000

1*,J00

c

2,600

1*30

D

1*6,$00

25,000

In all instances the Endo medium gave a higher count than the M.P.N.
This would indicate a supression of the coliform by the "normal" flora
even in lauryl sulfate tryptose broth*
The presence of coliform organisms in soluble oil emulsions obtained
from industrial sources tends to substantiate the results of the experi­
ments concerned with antagonisms*

Of the 22 samples examined by the

M*P*N* method, only 11 contained any coliforms and these were present
only in very small numbers as is shown in Table

7 *

-29
Table 7
Coliform bacteria in industrial samples of soluble oil emulsions
Total Bacteria
per ml

1
3
5
6
Hi
15
17
18
19
20
22

2,350,000
65o,ooo
300,000
11,300,000
105,000
7,650,000
5,575,000
i7,5oo,ooo
6,870,000
li3,Uoo
20,510,000

Coliforms
per al

Per cent
Colifarms

2,500
250
1*5
92
Ui.7

•1
•oil
•0005
*002
.01
*003
•2
•005
.001
5.8
*0005

CM

Sanple
Number

10,000
920
92
2,500
92

The presence but not the number of coliforms was determined in eight
samples of emulsions obtained from one factory*

The eight sampless in­

oculated into lauryl sulfate tryptose and brilliant green bile lactose
broths, all gave presumptive evidence, and this was confirmed for the
lauryl sulfate tryptose medium by streaking on easin methylene blue agar*
It may be concluded that coliform bacteria are capable of growing
in the presence of bacteria normal to soluble oil emulsions when both are
introduced into fresh emulsion at the same time*

But the normal flora

acts antagonistically towards the coliform and, regardless of the ratio of
coliform to non-coliform bacteria at the beginning, they cause the c o n ­
form to die out within a few weeks to a few months*

-5 0 -

PART IV.

DERMATITIS AND CUTTING OIIS
Review of Literature

The history of cutting fluids is paralleled by a history of Indus­
trial dermatitis.

Schwartz ( 49) has stated that a study by the U.S.

Public Health Service showed that 13.8 per cent of lil,628 cases of occu­
pational dermatosis were due to petroleum oils and Weslager ( 61 ) has
said that 6$ per cent of all occupational diseases reported are caused
by or related to dermatosis.

This would indicate that approximately 12

per cent of all occupational diseases are due to petroleum oils.
Annually, one per cent of the workers in the basic industries suf­
fer from dermatitis at an estimated total cost of $200 per case (19 ).
The contribution of cutting fluids to the overall cost of industrial
dermatosis is considerable.
The Committee on Healthful Working Conditions, National Association
of Manufacturers ( 1 9 ) defines industrial dermatosis as any disease of
the skin due to industrial work, and they call dermatitis any skin dis­
ease which results in an inflammation cf the skin.

Dermatitis is the

most frequently encountered of all skin diseases.
The dermatitis found bn workers exposed to cutting fluids may be of
two general types, the non-infect^3Ms§id the infect^.P* s*The former, may­
be in the fora of a vesicle, and this may, at a later stage, become in­
fected.

Or dermatitis due to infection may result from the entrance of

pyogenic organisms into minute abrasions, skin cracks or malfunctioning
hair follicles.

-51'

The research staff of the E.F. Houston Company ( 32 ) recognized
four types of infection which involved especially the extensor surface
of the arms and perhaps the backs of the hands and the skin between the
fingers.

They were: (a) A leathery condition of the skin showing come­

dones (black heads) thickly dotted over the skin's surface; (b) Red
blotches on the skin varying in size from 1/6U inch to a quarter-of-e—
dollar, which became elevated; (c) Progression of the red blotches to
pustules; and (d) The true furunculosis or boil stage, usually exhibiting
several lesions.

Schwartz ( 47 ) stated that the acute stages of indus­

trial dermatitis were characterized by erythema, edema, papules and ves—
iculation, tut that pus formation and parasitic infection might entirely
change the picture of the original disease.
Dermatitis of persons exposed to cutting fluids may be caused by
one or more of the following: (a) primary irritants, (b) sensitization,
(c) folliculitis, (d) defatting of the skin and (e) cutting and punctur­
ing the skin.
Primary irritants in cutting fluids may be of several types.

Adams

and co-wrorkers ( 1 ) lune found that several chlorinated hydrocarbons
and even non—chlorinated petroleum cils can cause acneform dermatitis in
man, and they believed that these coopounds might be the cause of re­
lated papular and warty eruptions.

Schwartz and Barlow ( 50 ) stated

that workers exposed to the mists of chlorinated catting oils developed
lesions, which resembled chloracne both clinically and microscopically
on their face and other parts of their body.

Schwartz (48

) stated

-32-

tliat the chlorine or sulfur content of cutting oils may be sufficiently
high to irritate the skin; or that hydrogen sulfide or sulfur dioxide
formed by the heat of cutting operations might cause dermatitis*

He

did not believe (49 ) that alkaline coolants such as solutions of soaps
and/or sodium carbonate -were often the cause of dermatitis.
Disinfectants have been occasionally incriminated as primary irri­
tants.

Schwartz ( 49 ) stated that excessive amounts of germicides

which contain phenol or formaldehyde are more conducive to dermatitis
than the bacteria in the oil, and he reported ( 48 ) finding as much as
five per cent of a phenolic disinfectant in cutting fluids.

Duckhaa

( 24 ) also blamed excessive concentrations of germicides in cutting
fluids as causes of dermatitis.

Duckhan did not believe that petroleum,

per se, caused skin trouble because in 1*0 years he never found a single
case of skin irritation among workers in crude oils in the oil fields or
in persons handling fuel oils.
Sensitization causes trouble only among susceptible individuals,
whereas primary irritants are likely to affect all workers equally (19 ).
Sensitization, leading to allergic dermatitis, only occurs when an in­
dividual 's skin is supersensitive to a particular substrate even in weak
concentration.

During the sensitization period there is usually no derm­

atitis, but skin disorders appear on contact subsequent to the sensitiza­
tion.

Schwartz ( 49 ) stated that such conpounds as phenols, cresols and

nitrobenzene may act as sensitizers and cause allergic eczema.

In higher

-33
concentrations than are present in cutting fluids, they would function
as primary irritants*

Lee and Chandler ( 34 ) did not believe that

dermatitis was generally of the allergic type*
Acne end folliculitis due to the plugging of the sebaceous glands
and the ducts supplying the hair follicles is thought to be one of the
most common causes of dermatitis among workers exposed to cutting fluids
( 21 )• The resulting irritation furnishes conditions suitable for in­
vasion by infectious bacteria*

Schwartz ( 49) has stated that these

types of dermatitis occur especially where oil-soaked sleeves and trou­
sers contact the skin*

Folliculitis occurs more frequently on hairy

workers than on workers with little hair*
Defatting of the skin by the solvent action of the cutting fluid
has been mentioned as a factor contributing to dermatitis (31 ,40, 6 )#
The defatted skin cracks more readily thus allowing bacteria to enter*
As a result, people with dry skins are less resistant to cutting fluid
dermatitis than people with oily skins*
The cutting and puncturing of the skin by metal particles in the
cutting oils or in dirty towels and cotton wastes lays it open to infec­
tion (

49, 32, 21, 48, 40, 26

)•

Besides the dermatitis arising from exposure to cutting fluids,
there is also dermatitis which results from the harsh cleaning methods
used by some workers to remove the cutting fluids from their bodies*
Such treatment as washing hands in gasoline or kerosene, use of sand
soaps or high-alkali soaps, bleaching powders, and other harmful agents
often results in dermatitis which is attributed to the cutting fluids*

-3U-

Bacteria responsible for dermatitis among workers exposed to cutting
fluids have been the subject of some Irresponsible comment*

Some workers

have blamed the indigenous flora growing in soluble oil enulsions for in­
citing dermatitis9 and others have postulated that contaminated water
used for making enulsions has carried the responsible organism*

However*

Schwarts ( 48 )» former Medical Director of the United States Health Ser­
vice and outstanding worker in the field of industrial dermatitis* be­
lieves that the most common infective agent in cases of oil dermatitis is
Micrococcus aureus found on the healthy skin or in ordinary boils or in
cellulitis*

Streptococci have been isolated ( 41 ) from lesions in mach­

ine shop workers* but such occurrences are not common*
The role of cutting oils in the distribution of Micrococcus aureus
haw received considerable attention though there is no evidence that
Micrococcus aureus can grow in cutting fluids*

Schwartz ( 48 ) tasc

stated that the United States Public Health Service found no significant
numbers of staphylococci or streptococci in straight cutting oils used by
workers with cutting oil dermatitis* and Davis (20 ) reported that reg­
ular checks in a very large plant over a period of years found pyogenic
bacteria on only two occasions and then only in negligible numbers*

In

contrast to the above opinions* Weirich ( 59 ) stated that streptococci
have frequently been found in grinding compounds, especially in the win­
ter* and mist from grinding may cause sore throat infection*

Albau.gh

( 1 ) believed that cutting compounds were carriers of pus—farming bac­
teria and were responsible for spreading the wound infections from worker

-35-

to worker*

He found 110*000 organisms per gm of oil —

culture of Staphylococcus aureus*
9,40,39

almost a pure

This opinion was shared by others ( 28 ,

)• Shie ( 52 ) also found pyogenic organisms in "vast num­

bers" in the cutting confounds though he found none in the straigit oils*
Guinea pigs inoculated with isolates from the conpound and with cultures
obtained from boils of infected workers displayed identical syndromes*
Disinfection of the cutting compounds with cresol reduced incidence of
boil affliction from five per cent to 0*5 per cent in this particular
group*

Another extensive outbreak of furunculosis ( 39 ) in a machine

shop was curbed by centrifuging the fluid to remove metal chips and pas­
teurizing to destroy the bacteria*

Two years after this practice was

begun* an examination of U00 men failed to show any furunculosis*
Besides dermatitis* a less frequent type of infection may be incited
by bacteria in cutting fluids*

Weirich (59 ) stated that minor conjunc­

tivitis outbreaks occur in machine shops*

He mentioned the possibility

that Bacillus pyocyaneus* growing in oil emulsions* might be the cause of
such outbreaks*

Recently* an outbreak of conjunctivitis in a Canadian

machine shop was attributed to M* aureus * the same strain having been iso­
lated from *>ii cases (38 )• However* no additional information concern­
ing this subject has been encountered*
The methods of control of cutting fluid dermatitis are numerous*
Possibly all are necessary for a well regulated safety program*

-56 -

Scrupulous cleanliness with adequate -washing facilities for -workers
appears to be the most important*

Schwartz ( 48 ) recommended that the

workers be supervised during their ablutions to ensure adequate personal
cleanliness*

He believed that a soap of sulfonated castor oil with two

per cent fatty alcohol sulfate was desirable as a cleansing agent* Harsh
cleaning methods such as washing with gasoline, kerosene, sand soaps $
high-alkali soaps or bleaching powder should be prohibited*

Clean cloth­

ing at frequent intervals and protective clothing such as iiqpervious
aprons to prevent the bocjy from becoming soaked with oil were recommended*
Clean cotton waste and towels must be available in order that workers
need not be subjected to the hazards of wiping their hands with rags im­
pregnated with metal slivers.
Bland protective ointments should be applied to the skin before ex­
posure to cutting fluids.

Susceptible workers should be transferred to

departments where they would not be in contact with cutting fluids*

Per­

sons with dermatitis should be removed from contact with the oils, and
this is especially important where infections such as boils may contami­
nate the oils*

The metal chips and slivers in the cutting fluids should

be removed constantly by centrifugation or settling*
Fluids should be pasteurized frequently or should contain disinfect­
ants in sufficient amount to control the bacteria but not in such concen­
trations that they would act as primary irritants*
Prohibition of expectoration into the cutting fluids by the machin­
ists has been advocated by most workers*

-37-

Exp erimental and Results
The foilerring exp eriments were designed to investigate the effect
of soluble oil emulsions on a few common pathogens $ some of which are
known to cause cutaneous and wound infections*

More specifically it

was desired to find out whether the organisms could grow or could be
adapted to grow in soluble oil emulsions*
Four cultures were obtained from the stock culture collection of
the Department of Bacteriology at Michigan State College#

They were M*

aureus* alpha and Beta hemolytic streptococci and Pseudomonas aeruginosa#
Another culture of M# aureus was obtained from the Ontario Department of
Health#

It had been isolated from several cases of conjunctivitis which

had occurred in a Canadian machine shop ( 38 )* and it was thought that
the organism may have been disseminated in the soluble oil mist which
arises during grinding operations#

To check this possibility* eight

samples of soluble oil emulsions were obtained from the factory in ques­
tion#
Media used were made with Difco products and C#P# chemicals#

The

soluble oil was a petroleum oil with a petroleum sulfonate emulsifier#
Soluble oil emulsions and nutrient solutions were made with tsp water#
Unless otherwise indicated* M/20 phosphate buffer at pH 7*0 was used as
a diluent and brain heart infusion agar was used as a plating medium# Col­
onies were counted after two days at 37*C#
All growth experiments were carried out in 5>00 ml Erlenmeyer flasks
containing 150 ml of solution or emulsion#

Flasks were incubated at room

GROWTH OCCURRED

10

Flow a h N t of tho adaptation of
to proaoneo of soluble oil*

pyogonoa t m

-5 8 -

tenperature (25-27*C) to simulate conditions -which would be obtained in
industry#

Immediately before plating* the flasks were shaken for five

minutes on a reciprocating shaker travelling at 108 strokes per minute.
Inoculation of the four organisms from the Michigan State College
collection into a one per cent soluble oil emulsion and into the same
emulsion containing 0.1 per cent each of peptone and dextrose showed
that the streptococci were quickly destroyed by the medium.

None were

found in the first plating although they were in contact with the oil
less than 30 minutes*

21mc M. aureus was present at the first plating

but not at two days. sOus Ps» aeruginosa, however, not only tolerated
the oil but grew well, after a period ox adaptation, in the emulsion
without added nutrients.

The results are shown in Table 8
Table

•

8

Growth of Ps. aeruginosa in soluble oil emulsion
Medium

Bacteria per ml x 10s
0

2

days

One per cent oil emulsion

1*60

l.U

One per cent oil emulsion
with 0.1 per cent peptone
and dextrose

1*90

14*0

7

days

93
6,000

1U

days

6,187
1*5,000

Since Ps. aeruginosa readily adapted itself to grow in soluble oil
emulsion it was not included in any further experimentation.
The next step was to increase the concentration of nutrients and
decrease the concentration of oil.

M. aureus and the streptococci were

inoculated in 5*7 per cent brain heart infusion with and without 0.1 per

-39
cent of soluble oil*

Table 9

shows the very immediate lethal effect of

as little as 0*1 per cent oil on the streptococci and the lack of inhibi­
tion of M* aureus in the oil-containing medium*
Table

9

Effect of soluble oil on growth of pathogens
in brain heart infusion medium
B
Bacteria per ml x 10;

Culture

Brain heart infusion

Brain heart infusion* oil

0 days 2 days 7 days 0 days 2 days 7 days
M* aureus

970

26,300 90,000 i,oUo

Alpha streptococcus

960

2U»Uoo 8U,000

80

0

0

Beta streptococcus

560

8,100 73,000

30

0

0

10,000 95,000

Further work was concerned only with the two strains of M* aureus*
However, since the two strains responded in an almost identical manner to
.obtained from
the presence of soluble oil, only the results/ ' the culture isolated from
the conjunctivitis outbreak are reported.
To facilitate understanding of the experimental procedure carried
out in adapting M* aureus to grow in the presence of soluble oil a flow
sheet is given (Fig* 10 ) and the results are tabulated in Table

10 *

M* aureus, inoculated from a nutrient agar slant (A) grew well in the
bacteria after
presence of 0*5 (B) but not in 1.0 (C) per cent oil* However, thy growing
in 0*5 per cent oil grew readily when transferred to 1*0 per cent oil (D).
Increasing the oil concentration (E) or decreasing the brain heart infu­
sion (F, 0, H) did not prevent inoculum from D from growing*

Inoculum

from H (0*57 per cent brain heart infhsion and 1*0 per cent oil) grew

-AiO-

readily in the presence of 2.0 per cent oil only if appreciable brain
heart infusion were present.

Growth on nutrient agar slants inoculated

from contaminated soluble oil emulsions could not serve as a substitute
for brain heart infusion (L, M) nor would small concentrations of brain
heart infusion support growth. Sixty_day—old emulsion with organisms
"normal” to soluble oils did not supply the necessary nutrients when
sterilized with the growth medium. However* inoculum from H into media
L to P survived for two days but not seven days.
In a search for M. aureus in eight emulsion samples taken from the
site of the conjunctivitis outbreak Chapman and Stone and M. aureus
media ( 22 ) were used. These media are selective for micrococcus while
inhibiting most other bacteria. However* $ram stains of the organisms
which did grow showed only sporulating rods.

Gram stains of suspensions

washed from growth of these emulsions on nutrient agar plates also gave
no evidence of micrococci. Plating the emulsions with brain heart infu­
sion agar containing 0.02 per cent sodium aside resulted in the growth
of many yeasts* and there was evidence that one sample might contain mi­
crococci. Enrichment of this sample in dextrose azide broth with subse­
quent inoculation on to blood agar plates containing 0.02 per cent sodium
azide ( 46 ) furnished many colonies of gram positive cocci or coccobacilli which were non-hemolytic• Since the strain isolated from the
conjunctivitis outbreak was hemolytic* it was concluded that the non­
hemolytic strain could not have been the cause of this outbreak.

Table 10

Adaptation of M. aureus to presence of soluble oil

Flask
A
B
C
D
£
F
G
H
I
J
K
L
M

N
0
P

Pep cent
brain-heart
infusion
3.7
3.7
3.7
3.7
3.7
1.85
0*92
0.37
1.85
0.92
0.37
0.037
0
0
0
0

Per cent
oil

Other
constituents

0.1
0.5
1.0
1.0
2.0
1.0

•
-

1.0
1.0

2.0
2.0
2.0
1.0
1.0
1.0

1.0
2.0

m

-

bacteria*
bacteria*
old emulsion#*
-

Bacteria per ml x 10
0 days
2 days
7 days

Inoculum
Agar culture
Flask A- 2 days
flask A- n
Flask B- 9 days
Flask D- «
N

n
n
Flask Hn

it
it

n
it

k

9

1.6
0.8
1*30
UC

69
17U
2U
0.59

it

0.59

n
n

n
«
n
n

it

it

11

11

0.59
0.59
0.59
0.59
0.59
0.59

N

it

295
ljllOjOOO

0
1,550
275
205,000

75,000
1U8
6
7
30

0.70
0.80
0.50
0.25
0.60

* suspension of mixed organisms from oil emulsion grown for tiro days on nutrient agar#
** 25 mL of an emilsion which had been inoculated 60 days previously with emulsion from
a machine shop#

1,035,000
920,000
0
3$,000

8,000
220,000
315,000
885
1,250
500
121
0
0
0
0
0

-U2-

Discussion
There hasn't been sufficient work done with Micrococcus aureus to
state definitely whether or not it will grow in soluble oil emulsions
without additional nutrients*

Perhaps fatty oils such as lard oil would

support growth even though the petroleum oils used in this experiment
did not*
It has been conclusively demonstrated that M, aureus is capable of
growing in 0*37 Per cent brain heart infusion broth in the presence of a
soluble oil emulsion containing two per cent of soluble oil and that this
growth takes place readily at room temperature#

It has also been shown

that a strain adapted to the presence of soluble oil can exist in a two
per cent soluble oil emulsion without brain heart-infusion for at least
two days*
The two strains of streptococci were so susceptible to the toxic
action of 0*1 per cent soluble oil that they do not appear to be capable
of withstanding the usual concentrations of soluble oils which may vary
from 2 to 1*> per cent*
The growth of Ps* aerugenosa in soluble oil emulsions confirms the
work of Weirich ( 5 9 ) who isolated this organism from industrial saisples*

-445part

V.

DISINFECTION OF SOLUBUS OIL EMULSIONS

There are two reasons why disinfection of soluble oil emulsions are
of considerable interest. These reasons are health and economic.
From the health viewpoint, proof is lacking that pyogenic organisms
can not be disseminated by cutting fluids* And the work of Shie (52 )
and Albaugh ( 2 ) furnishes evidence that cutting confounds can act as
carriers for Micrococcus aureus* Also, there is no proof that M* aureus
will not grow in at least some of the several hundred different soluble
oils which are available*
Economic reasons for disinfecting the oil are several*

Growth of

bacteria in the emulsions results in acid and hydrogen sulfide production*
The acid must be neutralized frequently at some expense.

If it is not

neutralized adequately, the pH drops to the point where the emulsion
breaks and the whole batch must be thrown out* The hydrogen sulfide and
other disagreeable odors are objectionable to the workers and unless
steps to eliminate such odors are successful, labor-management difficult­
ies occur* Such steps which have had success are aeration of the emul­
sions and frequent pasteurization* Both are expensive as regards initial
cost of installation, and pasteurization is expensive to carry out* More­
over, the efficacy of pasteurization is sometimes in doubt unless very
thoroughly performed*
Disinfection of soluble oil emulsions has been the object of consid­
erable comment* Some workers advocate it with reserve and some think its

value is questionable*

Few think that it is a panacea.

Schwartz ( 49 )

stated that if disinfectants were added they should be added strictly
according to the manufacturer’s specifications.

He said that the germi­

cides were usually either phenolic or contained formaldehyde and that
excessive concentrations of these were more conducive to dermatitis than
the bacteria in the oil.

He cited an instance (48

) of finding five

per cent of a phenolic disinfectant in a cutting fluid.
added from time to time over a long period.

It had been

Ballard ( 9 ) was of the

opinion that disinfectants should be added only on the advice of the
plant physician.

Beard ( 11 ) stated that automobile plants used phenol

type disinfectants in an initial concentration of 1 to U00 and maintained
the disinfectant by adding one gallon per day for each 1000 gallon of
cutting fluid.

The Department of Scientific and Industrial Research in

Great Britain ( 21 ) advocated 0.5 per cent of water soluble disinfect­
ants for emulsions and this has been advocated by others ( 6

).

Varley

( 56 ) stated that cutting oil disinfectants were usually high boiling
cresylic acids in combination with suitable coal-tar hydrocarbons.
recommended a 1 to 200 to a 1 to U00 use dilution.

He

Weirich ( 59 ) stated

that heating the entire body of oil at lUo-l60°F for 5° ®in. would de­
stroy the bacteria, but he cautioned about the dangers of recontamination
and recommended the use of disinfectants which would act under operating
conditions.

Westveer ( 62 ) recommended sodium o-phenylphenate in con­

centrations of 0.1 per cent as an adequate disinfectant in soluble oil
emulsions and the Dow Chemical Company (23 ) recommended 0.1 per cent of

-U5-

either 2, 3* U* 6 tetrachlorophenol, sodium o*phenylphenate or sodium 2,
3 , h» 6 , tetrachlorcphenate#

They stated that the sodium tetrachloro-

phenate could be used alone or in combination with the other tiro but ad­
vised against using it in emulsions which would be in contact with the
workmen# Lee and Chandler (34 ) recommended resorcinol as a suitable
disinfectant and stated that as little as 15 lbs per month in a $00 gal#
tank would prevent spoilage of the emulsion under conditions of use#
Larger quantities were necessary for sterilization#

They found that

coal tar disinfectants would kill bacteria in soluble oil emulsions in
the laboratory although the same concentrations were ineffective in the
factory# Chlorine as a gas or as chlorine water destroyed the bacteria
tut also broke the emulsion#

Iodine sterilized the oil but on re—inocu­

lation, the bacteria grew. Acriflavine in 0.15 per cent concentration
was not effective, and boric acid in high concentrations was also inef­
fective. Copper sulfate broke the emulsion# Liberthson ( 36 ) stated
that formaldehyde was satisfactory for rendering the emulsions stable to
bacteria, but it was subject to volatilization and loss in hot summer
weather#
On the other hand, some think that disinfection was of little value#
Yates ( 63) thought that personal hygiene and keeping the oil free from
contamination were more inportant in the prevention of dermatitis than
heat sterilization or disinfection# Albangh (2

) concluded that chem­

ical disinfection was not practical because of the following reasons:

-1 *6 -

lack of effectiveness of the disinfectant in the emulsion; the disinfect­
ant had to be so strong that it would act as an irritant; objectionable
odors of the disinfectants*

Ke recommended heat sterilization*

Paine

( 40 ) stated that germicides were not too practical because some were
corrosive and phenols were irritating.
ization*

He also recommended heat steril­

Contrary to these beliefs, Shie ( 52) found that disinfection

of cutting oils with cresol reduced infection of workers by more than 90
per cent*
Methods of laboratory testing the effectiveness of disinfectants in
soluble oil emulsions have not occupied the attention of many workers*
Lee and Chandler ( 34 ) used plate counts to determine the effectiveness
of added disinfectants*

Westveer (62

) inoculated 0*1 mi of a 21* hr

culture of an organism isolated from soluble oil emulsion into $0 mL of
emulsion which contained the disinfectant*
at 37 °C, plates were streaked*

After incubation for 21* hrs

No growth on the plates after 21* hrs in­

dicated an adequate concentration of the disinfectant*
Experimental
The experimental work reported here was designed first to find a
suitable screening method for eliminating useless compounds: and then to
subject the compounds which had appeared suitable in the screening test
to conditions similar to those obtained under actual use*
The screening method was devised after considerable work with changes
in oxidation-reduction potentials of mixed cultures growing in soluble oil

-4*7

emulsions#

The oxidation—reduction potential curves were followed with a

Model G Beckman potentiometer using platinum electrodes and a calomel
half cell, ( 55 )• The experiment was set up in the following manner*
Five-hundred ml florence flasks were filled to within two inches of the
top of the neck with a four per cent soluble oil emulsion and three gm
of iron chips were added*

The flasks were then inoculated with one ml

of a mixture of several soluble oil samples obtained from industrial
sources*

Then a platinum electrode and a potassium chloride agar bridge,

held in a rubber stopper, were put into the flask, and Eh readings were
taken*
The potential dropped 51*0 mv in two days changing from +53^ to -205
mv with respect to the hydrogen half cell* When methylene blue thioqyanate was used in the concentration recommended for testing milk ( 3 )
there was a change in redox potential from +310 mv to -90 mv and the
methylene blue thiocyanate was reduced to the colorless state* There
was little change in the above values after six days indicating a pois­
ing action of the methylene blue# However, this poising action was in­
sufficient to interfere with reduction of the indicator*
When methylene blue thiocyanate is used in dairy bacteriology, one
ml of a 1:200 concentration is added to 10 ml of milk, and the approxi­
mate number of bacteria per ml of milk can be determined by the length
of time required to reduce the methylene blue to its colorless or leuco
form* However, when methylene blue thiocyanate was added in the above
concentration to twelve emulsions, which had been inoculated 11 days pre—

-usviously with a nixed inoculum from machine shop sources, there was no
appreciable reduction after 2.5 days even though these emulsions con­
tained from several hundred thousand to a few million bacteria per ml.
In these experiments tubes containing 0*3 gm of iron chips to 5 ml of
emulsion were used as well as tubes which contained emulsion only.

Ex­

perimentation eventually showed that if a sterile emulsion containing
methylene blue and iron chips were inoculated with a mixture of organ­
isms normally found in soluble oil emulsions, the methylene blue would
be reduced, but, if no iron were present, reduction would occur slowly
or not at all*

In this work the methylene blue test was not used as an indicator
of the number of bacteria per ml of emulsion. Rather, it was used only
to indicate whether or not growth of an inoculum was taking place in an
emulsion which contained a disinfectant. If the methylene blue were re­
duced, the disinfectant was considered ineffective.

If no reduction oc­

curred, the disinfectant appeared to be useful in the concentration used
and further experiments were carried out. In short, it was used only as
a screening test.
Details of the test were as follows:
The culture tubes, 16 mm x 150 mm, containing about 0*5 gm of cast
iron chips (approximately £0 chips per gm), were plugged with cotton and
sterilized in a hot air oven.

-U9-

Methylene blue thiocyanate solution was prepared by adding one tab­
let to 200 ml of sterile distilled water which had been heated to boiling
immediately before addition of the indicator*
The soluble oil used was an oil emulsified with petroleum sulfonate*
A four per cent emulsion of this oil was made with tap water and steril­
ized in the autoclave at 15 lb pressure for 20 min*
The inoculum was 0*5 ml of a mixture of soluble oil emulsions ob­
tained from industrial sources*
The disinfectant was in a one per cent solution made by dissolving
0*1 gm of the disinfectant in 10 ml of four per cent soluble oil emulsion
contained in a dilution bottle*
Ten ml of the autoclaved emulsion was pipetted aseptically into each
tube and this was followed by one ml of the methylene blue thiocyanate
solution.

Then the disinfectant solution was pipetted into duplicate

tubes to give concentrations of 0*1, 0*05 or 0,01 per cent*
The tubes containing the disinfectants were held for one week at
room tenperature with occasional shaking before they were inoculated* and
then one tube was inoculated with 0*5 ml of the inoculum while the dupli­
cate tube was held as a sterile control*

This control was necessary be­

cause occasionally a disinfectant reduced the methylene blue* A second
control was always used to check the organism and the method* This was
done by inoculating a tube which contained no disinfectant*

-5 0 -

The tefets were held at room temperature (25 - 27°C).

The methylene

blue in the inoculated control, which contained no disinfectant, was re­
duced in one to one and a half days*

Disinfectants which inhibited

reduction for one week were considered for further tests#

If the c^e was

reduced by the disinfectant (sterile control) then plate count methods
were used to study the disinfectant#

In this work, although 0#5> ml of a mixed growth in soluble oil emul­
sion was used, it was found that as little as 0«1 or as much as 1«0 ml
would work#

The age of the inoculum and the exact number of bacteria

per ml was of little consequence:

emulsions stored several months in the

refrigerator served very well as inoculum# Pure culture studies with 90
different organisms showed that 92 per cent of them reduced the dye to
same extent under the conditions of the test but only 35 per cent of the
organisms brought about couplete reduction#

However, when a mixed inocu­

lum from industrial sources was used, reduction was always complete and
rapid#
The promising disinfectants were subjected to more severe tests#
These tests, designed to simulate conditions of use in industry, included
aeration of the emulsion containing the disinfectant and circulation of
the liquid through iron chips# Also, the emulsion was subjected to re­
peated inoculation with contaminated soluble oil emulsions obtained from
industrial sources#

-51-

The test was run in the following manner:
in Fig* 3

The apparatus described

was filled with 3600 ml of two per cent soluble oil emulsion

and sufficient disinfectant was added to furnish the concentration re­
quired*

The emulsion was circulated for one week and then inoculated

with 10 ml of a mixture of contaminated soluble oils obtained from in­
dustry®
Plate counts were made on the inoculum to determine the resultant
concentration of organisms in the flask*

Plate counts were also made

one day after the initial inoculation using 0*1 n£L of the emulsion di­
rectly and decimal dilutions to 1:100,000,
made at 10 and 18 days*

Further plate counts were

Those emulsions found to be sterile at 18 days

were reinoculated at 22 days with 10 ml of the inoculum and a final plate
count was made at 29 days*

In a few instances plate counts were also

made at UU days®
Results and Discussion
Many compounds were screened by the methylene blue reduction tech­
nique , but only a few were considered sufficiently effective to be tried
in the circulation apparatus®
Compounds which failed to inhibit reduction of methylene blue under
conditions of the test when used in 0*1 per cent concentration were or­
thophenyl phenol and its sodium salt; 2, h* 6 trichlorophenol; chloro-2—
phenylphenol; 2 chloro-4i-phenylphenol; 2 bromo—U.-phenylphenol; 2 , $, U>

6- tetrachlorophenol, technical grade, and its sodium salt; pentachlorophenol and its sodium salt; mixture of H-chloro- and 6-chloro-2-phenylphenol; parachlorometaxylenol; dehydroacetic acid and its sodium salt;
trichloroacetic acid; sodium benzoate; methyl parahydroxybenzoate; paracresyl benzoate; benzoyl peroxide; alpha alanine; beta alanine; sorbic
acid; raalonic acid; thiourea; sodium tetraborate; hexylene glycol; Hycol
(mixture of phenols, coal tar neutral oils and soap sold by the Creolin
Co*, N.J.); octyl phenol; nonyl phenol; dinonyl phenol; dodecyl phenol;
orthobenzyl parachlorophenol; succinchlorimide; 1, 3-dichloro 5>, 5-dimethyl
hydantoin; dodecylamine; tetradecylamine; U, U—dichlorobenzophenone.
The following compounds appeared promising: 2,

5* trichlorophenol

and its sodium salt in 0.1 per cent concentration; parachlorometacresol
in 0.1 per cent concentration; nitromersol (U-nitro anhydro-hydroxy - mercuri ortho eresol) in 0.01 per cent concentration; mixture of sodium salt
of 2-raercaptobenzothiazole (58 per cent) and lauryl pyridinium salt of 2
mercapto thiazole (U2 per cent) in 0.05 but not 0.01 per cent concentra­
tion; bis 3 > 5>j 6-trichloro-2 hydroxy phenyl methane in 0.1 per cent con­
centration; and chloramine T and dichloramine B in 0.1 per cent concentra­
tions.
Several compounds were tried but were abandoned for various reasons.
with anionic constituents of soluble o
Quaternary ammonium compounds were incompatible' as was expected. Conpounds
of iodine such as potassium iodide and biniodate broke the emulsion as did
copper acetate. Some conpounds were not soluble.

The confounds -which appeared promising in the methylene blue test
were subjected to the circulation test*
test are shown in Table 11

The results of the circulation

*

It is obvious that ability of the disinfectant hr© inhibit reduction
of methylene blue did not necessarily mean that the disinfectant would
be suitable under more severe conditions.
amine T in 0.05, and

As shown in Table 11

Chlor-

S» trichlorophenol in 0*1 and G.C5 per cent

concentrations had little or not inhibitory effect on bacterial growth*
Dichloramine 3 and nitromersol caused immediate decreases in the bacter­
ial populations, but their action was not sustained*

Farachlorometa—

cresol in 0*05 per cent concentration killed the initial inoculum but
not the inoculum which was added at 22 days*

Bis

St

6 , trichloro, 2

hydroxy phenyl methane gave sustained killing action in 0*1 per cent
concentration and inhibited growth even in 0*05 and 0.01 per cent con­
centrations*
The use of a disinfectant in soluble oil emulsions is governed by
many factors.

Besides being effective under the conditions of use, it

must be non-toxic to the workers and inexpensive*

It must be long last­

ing or else an easy analytical method must be available to the plant
chemist in order that he can maintain the desired concentration of dis­
infectant*

Of the confounds tested only bis $, 5, 6 trichloro, 2 hydroxy

phenyl methane was effective for a long period of time.

Since this com­

pound is a conponent of toilet soap, it must be suitable from a toxicological viewpoint*

If its cost were not prohibitive, it might be valuable

-suin industry*

Parachloromet acre sol might have limited activity under con­

ditions of use*

Hie others did not appear of much value riien tested by

the above methods*

"55Table 11
Effeot of disinfectants on the bacterial populations of
soluble oil emulsions aerated and circulated through iron chips
Per cent
cone#

Disinfectant
Nitromersol
Chloramine T
Parachlorometacresol
Dichloramine B
2,1*,5 trichlorophenol, sodium salt
n
n
it
n
bis 3,5*6 trichloro, 2 hydroxy
phenyl methane
n
it

Control-no disinfectant

.05

Bacteria per nil x IQ3
^0 day* 1 day 10 days 18 dqys 22 days* 29 days 14* days

.05
.05
.05
.1
.05

15
15
15
118
118
118

•l

118

.05
.01
-

2.8
2.3
2.8

0
38
0
6

950
0

25
930 6,1*00
6,700 7,000
0

0

mm

0

260
3*Uoo
3,000
0

0
123
8.7
2,700
680
l*5o
5,700 U,900
l*,l*oo

* Inoculum added at these days#
- No data obtained
^ The various compounds were investigated in three different
groups as can be seen by the bacteria per ml at 0 days#

260

m
-

-

-

0.1

13

60

•»

m

mm

-

-

-

-

at

80

0

-

-

30

-

-

135
i,5oo

-

-56-

PART VI.

NUTRITIONAL STUDIES OF THE BACTERIA IN
SOHJBIE OIL EMULSIONS
Review of Literature

The chemical composition of soluble oils may be extremely variable
because the molecular structure of the petroleum component is unknown:
and the petroleum may be made water soluble with a large number of emul—
sants whose chemical composition may or may not be known. Also many of
the soluble oils contain a coupling agent and a water softener.
The oil portion of the soluble oil may be either a petroleum oil, a
fatty oil, or a mixture of the two. The petroleum oil may be a paraffin,
a napthene, or a mixture of the two, while the fatty oil may be lard oil
or one of several vegetable oils.

Both types of oil may be untreated or

they may be treated with such materials as sulfur or chlorine. Even the
method of treatment may vary, e.g. lard oil sulfurized with flowers of
sulfur by a hi^i temperature, short-time process replaces hydrogen with
sulfur while a low temperature, long time process results in sulfur add­
ing at the double bond ( 45 )•
The confounds used for emulsifiers and stabilizers Are numerous.
Schwartz and Perry ( 51 ) stated ^The usual emulsifying agents for soluble
cutting oils include the alkylolamine soaps of fatty acids, rosin acids,
and naphthenic acids, the mahogany sulfonates, and the sulfated oils.

-

57 -

The3e are most often used in mixtures rather than singLy, and the mahogany
sulfonates are probably used in larger quantities than the other products
mentioned.

Other emulsifying agents that have been used are the Igepon A.

class of sulfonates, the soaps of carboxylic acids from oxidized paraffin
wax, and the polyethylene oxide type of non-ionic agent.’1 Besides the
above-mentioned emulsifying agents the sludge base extracts from the acid
refining process of petroleum ( 2 8 ) and tall oil ( 13 ), a by-product of
the sulfate process for making wood pulp, are often used.
In addition to the emulsifying agents most soluble oils contain a
coupling agent or mutual solvent to aid in solubilizing additives.

Such

coupling agents may be glycol ethers, terpene solvents, butyl and amyl
alcohols, etc. (51

)•

Obviously the composition of any soluble oil emulsion is so complex
that it eludes exact chemical definition.

Perhaps a combination of the

definitions of Schwartz and Perry (51 ) and Philipp ( 42 ) are as com­
prehensive as we can expect.

Schwartz and Perry stated ’’Soluble oils are

usually mixtures of oil and a surface active agent together with a blending agent or mutual solvent which serves to give a homogenous prepara­
tion", and, concerning the emulsion, Phillips sajLd, "Der Qrad der Emulgierbarkeit erstreckt sich vom volligen Klarbleiben beim Verdunnen mit
beliebigen Mengen Wasser uber das Opalisieren der Losung hinweg bis zur
Bildung einer milchigen Suspension von grosserer oder geringerer Best-^nxutdigkeit, die also mehr octer weniger zum Aufrahmen neigt”.

-58-

The gronrrth of bacteria in soluble oil emulsions has been demonstrated
but there has been little attempt to study the nutritional factors affect­
ing such growth.

Lee and Chandler ( 34 ) found that the bacteria isolated

from soluble oil emulsions (presumably Ps* oleovorans) could grow in
Locke's Basal Solution containing one per cent of crude naphthenic acids
only if a nitrogen source w a s ; added.
nitrate.

They U3ed 0.1 per cent potassium

The bacteria did not grow in a medium containing only carbohy­

drate as a carbon source thus proving that the crude naphthenic acids
acids were utilized by the bacteria as a carbon source*

Lee and Chandler

were unable to concentrate the oil used in their experiments sufficiently
to demonstrate any nitrogen in it but they believed that the nitrogen
source was nitrogenous compounds in the oil itself.

Such compounds have

been discussed by Ellis (27 )•
The debris which frequently finds its way into the soluble oil emul­
sions used in factories may serve as a source of nutrients.

Liberthson

( 36 ) reported finding metal filings, powder, abrasives and such organic
contaminants as tobacco juice, remnants of abandoned lunches and other
putrefieable materials.

And the C*B. Dolge Conpany (16 ) stated that

feces and other body discharges have been found in emulsions taken from
some shops.
Work with petroleum bacteriology, outside the field of soluble oil
emulsions, has been more abundant and fruitful.

Recently it has been

the occasion for two very excellent reviews ( 65 » 66 ) which may be sum­
marized briefly.

-59Contrary to popular belief the ability of microorganisms to attack
hydrocarbons is quite general, over 100 species representing about UO
genera of yeasts, molds and bacteria having been shown to possess hydrocarbonoclastic properties*
Almost all types of hydrocarbons are susceptible, aliphatic, olefinic,
naphthenic and aromatic confounds having been investigated*

Aliphatic

compounds are generally more easily attacked than aromatics and some or­
ganisms attack aliphatics but not aromatics of similar molecular weight*
If dispersed adequately long-chain hydrocarbons are more susceptible to
attack, within certain limits, than short—chain compounds#

Unsaturation

and branching enhance the vulnerability cf the molecule to attack* Nearly
100 pure hydrocarbons ranging in complexity from methane to dibenzanthra-

cene and several hundred mixtures of hydrocarbons have been investigated#
'•The latter include natural gases, ligroins, gasolines, kerosenes, ben­
zines, lubricating oils, fuel oils, paraffin waxes, tars, crude oils,
asphalts, asphaltenes, synthetic and natural rubbers, etc#" ( 66 )*Even
coapounds commonly used as bactericides, eg* phenol, cresols and toluene,
are metabolised in loir concentrations by some bacteria*

Experimental
The experiments described in this section were designed to stuctysome of the nutritional factors involved in the growth of bacteria in
soluble oil emulsions#

Four projects were carried out (a) the effect of

soluble oil concentration on growth curves (b) the effect of added pep-

-60-

tone on growth curves (c) determination of the organic nitrogen content
of soluble oils and (d) the growth of pure cultures in various components
of soluble oil emulsions*
The effect of the concentration of oil was studied by inoculating
emulsions containing 10, 1, 0.1, 0.01 and 0.0 per cent of soluble oil and
following the growth curves.

The medium consisted of the above concen­

trations, soluble oil, tap water and iron chips.

The soluble oil was a

light- mineral oil emulsified with petroleum sulfonates.

Three hundred

ml of the different emulsion concentrations was dispensed into one—litre
flasks and autoclaved at 15 lbs for 20 rain.

To each flask was added a—

septicsilly three gm of oil—free, heat-sterilized cast iron chips.
The inoculum was prepared by growing a mixed culture (mixture of 20
emulsions obtained from industry) on nutrient agar containing one per
cent of a soluble oil, for two days at

The growth was harvested

in M/20 phosphate buffer at pH 7.0* shaken

min at 2H0 strokes per min,

centrifuged, and resuspended in fresh buffer.
carried out three times.

The final suspension was plated, after which

it was refrigerated at 0°C.
cubated and counted.

The washing process was

The plates made from the suspension were in­

Sufficient suspension was then added to each flask

to give a concentration of about 10® cells per ml of emulsion.
The flasks were incubated at room temperature (23 - 27°C) and plate
counts were made at intervals.

Immediately before withdrawing the samples

for plate counts the flasks were shaken at 108 strokes per min for five
min.

-61-

The effect that added peptone had on the growth curves of bacteria
in soluble oil emulsions was determined by growing an inoculum of the
abo^e-flientioned bacterial mixture in a four per cent emulsion of the
above-^nentioned soluble oil containing Difco peptone*
Three runs were made*

In two of them concentrations of 0.5, 0.05,

0.00$ and 0.0 per cent peptone were used.

And in the third run concen­

trations of 0.5, 0.25, o.l, 0.05, 0.01, 0.005, O.OOl and 0.0 per cent
were used*
The apparatus and methods enployed were the same as those used for
determining the effects of soluble oil concentrations on growth curves*
The kjeldahl nitrogen was determined on duplicate samples of four
of the oils by the Kjeldahl-Gunning method (8

). A slight modification

was necessary which used only about 0.5 gm of oil due to the difficulty
of digesting larger amounts of the oil by the described method.

Also,

the ammonia content of one sample of soluble oil was determined.
The ability of bacteria to utilize the various materials which have
been reported to be found in soluble oils was investigated.
were four pure cultures isolated from soluble oils.
in Part 7*

The bacteria

They are described

The soluble oil conponents were six compounds commonly used

as emulsifiers and a light paraffin oil which was representative of those
used in industry as the main component of soluble oils. The emulsifiers
were sodium naphthenate designated as A (about a Uo per cent mixture of
naphthenate in mineral oil); polyoxyethylene sorbitan trioleate (Tween 85),
B; mahogany soap (petroleum sulfonates), C; triethanolamine, D; tall oil

•*
K*
c*

LOGARITHM OF BACTERIA PER ML
0>

&

!
o>
I

i*

«

*
3

01

i.

S'

fe

0

1

3
ft

>1

flD

-62-

soap , E; sodium salt of abietic acid, F*

Besides the components of sol­

uble oils another compound, G, used in place of soluble oils for machine
shop work, was investigated*

G was the sodium salt of an alkylated gly­

cine having the formula RNHCHsCOONa where R is a

to

hydrocarbon

chain*
Three media were used —

tap water; tap water plus C.l per cent of

compound A to G; and tcp water plus 0*1 per cent of compound A to G plus
one per cent of ligjit paraffin oil*

The media were agitated in a Waring

Blendor, dispensed in 10 ml quantities in dilution bottles and autoclaved*
The inocula were the four previously mentioned pure cultures*

They

were prepared by suspending a small amount of growth from a soluble oil —
nutrient agar slant in sterile distilled water*

Each bottle containing

medium was inoculated with 0*1 ml of bacterial suspension to give an in­
itial bacterial population of 102 to 103 cells per ml*

A plate count was

made on tho inoculum in order to determine the bacteria per ml of medium
immediately after inoculation*
The inoculated bottles were incubated at room temperature and shaken
four hours daily on a reciprocating shaker running at 120 strokes per
minute*

After five days the bacterial population of each medium was de­

termined by the plate count method*
Results and Discussion
Fig* 11 shows growth curves prepared from the averaged results of
two identical experiments, both of which showed identical trends*

-63-

X*fc can be seen that an increase in soluble oil concentration resulted
in an increased growth rate*

As shown below the mean generation times

during the first 2h hrs decreased as the oil concentration increased*

Per cent oil

Mean generation time
in minutes

10.0
1.0

173

0.1

2U7

0.01

289

0.0

367

193

The mean generation time of 173 minutes is considerably greater than
that obtained in work with enteric organisms and many other groups of
bacteria in laboratory media®

It would appear that something in the sol­

uble oil itself was responsible for the stimulation of growth because of
the inverse relationship cf oil concentration and generation time but it
is very unlikely that this stimulant was a hydrocarbon*

The oil was very-

well dispersed, globules being generally only a few microns in diameter,
and therefore the hydrocarbon would not be the limiting factor*

The ni­

trogen present in the soluble oil might be the limiting factor because
nitrogen was present to only a very small degree*
The ability of increased concentrations of soluble oil to maintain
increased bacterial populations is apparent from an examination of the
curves for 10, 1*0 and 0*01 per cent concentrations*

The decrease in

A

-

64 -

bacterial population with time found in 0*1 per cent concentration of
olie oil can only be explained by the rapid production of antagonistic
conditions, perhaps pH*

A higher concentration of oil might have ex­

erted a buffering action while a lower concentration would not have
sufficient oil to cause much change when metabolized*
The effect of added peptone is shown in Fig*s* 12 and 13*

Fig* 12

shows growth curves prepared from the averaged results of two identical
experiments and Fig* 13 was obtained by using the bacterial populations
per ml at four days for the above two experiments, and also a third ex­
periment which used eight concentrations of peptones*

In all instances

an increased peptone concentration resulted in maintenance of a greater
bacterial population but this population increase was relatively greater
with small concentrations of peptone*

The data represented in Fig* 13*

when plotted as logarithm of bacterial population against logarithm of
peptone concentration, gave a linear relationship between 0*005 and 0*25
per cent peptone concentrations.

The curve had the formula y - l * U 7 x

where y was the logarithm of the bacterial population at four days and
x

was the logarithm of the peptone concentration in the oil emulsion*
The addition of peptone to the soluble oil emulsion resulted in the

production of foul odors*

At four days oils containing 0.5 per cent pep­

tone possessed very objectionable odors while those containing 0*05 per
cent peptone possessed the same odor but it was less pronounced*

Emul­

sions containing 0*005 per cent peptone smelled like the control which
contained no peptone*

5%

ML

.05%

OF BACTERIA

PER

005%

UOGAftTHM

NONE

Fig. 12

Gbrowth aurraa of alxed cultures in soluble oil containing
Tsrious Mounts of p«ptont«

ml
of b a c t e r ia per

LOGARITHM

* mixed culture grown for four days in soluble oil emulsion*

-

65 -

The kjeldahl determinations of four soluble oils shewed that
contained some amino nitrogen.

1

Oils designated 1, 3, 5 and 9 contained

0.0276, 0.0672, 0.1US8 and 0.1306 per cent kjeldahl nitrogen respectively.
It is interesting to note that growth curves in oils 1, 3 ?nd 5 were veiy
similar while oil 9 supported about
that the other three oils did.

2*5 times the bacterial population

Oil 5 contained the greatest amount of

nitrogen and it was known to. contain triethanolamine. Data of experi­
ments not given here in which eight different concentrations of triethan—
olamine were added to soluble oil emulsions showed that triethanolamine
did not cause an increase in growth.

This may be the reason why oil No. 5»

with the greatest nitrogen content, did not support as much growth as oil
No. 9.

Oil No. 9 did not contain triethanolamine but the kjeldahl nitrogen

which it did contain evidently was available to the bacteria.
The effect of the several components of soluble oils on the growth of
the four pure cultares is shown in Table 12, which is based on the aver­
ages of two identical experiments.

The bacteria per ml of tap water far

cultures 6, 12, 72 and 9U at five days were 15.5*
respectively*

million

The bacterial populations in the mixtures of water and em­

ulsifier or water, emulsifier and oil at five days are reported in Table
12 as percentages of the populations in water.
It can be seen that the four cultures did not grew equally well in
all of the soluble oil components.
cultures 6 and 12 but not 72 and 9U.

Sodium naphthenate enhanced growth of
Tween 85 appeared to cause increased

growth of culture 72 but not the others.

Mahogaqy soap did not affect

-66cultures 6 and 12 to any appreciable extent but it "was toxic to 72 and 9h
when used alone or frith oil*

Triethanolamine -with water, or -with water

and oil had little effect on cultures 6 and 9U but was toxic to 12*

Tall

oil soap alone had little effect on cultures 6 and 12 but when oil was
added to the mixture growth of these two organisms was stimulated*

In

contrast when oil was added to the sodium naphthenate — water mixture
growth of 6 and 12 was inhibited*
culture 72 but it was toxic to 9U#

Tall oil soap had little effect on
Sodium abietate stimulated cultures

6, 12 and 72 but the addition of oil caused little additional stimulation
to cul tux ‘j C and suppressed 12 and 72*
ture 9U*

Sodium abietate was toxic to cul­

'it: compound RNHCH2 COONa had no effect except on culture 12*

Apparently cultures 6 and 12 were stimulated ftor more than cultures 72 and
9h by the compounds investigated ard culture 9h was frequently destroyed

by compounds which were stimulatory to 6 and 12*

-

67-

Table 12
Growth of pure cultures isolated from soluble oil emulsions
in components of soluble oils after five days
Growth expressed in terms of per cent of growth in tap water*

Soluble oil
component

Culture 12
Culture 72
Culture 6
Culture 9b
Emulsifier Emulsifier Emulsifier Emulsifier Emulsifier Emulsifier Emulsifier Emulsifier
+ oil
+ oil
+ oil
+ oil
387

86

208

159

h9

135

96

rween 85

72

19h

1ia

0

a6

0

151

Mahogany soap

Sh

108

1U7

20U

0

0

0

0

Criethanolamine

97

119

0

0

-

-

86

U8

Tall oil soap

1U8

hOk

58

269

U3

7h

0

0

Sodium abietate

175

197

163

86

255

63

0

0

MICH2C00Na

H3

61

226

-

69

7U

1U7

157

Tap water-control

IOC

100

100

ICO

100

100

100

ICO

Sodium naphthenate

0

5 >ioo,ooo

0

9,300,000

rc\
—

15>5oo,ooo

*

CO

<- Bacteria per ml in
tap water-control

99
78

,000

-

PART VII.

68 -

BACTERIA IN SOLUBLE OIL EMULSIONS
Review of Literature

Page and Bushnell

(41) found no obligate anaerobes by

any of the several methods which they used.

They isolated

Bacillus a e r o g e n e s . Bacillus coli c o m m u n i s , and an organism
similar to, but not identical with, Proteus vul garis.

They

found no staphylococci or spore formers.
Lee and Chandler

(34) found a short motile rod with a

single polar flagellum, which did not ferment sugars, liquefy
gelatin, or produce indole.
was hydrolyzed.

Nitrates were reduced and starch

Growth in stab culture was only to about

two-thirds of the depth of the stab.

One of the outstanding

characteristics of the organism was that it produced no
water-soluble

fluorescent pigment though the colonies them­

selves were markedly fluorescent.
present in almost pure culture

This organism which was

in all the machine shop

emulsions wnich they investigated was named Pseudomonas
ole o v o r a n s .
Duffett,

Gold and <*eirich (25) examined the flora of

thirty samples of soluble oil emulsions.

The predominating

colonies on most pour plates produced greenish or reddish
iridescence by transmitted light without the production of
true pigment.

Unlike Lee and Chandler they found several

species and genera contained in single samples of oil.

The

-

predominating flora *®8

69 -

members of the genus Pseudomonas,

Ps. oleovorans and six new species of Pseudomonas being most
common*

They also found Ps * aeruginosa. Aerobacter aerogenes.

Escherichia coll . Bacillus alvel. and two species of uniden­
tified pink-producing gram-negative rod bacteria, as well as
yeasts and molds.

Unfortunately there was no published des­

cription of the six new species and the original experimental
data were not available

(60).

In order to gain additional knowledge of the flora of
soluble oils the following work was undertaken*
Experime ntal
Methods employed for pure culture study were generally
those advocated by the Society of American Bacteriologists

(53).

However, deviations from these methods were occasionally
nece ssary.
Ninety cultures were isolated from the previously
mentioned samples obtained from industries in Michigan,
Illinois, Wisconsin and Ontario, a total of 14 samples being
investigated.

After plate counts had been made on the refri­

gerated samples the emulsion was diluted and quantities plated
in duplicate so as to obtain 30, 40 or 50 colonies per plate.
Colonies were fished then to nutrient agar slopes and after
growth had occurred loop inoculations were made into about
50 ml of sterile water*

This suspension was shaken 225 times

-

70 -

per minute for 30 minutes and then streaked on nutrient
agar plates.

'..'ell separated colonies were fished and the

process repeated until at least two successive platings
showed only one type of colony.

Final isolates were

stabbed into nutrient agar and held in the refrigerator for
identi fication.
All work, except microscopical studies, was done at
least twice.

Gram stains were made at one, two, four and

six days; capsule and acid-fast stains at six days; flagella
stains at 18 to 24 hours and at two days.

Flagella stains

were made by the Leifson method (35).
Colonies on agar plates incubated at 30° and gelatin
plates at 20° were examined at one, two, three and seven days
with the aid of a dissecting microscope

(X43) and at seven

days only with a "black daylight" ultra violet

(U/V) lamp.

Only where marked fluorescence under ultra violet was observed
is it discussed in the results.

Fluorescence by transmitted

light (T/L) was observed on the Quebec colony counter; by
holding the plates or tubes between the eye and a 150 watt
frosted electric light bulb; and by the method of Huddleson (31).
It was necessary, in observing fluorescence,

to move the

investigated material until the light struck it at an oblique
angle because if the plate w*e

on a straight line between

-

71-

the frosted electric light bulb and the eye colonies rarely
appeared fluorescent.

Observations by reflected light

(R/L)

were made under the above-mentioned electric light bulb or
by daylight.
Agar strokes, nutrient broth and gelatin stabs

(10 per

cent Difco gelatin in distilled water) were examined at one,
two, three and seven days.

The gelatin stabs were also

examined at intervals up to eight weeks.
I/iotility medium

(22) was incubated at room temperature,

30° and 37© and observed at one, two and eight days.
tation as well as motility was observed.

Pigmen­

True pigment pro­

duction was observed in many organisms on this medium but not
on other media.
Potato slants,

incubated at room temperature were

observed at one, two,
Peptone

four and seven days.

iron agar, incubated at room temperature, had a

lead acetate paper strip inserted with the cotton plug.
Litmus milk, nitrate broth,

indole test medium, Voges-

Proskauer test medium and citrate broth were Difco products.
All were incubated at room temperature.

Litmus milk was

examined at frecuent intervals up to 28 days; nitrate reduc­
tion was tested at one, two, four,

seven and 21 days; indole

production was tested at one, two and four days; methyl red

-

72 -

and Voges-Proskauer tests, when made, were tn&dae at one, two
and four days.

Citrate broth was examined at one, two, four

and ten days.
All cultures were grown in Uschinsky's solution which
was found by Clara

(18) to be very reliable for elaboration

of water-soluble green pigments and also in Georgia and Poe's
medium.

However, after three weeks at room temperature no

green or blue water soluble pigments had been observed.
Results and Discussion
All cultures except two were gram-negative rods.
were acid fast or capsulated.
99, 101, 103)

None

Five of them (No's. 81, 97,

fermented lactose with the production of acid

and gas and are members of the Enterobacteriaceae.
a gram-positive Uicrococcus
one a Flavobaoterium

One was

(No. 4), one a Sarcina (No. 105),

(NoJ.06) . and one a Vibrio

(No. 55).

The remaining 31 organisms were almost all members of the
genus Pseudomonas because of the presence of polar flagella
although it was not possible to state definitely whether some
of the cultures belonged to this genus.

Twenty of the 81

cultures regularly produced opaque, white daughter colonies
within the translucent,

fluorescent parent colonies and

because it was impossible to secure cultures free from these
variants, regardless of the number of successive platings
employed, they are not described.

No organisms studied could

-

73 -

be classified as Pseudomonas oleovorans because this organism
hydrolyzes starch and the few organisms of our collection
which did hydrolyze starch differed considerably from P s .
oleovorans in other respects.

Cultures No's. 33, 46, 87 and 93

may belong to the genus Phaeomonas proposed by Kluyver and
Van Niel

(33) because of the production of a water soluble

brown pigment.

However, Bergey

which resembles this group.

(14) describes no bacteria

Cultures

(No's. 83, 88, 94)

’
were bright pink in all media tested and this characteristic
was maintained in culture for over a year.
flagellated and produce a yellowish,

They are polar-

florescent, water

soluble pigment in motility medium,Uschinsky's solution and
gelatin stab but this pigment is not noticeable in any other,
media.

Undoubtedly this group and the group of organisms

producing a brown water soluble pigment will be classified
in the genus Pseudomonas if additional work shows that they
are stable.

Cultures No's. 12, 72, 75, 76, 77, 89, 92

reduced nitrate to nitrogen gas and in many ways resembled
ficans.

However,

they differ from this organism

in several respects and actual comparison with P s . denitrificans
will be made in order to determine whether these organisms
may be classified as P s . denitrificans.

Cultures No's. 1, 2,

3, 4, 5, 6, 7, 9, 10 resemble P s . oleovorans but whereas P s .

-

74 -

oleovoraas is described as reducing nitrates and hydrolyzing
starch these organisms produce only slight amounts of nitrite
and that only after prolonged incubation and they do not
hydrolyze starch.

Here again actual comparison with P s .

oleovorans must be employed to determine whether the difference
is apparent or real.
Many of the cultures do not resemble exactly any des­
cribed species and have so few outstanding characteristics
that they can not be grouped together.
18, 21,

Cultures Mo's. 13,

32, 35, 37, 40, 41, 43, 47, 49, 50, 51, 54, 56, 59,

65, 66,
68, 71, 73, 86, 90 are tentatively identified as
^ eudoinonas,,
v
P s , desmolyti cum Gray and Thornton (29), a species which
may or may not reduce nitrates and ferment dextrose.
eudomonas
Culture
Mo. 27 resembles Ps^ dacunhae Gray and Thornton.
Culture

No. 55 resembles Vibrio percolans Mudd and Ti/arren

(14)

but it was non-raotile and no flagella were observed.
Culture No.

82 produced no water soluble pigment in motility

medium and gave no indication of motility.

It is not similar

to any organism described in Bergey and is probably a hither­
to unidentified A c h r o m obacter.
Cultures No's. 11, 15, 16, 17, 49, and 50 which do not
resemble any organisms described in Bergey could be paired
according to their characteristics into No's. 11 and 15;
16 and 17; and 49 and 50.

No's.

11, 15, 49 and 50 possessed

-

75 -

a single polar flagellum and are members of the genus
Pseudomonas.

Numbers 16 and 17 were non-fluorescent, did

not produce water soluble pigment and were not flagellated.
However,

insufficient work was done to determine whether

they belonged in the genus Bacterium.

They resembled none

of the Achromobacteria described by Bergey.
Culture No.

8 resembles P s . oleovorans in many respects

but it turns litmus milk alkaline and does not hydrolyze
starch.
No. 98 slowly produced a small gas bubble in lactose
broth indicating that it may be a coliform.

However, its

predominating flagellation is polar indicating a Pseudomonas.
Further work must be done to determine to which genus this
organism belongs.
No. 100, a member of the genus Pseudomonas, has several
very distinctive characteristics.

It is not described in

Bergey and if a few more cultures can be isolated it may be
named as a new species.

Cultures No

.'a. 102 and 104 are also

Pseudomonas the species of which differ from each other and
also from No. 100.

They,

too, may be new species if further

work and additional isolates substantiate

the work which

has been done already.
In general it may be concluded that soluble oil emul­
sions contain a large number of species hitherto not des­
cribed and that at least five genera are represented.

This

-7 6 -

work confirms the findings of Duffett,

Gold and 7/eirich (25)

and goes further in that it gives accurate and detailed
descriptions of at least a few of the species.

It also

presents several genera not found by these workers*

It

is expected that future work will result in naming these
organi sms.
Cultures No's. 81. 97, 99, 101, 103
These five cultures could be classified as Escherichia
intermedlum

(Werkman and Gillen) Vaughn and Levine.

gave a positive Voges-Proskauer test.

They

On eosin methylene

blue agar all five cultures fitted descriptions of Vaughn
and Levine's

colony types 1 or 2 (57).
Culture No.

Vegetative cells:
mainly in clusters,

44

Gram-positive cocci, 0.6 u diameter,

occasionally in pairs or chains of three

or four cells.
Agar colonies:

Punctiform,

smooth,

entire,

convex,

translucent at one day enlarging to 1.0 - 1.5 mm at two days
and 3.0 mm at seven days.

Colonies are greyish becoming

whitish by seven days.
Agar stroke:

Moderate,

Nutrient broth:

filiform,

Slight clouding.

glistening,

greyish.

Scanty granular

sediment at two days becoming viscid by seven days.

-

Gelatiri stab:

Uniform,

77 filiform growth with slow

saccate liquefaction becoming stratiform.

Three-quarters

liquefied at eight weeks with no sediment.
Motility medium:
Litmus milk:
Potato:

No pigmentation.

Acid by one week but no curd at four weeks.

Creamy growth on one potato,

colorless on

another.
Nitrate:

Nitrates not reduced.

Indole and hydrogen sulfide negative.
Starch not hydrolyzed.
fructose, galactose, lactose,

Acid but no gas from dextrose,
sucrose, maltose.

acid from dextrin at 21 days.
raffinose,

Very slight

No action on arabinose, xylose,

inulin, mannitol, dulcitol,

salicin, aesculin.

No growth in citrate broth and Uschinsky's medium.
Discussion:

Similar to M. candldus but differs with

regard to gelatin liquefaction,

color and size of colonies

and growth on potato.
Culture No. 105
Very similar to KecKg9C3^xdcac3c<xrckpcAacooxxxft Sarcina flava.
DeEarry ,except that litmus milk does not become alkaline.
Culture No. 106
Very similar to Ba®cgey5cacx«tacac«ixi{prtck<xacxoclt Flavobacterium
b r e v e . (Frankland and Frankland).

-

73-

Cultures No's* 38 and 46
Vegetative

cells:

0*6 by 1.0 to 1.5 u shortening to

0.3 to 1.0 u at two days with no change from two to six days.
No. 46 is motile at two days at 30° and 37° while No. 38
is doubtful at 37° after eight days and non-motile at 30°.
Single undulate polar flagellum in No.
on No. 38.

None observed

Gram-negative.

Agar colonies:
convex,

46.

At one day, punctiform,

translucent,

smooth, entire,

greyish enlarging to 1.5 mm at two days

and 2.0 to 3.0 mm at seven days.

Fluorescent by T/L.

No. 38,

colonies become rugose and centres drop precipitously at two
days.

Medium becomes browned,

especially in the case of

No. 38.
Gelatin colonies:
blue by T/L.

Punctiform,

convex,

entire,

Medium browned, especially by No. 38.

smooth,
Colonies

increase to about one mm at seven days.
Agar stroke:

No. 38, abundant filiform,

brownish growth, with medium becoming browned.
abundant,

filiform changing to echinulate,

glistening,
No. 46,

glistening,

grey­

ish, medium not browned.
Nutrient broth:

No. 38,

viscid sediment by seven days.
sediment.

Medium brown.

strong clouding with scanty,
No. 46, like 38, but abundant

-7 9 Motility medium:

Profuse brown pigmentation throughout

medium at two days at 30° and 37°.
Litmus milk:

No. 38, slowly alkaline; No. 46, no

change.
Potato:

In one potato medium both cultures made the

medium reddish brown and there was glistening, brownish
growth.

On another medium the growth was very sparse and

the potato became tan.
Nitrate:

Reduced to nitrite.

Indole and hydrogen sulfide negative.

Peptone iron

agar becomes dark brown in upper half.
Starch hydrolyzed to achroodextrin by 10 days by No. 46
but not No. 38.

Dextrose not fermented.

No growth in citrate broth.
Culture No's. 87 and 93
Vegetative cells:

0.5 by 0.6 u, with rounded ends*

No change in size or shape from one to six days.
two days at 30 and 37° in motility test medium.

Motile in
Single

undulate polar flagellum.
Agar colonies:
translucent,

Punctiform,

smooth, entire, convex,

greyish to brownish at one day enlarging to 1.0

to 2.0 mm by two days*

At seven days colonies of No's.

87

and 93 were 4.0 and 2.0 mm respectively and were brown.
Colonies were translucent,

fluorescent by T/L; and by U/V

-

80 -

bo th colonies and media fluoresced yellowish green.

Medium

became brown.
Gelatin colonies:

Punctiform,

convex, entire,

smooth

greyish colonies at two days enlarging to 1.0 mm at seven
days and No. 93 becoming brown.

Colonies were translucent

and fluorescent by T/L and by U/V both colonies and media
were yellow.

Media became brown.

Agar stroke:

From one to seven days abundant,

glistening greyish growth,

filiform,

strongly fluorescent by T/L.

Medium is brown.
Nutrient broth:

Strong clouding with abundant viscid

sediment by seven days.

Medium is brown at seven days.

No. 93 has a pellicle at three days.
Gelatin stab:

Filiform growth, medium becoming brown

by 14 days.
Motility medium:
Litmus milk:

Brown at 30° and 37°.

Becomes very slightly alkaline by three

weeks with reduction by four weeks.
Potato:
potatoes.

Variable pigment production on different

No. 94 did not grow on one potato medium.

Nitrate is not reduced.
Indole and hydrogen sulfide negative.
Citrate:

No.

87 did not grow, No.

93 did.

81 -

-

Culture No's. 83, 88, 94
Vegetative cells:

0.5 by 0.6 u, with rounded ends.

No change in size and shape by six days.

Motile in motility

test medium at one day at 30° but not at eight days at 37°
although there is good growth at 37°.

Single polar flagellum

may be quite straight or slightly undulate.
Agar colonies:
translucent,

Circular, pink,

smooth, entire, convex,

strongly fluorescent by T/L, 1.0 to 1.5 mm at

one day increasing to 7.0 to l£.0 mm by seven days.

At

seven days, by U / V , colonies fluoresce olive green.
Gelatin colonies:
at seven days.

Considerable cup-shaped liquefaction

By U/V colonies fluoresce yellowish, media

silverish.
Agar stroke:
growth.

Abundant,

filiform,

glistening, pink

Strongly fluorescent by T/L.

Nutrient broth:

Moderate clouding in 83 and 88 at

three and seven days.
pellicle at three days.

No. 94 shows strong clouding and a
No. 83 has scanty sediment, No. 88,

abundant and viscid, and No. 94, abundant but not viscid at
seven days.
Gelatin stab:

Filiform growth, best at top with slight

napiform liquefaction in No*s. 83 and 88, crateriform to
saccate in No. 94 at two days.

At eight weeks No's. 83

and 88 have stratiform liquefaction,

two thirds completed

-

32 -

with the unliquefied medium yellowish and the line of stab
black.

At two weeks No. 94 was two-thirds liquefied with

stratiform liquefaction and liquefaction was completed before
the observation at eight weeks.
Motility medium:

Fronounced yellow in the medium at

30° for cultures 83 and 88 and at 37° for culture 94.
Cultures 83 and 88 caused a brownish pigmentation at 37°
and culture 94 produced only a slight yellowing at 30°C.
Litmus milk:

All cultures reduced litmus milk within

16 hours and No's. 88 and 94 caused noticeable proteolysis
within 10 days.

No. 83 did not cause noticeable proteolysis

by 21 days.
Potato:

These cultures gave variable characteristics

on the two tests, perhaps due to the difference in potatoes.
All grew but No. 94 grew more profusely than the other two.
Nitrate:

All reduced nitrate to nitrite, No. 94 more

slowly than the others.
tests for nitrogen,

At 21 days but not at seven days

nitrite and nitrate were negative.

Indole negative.
Hydrogen sulfide was produced in the medium in two to
four days by No's. 83 and 88 and in one day by No. 94.

Lead

acetate test strips were blackened by No. 94 in one day but
the other cultures caused little blackening even after 28 days.
Starch not hydrolyzed and glucose not fermented.
No growth in citrate broth.

-8 3 -

Cultures 12, 72, 75, 76, 77, 89, 92
Vegetative

cells:

0*5 x 0.6 u, with rounded ends.

not change from one to six days.

No. 72 motile in motility

test medium at 30° at eight days.

Not motile at 37°.

motile in two days at 30° and 37°.
one to three very long,
polar flagella.

slender,

Do

No. 72

All except No. 75 had

straight to slightly curved

No's. 12, 89 and 92 had occasional cells

with a flagellum at each end.
Agar colonies:

Gram negative.

24 hr. colony is punctiform,

greyish,

smooth convex and translucent with edges which may be entire
or erose.

At two days colonies are 1.0 to 2.0 mm in diameter

and are rugose or

(X43) coarsely granular.

At seven days

all colonies have yellowish-grey central areas.

No's. 12, 72,

76, 89, and 92 have wide, effuse transparent marginal zones
of about five mm radius while the central portion of the
colony has a radius of 1.0 to 1.5 mm.

No's. 75 and 77 are

3.0 to 4.0 mm diameter with small or no transparent marginal
zones.

All

colonies except No. 12 fluoresced by T/L, especially

one to three day old colonies.
Gelatin colonies:
smooth,

translucent colonies,

fluorescent by T/L.
in diameter,
convex,

At two days punctiform, convex, entire

At seven days colonies were 1.0 - 1.5 mm

coarsely granular

translucent,

circular to slightly irregular,

(X43), smooth, entire to erose,

blue to bluish green by T/L.

m

-

Agar stroke:
with parts of the

Abundant,

84 glistening,

filiform growth

streak exhibiting spreading growth. By

seven days some cultures have yellowish grey growth.
Nutrient broth:

Moderate

clouding,

no sediment at one

day becoming strongly clouded with abundant viscid sediment
by seven days.
Gelatin stab:

Uniform stab,

filiform at one day be­

coming villous to arborescent by seven days.

At eight weeks

growth is best at top and has spread throughout the medium
to give a cloudy appearance for about two-thirds of the
diameter of the tube.
tate.

Edge of the diffuse growth is echinu-

No's. 12, 89 and 92 did not produce spreading growth

and No. 12 liquefied the gelatin slowly with an infundibuliform to saccate liquefaction.
Motility medium:

After eight days at 37° upper half

of the medium is moderately yellow while the surface growth
of No's. 75 and 77 is faintly pink.
Litmus milk:

No change for about two weeks then becomes

moderately to strongly alkaline.
Potato:

Glistening pinkish orange growth by seven days

with the potato becoming definitely grey.

None of these

cultures grew on one potato medium.
Nitrate is reduced to nitrogen gas with no detectable
nitrite or nitrate left at seven days.

-

85 -

Indole and hydrogen sulfide negative.
Starch hydrolyzed to reducing sugars by 10 days by all
except No's. 12,

76, 89 and 92 which gave a positive test

for erythrodextrin at seven days and achrooaextrin at 10
days.

Acid hydrolysis of these four at 10 days gave reducing

sugars.

Dextrose

not fermented.

Good growth in citrate broth at 10 days.
Culture No. 82
Vegetative cells:

0.5 by 0.8 u, with rounded ends.

Mo change in size or shape up to six days.
no flagella observed.
Agar colonies:

Non-motile and

Gram-negative.
Punctiforra,- smooth, entire,

convex,

greyish, translucent at three days becoming glistening white,
pulvinate, 2.0 to 2.5 mm in diameter by seven days.

At

seven days they were pale violet by U/V.
Gelatin colonies:
rtgar stroke:

No growth at seven days.

At seven days, moderate,

beaded,

glistening

creamy white.
Nutrient broth:

Slow growth.

At seven days there was

no surface growth or clouding and only a scanty sediment.
Gelatin stab:

After eight weeks very sparse, greyish

filiform growth in upper half of stab.
Litmus milk:
Potato:

No change.

No growth.

A

-

Nitrate:

86 -

Slight reduction to nitrite at 21 days.

Indole, hydrogen sulfide, starch hydrolysis,

glucose

fermentation negative.
No growth in citrate broth.
Culture No. 1
Vegetative cells:
ends.

0.6 by 1.0 to 1.3 u, with rounded

No change in size or shape up to six days.

in motility test medium at one day.
flagellum.

Notile

Single undulate polar

Gram-negative.

Agar colonies:

24 hr. colony is circular, about one mm

in diameter increasing to 1.5 to 2.0 mm at two days and 6.0
to 7.5 mm at seven days.

Colony is smooth, convex, translu­

cent with entire edges up to three days but seven day colony
has a convex slightly opaque central portion of 2.0 to 2.5 mm
diameter and a transparent effuse marginal zone of 2.0 to
2.5 mm which is somewhat undulate with finely erose edge.
At seven days colonies macroscopically show some radiate
ridging while microscopically
especially in central part.

(X43) they are coarsely granular,
By oblique transmitted light

(T/L) colonies are fluorescent, especially those one to
three days old while by reflected light

(IV^L) colonies are

greyi sh.
Gelatin colonies:

Invisible at one day but at two days

are punctiforra, circular in shape, convex, smooth, translucent,

-

greyish by R/L,

87 -

fluorescent by T/L.

At seven days colonies

are much like those at two days but are
irregular in

shape and

Agar stroke:

blue by T/L.

Abundant filiform growth becoming echin-

ulate at two days.
by T/L.

circular toslightly

Growth is greyish by R/L,

fluorescent

Fluorescence is pronounced up to three days but not

at seven days.
Nutrient broth:

Strong clouding which is persistent,

nbundant sediment which becomes viscid by seven days.
Gelatin stab:

Growth is best at top, filiform,

brown along line of stab.
Motility
the medium is
pink.

yellowish

No liquefaction.

medium: After eight days at 37° upper half
slightly yellow and surface growth

of

is light

At 30° medium is unchanged and surface growth is only

slightly pink.
Litmus milk:

Becomes slightly alkaline in about two

weeks and markedly alkaline by three weeks.
Potato:

No visible growth at one day but glistening,

tan

growth at seven days.

Potato becomes greyish

and

is tan at four and seven days.

at two

da^s

Nitrate is reduced to nitrite to a slight extent by
seven days and to a considerable extent by 21 days.
Indole and hydrogen sulfide negative.
Starch not hydrolyzed and glucose not fermented.
Good growth in citrate broth at 10 days but not at four
days.

-38Culture Mo. 2
Vegetative cells:
ends.

0.6 by 0.8 to 1.2 u, with rounded

Shortening to 0.8 to 1.0 u at two days with no further

change up to seven days.

Gram-negative.

Non-motile in

motility test medium at two days but shows doubtful motility
test at 8 days at 30°C and positive test at 37°.

Single

polar flagellum is undulate and is present in only a few cells
of an 18 hr. culture.
Agar colonies:
in diameter,
days.

24 hr. colony is circular, about one mm

increasing gradually to 3.0 to 3.5 mm by seven

From one to seven days colonies are circular, smooth,

convex and translucent with entire edges.
are greyish.
days.

By R/L colonies

Colonies are fluorescent by T/L at 1, 2 and 3

At seven days they have a yellowish centre and trans­

lucent bluish marginal zone by T/L and by R/L or T/L show
one to several wedge-shaped areas extending from the border
towards the centre.

These wedge-shaped areas possess less

optical density than the rest of the colony and microscopi­
cally

(X43) they appear as if a gouge had cut a concave,

smooth, amorphous,

shining channel in an otherwise finely

granular colony.
Gelatin colonies:

No visible growth at 24 hr. but two

day old colonies are punctiform,
entire edges.

convex, smooth, and with

They are greyish by R/L and translucent.

-

By T/L they are fluorescent*

89 Seven-day colonies are much

like those at two days but by T/L are blue.

They are circu­

lar to slightly irregular in shape.
h

gar stroke:

filiform,

Abundant,

glistening,

greyish, butyrous,

strongly fluorescent by T/L, from one

Nutrient broth:
at seven days.

to seven days.

clouding is slight at oneday, strong

Sediment is scanty at one and two days,

abundant at seven, and is viscid from one to seven days.
Gelatin stab:

Identical with culture No. 1.

Ivlotility medium: After eight days at 37° the
half

upper

of the medium is fairly yellow and surface is light pink.

No change in medium at 30°.
Litmus milk:
Potato:

Identical with culture No. 1.

Glistening creamy yellow growth at two days

becoming brownish by seven days.

Potato becomes tan by seven

days.
Nitrate:

No nitrite at seven days but slight nitrite

by 21 days.
Indole and hydrogen sulfide negative.
Starch and glucose negative.
Scanty growth in citrate at four days and fair at 10 days.
Culture No. 3
Vegetative cells:
Agar colonies:

Identical with culture No. 2.

Identical with description of culture

No. 2 except that tney were considerably smaller at one day

-90and slightly larger at seven days.
Gelatin colonies:
at seven days they

Identical with culture No. 2.

Nutrient broth:
at seven

growth at two days but

were identical with culture No. 2.

Agar stroke:

that

No visible

Identical with culture No. 2 except

days there was a slightly flocculent

as well

as a viscid sediment.
Gelatin stab:

Identical with culture No. 1.

Motility medium:

Identical with No. 2 except surface

growth is only slightly pink.
Litmus milk:
Potato:

Identical with No. 1.

Sparse tan growth by seven days.

Potato

becomes tan.
Nitrate:

No nitrite at seven days and doubtful results

at 21 days.
Indole, hydrogen sulfide,

starch and glucose identical

with No. 1.
Citrate broth:

Negative at 10 days.
Culture No. 4

Vegetative cells:
long.

0.6 to 0.8 u thick by 0.8 to 1.4 u

Longer cells are most numerous at one day while shorter

cells are most numerous at seven days.
ends.

Gram-negative.

Cells have rounded

Motile in motility test medium within

two days at 30° and 37°.

One or two undulate flagella on

one or both ends.
Agar colonies:

Are identical with colonies in culture

No. 2 except that seven-day old colonies are only 2 to 3 mm
in diameter.
Gelatin colonies:
Aga r stroke:

Identical with culture No. 2.

Identical with culture No. 2 except that

growth is filiform to echinulate.
Nutrient broth:

Identical with culture No.

2 except

that clouding is moderate at one day and strong at two days.
Gelatin stab:

Identical with culture No. 1.

Motility medium:

Identical with No. 3 except only

slightly yellow in top half.
Litmus milk:
Potato:

Identical with No. 1.

At seven days profuse,

glistening tan growth.

Potato becomes tan.
Nitrate:

Identical with No. 2.

Hydrogen sulfide:

Negative in medium but strongly

positive on lead acetate test strip at seven days.
Indole,

starch and glucose:

Identical with No. 1.

Culture No. 5
Vegetative cells:

Ovoid rods 0.6 to 0.8 by 0.8 to 1.0 u

changing little from one to six days.

Gram-negative.

Non-

motile in motility test medium at two days but motile at
eight days at 30° and 37°.

Single polar flagellum is straight.

-

Agar colonies:
the following:

92 -

Identical with culture No. 2 except for

smaller at one day.

By seven days a thin

effuse growth with undulate margin had spread out from some
of the wedge-shaped areas in the original colony.

This

growth was greyish by T/L.
Gelatin colonies:

No visible growth at 43 hr. but at

seven days colonies are one mm in diameter, circular to
slightly irregular,
entire edge.

convex, smooth, translucent and with

By T/L they are bluish.

Agar stroke:

Identical with colony No. 2.

Nutrient broth:
at two days.

Clouding is slight at one day, strong

Sediment is identical with culture No. 2.

Gelatin stab:

Identical with culture No. 1.

Motility medium:
Litmus milk:
Potato:
Nitrate:

Identical with No. 3.

Slightly alkaline at 28 days.

Moderate tan growth by seven days.

Potato tan.

Identical with No. 3.

Indole, hydrogen sulfide, starch and glucose identical
with No. 1.
Citrate:

Negative at 10 days.
Culture No. 6

Vegetative cells:

0.6 by O.S to 1.3 u at one day

shortening to 0*3 to 1.0 u by six days.
negative.

Rounded ends.

Non-motile at eight days at 30° and 37°.

Gram-

Single

-

93 -

straight polar flagellum is present on only a few cells of
an 18 hr. culture.
Agar colonies:
smooth,

circular,

Colonies from one to seven days are

convex, translucent, with entire edge.

Increase from one mm diam. at one day to eight mm at seven
days.

Seven-day colonies have wedges similar to those in

culture No. 2.

Microscopically the seven-day colony is

coarsely granular.

By T/L one to three day old colonies

fluoresce strongly while seven-day colonies have yellowish
centre and bluish marginal zone.

By U/V seven-day colonies

are a very pale rusty orange.
Gelatin colonies:
Agar stroke:

Identical with culture No. 6.

Nutrient broth:
at three days.

Identical with culture No. 2.

Clouding is slight at one day, strong

Sediment is scanty up to three days but at

seven days it is abundant and viscid.
Gelatin stab:

Identical with culture No. 1.

Motility medium:
Litmus milk:
Potato:

Identical with No. 2.

Identical with No. 1.

Moderate yellowish tan growth at seven days.

Potato tan.
Nitrate:
Indole,

Slight nitrite at 21 days but not at seven.
hydrogen,

sulfide,

starch and glucose identical

with No. 1.
Citrate:

Scanty growth at four days, fair at 10 days.

-94Culture No. 7
Vegetative cells:
0.6 to 0.8 by 0.8 to 1.0 u; with
rounded ends.
negative.

Do not change from one to six days.

Gram-

Non-motile at eight days at 30° and 37°.

Flagella

as in culture No. 6.
Agar colonies:

At one day colonies are one mm in

diameter, circular, smooth, greyish, translucent,
and with entire edge.

At two days colonies

granular and have darker centres.
several deep fissures.
from the centre.

convex,

(X43) are finely

Some centres have one to

The fissures may or may not radiate

At three days colonies

(X43) range from

convex to urabilicate and all have pointed,

transparent

bristles extending from an otherwise entire edge.
days colonies are 3 to 4 mm in diameter,

At seven

have shallow,

crater-like centres and (X43) have small colonies surrounding
and usually adhering to the parent colonies.
are more or less coarsely granular,

All colonies

greyish by T/L or R/L

and very pale orange by U/V.
Gelatin colonies:

At two days punctiform,

translucent colonies with entire edges.
fluorescent by T/L.

convex, smooth,

Greyish by R/L,

At seven days colonies were one mm in

diameter, circular to irregular, raised to convex, smooth,
translucent and with entire edge.

By T/L, blue and by R/L,

grey.
Agar stroke:

Identical with No. 2.

-

Nutrient broth:
Gelatin stab:

Identical with No.

4.

Identical with No. 1.

Motility medium:
Nitrate:

95 -

Identical with No.

5.

Identical with No. 1.

Indole, hydrogen sulfide,

starch,

glucose,

citrate:

Identical with No. 1.
Culture No.
Vegetative cells:
ends.

9

0.6 by 0.8 to 1.0 u, with rounded

No change in size or shape from one to six days.

Non-motile in motility medium at eight days but several
have

single, undulate polar flagellum.
Agar colonies:

Circular,

smooth,

Gram-negative.
entire,

convex, trans

lucent, one mm diameter at one day increasing to three mm
at seven day.

Greyish by R/L,

Gelatin colonies:
greyish colonies.

fluorescent by T/L.

Punctiform,

convex, entire,

smooth,

By T/L they were fluorescent at two days,

bluish at seven days.
Agar stroke:

Abundant,

filiform,

glistening strongly

fluorescent greyish growth from one to seven days.
Nutrient broth:

Slight clouding and no sediment at

one day becoming strongly clouded with abundant viscid
sediment by seven days.
Gelatin stab:

Identical with No. 1.

-9 6 -

Motility medium:
but not at 30°.

Surface growth slightly pink at 30° and 37°.

Litmus milk:
Potato:

Identical with No. 1.

Moderate tan growth.

Nitrate:
Indole,

Faint yellowing of the medium at 37°

Potato tan.

Identical with No. 2.
hydrogen sulfide,

starch, glucose,

citrate:

Identical with No. 1.
Culture No. 10
Vegetative

cells:

0.6 by 0.8 to 1.0 u at one day short­

ening to 0.6 by 0.8 u at six days.
test medium at eight days.

Non-motile in motility

Single undulate polar flagellum.

Gram-negati ve .
Agar colonies:
smooth, entire,
cent by T/L.

At one day circular,

convex, translucent,

one ram diameter,

greyish by R/L,

By two days many of the colonies'

fluores­

centres

have dropped to give a rugose crater-like depression and
colonial

size is 1.5 to 2.0 mm.

Fluorescent by T/L.

Seven

day old colonies are much the same as two day old colonies.
Gelatin colonies:
f o r m , convex, entire,

From two to seven days are punctismooth,

translucent,

greyish by R/L.

By T/L they are fluorescent at two days, bluish at seven.
Agar

stroke:

Abundant,

filiform,

glistening, greyish

growth fluorescent by T/L from one to seven days*

-

Nutrient broth:
three days*

97 -

Slight clouding at one day, strong at

At seven days there is a stringy surface growth.

Sediment is scanty up to three days but is abundant and
viscid by seven.
Gelatin stab:

Identical with culture No. 1.

Motility medium:
Litmus milk:
Potato:

Identical with No. 6.

Identical with No. 1.

Glistening tan growth.

Nitrate:

Potato tan.

Slight amount of nitrite at four days with

no increase by 21 days.
Indole,

hydrogen sulfide,

starch,

glucose, citrate:

Identical with No.l.
Culture No's. 11, 15
Vegetative cells:

0.6 by 2.0 to 2.4 u at one day

shortening to 1.0 to 1.5 u by six days.

No. 11 shows positive

motility at four days but No. 15 shows only doubtful motility
at eight days.

Single polar flagellum is undulate.

Gram-

ne gative.
Agar colonies:

Punctiform,

smooth, entire, convex, trans

lucent colonies increasing to one mm diameter at three days
at which time colonies are raised with a peak-like central
area and

(X43) are coarsely granular with many bristles ex­

tending from the edge.
at two days.

Colonies, macroscopically, are rough

They are not fluorescent by T/L.

-

Gelatin colonies:

98 -

Punctiform,

translucent, bright blue by T/L,
Agar stroke:

Moderate,

convex, entire, smooth,

greyish by R/L.

filiform,

glistening,

greyish,

non-fluorescent by T/L.
Nutrient broth:
day.

Slight clouding and no sediment at one

By seven days there is a ring, moderate clouding,

abundant sediment which rises as floccules when agitated
(No. 11) or has some viscid and some flocculent sediment,
No. 15.
Gelatin stab:

Uniform, beaded, brownish growth.

Liquefaction is stratiform to slightly cratcriform and is
very slow, about one-third of the medium being liquefied in
eight weeks.
Motility medium:

Moderate brown discoloration of

medium at 30° and 37°.
Litmus milk:
Potato:

Becomes alkaline after about two weeks.

Orange to reddish tan growth varying with the

medium.
Nitrate:

Slowly reduce nitrate to nitrogen gas without

detectable nitrite.
Indole and hydrogen sulfide negative.
Starch not hydrolyzed and glucose not fermented.
No growth in citrate broth.

-99Culture No's. 16 and 17
Vegetative cells:

0.5 by 1.0 to 1.2 u.

size or shape up to six days.

No change in

Non-motile and no flagella

observed.
Agar colonies:

Circular to slightly irregular, smooth

to rough, entire to undulate, convex, greyish, translucent.
One mm at one day increasing to 4.0 to 7.0 mm by seven days.
At seven days colonies are lobate and (X43) are coarsely
granular with amorphous marginal zones.
Gelatin colonies:

Greyish by T/L.

Little growth at two days.

At

seven days they are circular to slightly irregular, convex,
entire to slightly undulate, smooth, translucent and bluish
by T/L.
Agar stroke:

Abundant, echinulate, glistening, greyish,

non-fluorescent by T/L.
Nutrient broth:

Slight clouding with little sediment

at one day increasing to moderate clouding and abundant,
viscid sediment at seven days.
Gelatin stab:

Uniform, filiform growth.

Both cultures

produce feathery outgrowths from the line of stab, especially
in the top half of the medium but at eight weeks No. 16 has
spread completely throughout the top 5 mm of the medium.
Motility medium:
Litmus milk:
10 days.

No pigmentation.

No. 16 caused noticeable proteolysis by

No. 17 was moderately alkaline by 16 days and com­

-1 0 0 -

pletely reduced by 21 days.
Potato:

Variable pigment production.

At seven days

growth and potato were both tan.
Nitrate is not reduced, starch not hydrolyzed, dextrose
not fermented,

indole not formed, and no growth in citrate

broth.
Discussion:

Although these cultures were not found to

be motile under the conditions of testing and flagella were
absent it is felt that the growth in gelatin stabs precludes
any possibility of calling these organisms non-motile.
Cultures N o ’s. 49 and 50
Vegetative colonies:

0.6 by 0.8 to 1.0 u at one day

shortening to O.Q u at six days.
undulate polar flagellum.
Agar colonies:

Rounded ends.

Single

Gram-negative.

Punctiform at one day increasing to

four to six mm by seven days.

Circular,

smooth, entire,

convex, greyish, translucent, fluorescent by T/L losing the
fluorescent properties by seven days.

By U/V, yellowish to

rusty orange in large colonies.
Gelatin colonies:
convex, undulate,

At two days, punctiform, greyish,

smooth, translucent, fluorescent by T/L.

At seven days circular to slightly irregular, 1.0 to 1.5 mm,
smooth, convex, greyish, blue by T/L.

-1 0 1 -

Agar stroke:

Abundant,

filiform,

glistening,

greyish,

butyrous becoming decidedly viscid by seven days.
Nutrient broth:

Slight clouding and scanty sediment

at one day becoming strongly clouded with abundant viscid
sediment by seven days.
Gelatin stab:

Uniform,

filiform growth developing

feathery puffs from the stab line by two weeks.
Motility medium:
37° at eight days.

Slight yellowing of the medium at

Surface growth of No.

50 is slightly

pink at 30° and 37°.
Litmus milk:
Potato:
on another.

Becomes alkaline

slowly.

Creamy tan growth on one medium,

orange tan

Medium tan.

Nitrate reduced to nitrate in one day.
Indole negative.
Hydrogen sulfide on lead acetate strip but not in stab.
Starch not hydrolyzed and glucose not fermented.
Moderate growth in citrate by 10 days.
Culture No.
Vegetative cells:

8

0.6 by 1.0 to 1.6 u with shorter

forms predominating by six days.
motility medium at one day.

Rounded ends.

Motile

in

Single, undulate polar flagellum.

Gram-ne gative.
Agar colonies:

At one day colonies are circular,

smooth,

-102entire,

convex and translucent,

greyish by R/L.

scopically they are coarsely granular.

Micro­

Colonies increase

in size to 2.5 - 3.0 mm at seven days and from two to seven
days many of them have precipitously sunken centres which
are rugose.
Gelatin colonies:

Punctiform,

convex, entire, smooth

colonies at two days becoming irregular,

flat and lobate by

seven days.
Agar stroke:

Abundant,

ecninulate,

glistening, greyish

growth, fluorescent by T/L from one to three days*.
Nutrient broth:
Gelatin stab:

Identical with No. 4.
Identical with No. 1.

Motility medium:
but not at 30°.

Faint yellowing of the medium at 37°

No pink colour in surface growth.

Litmus milk:

Slightly alkaline at two weeks, strongly

alkaline at four weeks.
Potato:
Nitrate:
Indole,

Glistening tan growth.

Potato tan.

Reduced to nitrite in one day.
hydrogen sulfide,

starch,

glucose, citrate:

Identical with No. 1.
Culture No.
Vegetative cells:
change up to six days.

98

0.6 by 0.8, with rounded ends.

No

Motile in motility test medium.

Flagellation extremely varied, most common types being one

-

103 -

flagellum at one or both ends or from the side, or two
flagella from one end:
end,

more rarely five flagella from one

two from the side, or a variable number from one end

with one flagellum on one or both sides.
Agar colonies:

Gram-negative•

Circular, rough, entire,

convex, 1.0 -

1.5 mm at one day increasing to 2.0 to 2.5 ram at seven days,
greyish by R/L or T/L.
Gelatin colonies:

Circular,

smooth, entire,

convex,

punctiform at three aays and one mm at seven days.
rtgar stroke:

Abundant,

filiform,

glistening,

greyish,

becoming viscid by three days.
Nutrient broth:
Gelatin stab:

Strong clouding,
Filiform,

putrefactive odor.

growth best at top, with

numerous small colonies scattered tnroughout top cm of
medium at 14 days.
Notility medium:
Litmus milk:
Potato:

No pigmentation.

Acid at one day,

Abundant,

coagulated at three week

glistening creamy growth.

Potato

is reddish-brown at two days.
Nitrate:
Indole:

Reduced to nitrite at one day.
Negative.

Hydrogen sulfide:

Slight blackening of leaa acetate

test strip at 14 days.
Starch not hydrolyzed.

-104n.cid and gas in arabinose, xylose,

dextrose, fructose,

galactose, m a n n o s e , sucrose, maltose, mannitol.

Slow acid

production with slight gas bubble in lactose, raffin ose,
inulin,

dextrin,

dulcitol,

salicin and aesculin.

Culture No. 100
Vegetative cells:

0.9 by 0.7 u.

at one day at 30° and 37°.
is common.

Notile

in motility

Single undulate polar flagellum

Occasionally two or five polar flagella.

Gram-

negat ive.
Agar colonies:
greyish,

Circular,

fluorescent by T/L.

smooth, entire,

convex,

At seven days colonies are 1.5

to 2.0 mm, smooth to slightly rough, raised to convex*
Gelatin colonies:
entire,

smooth,

At one day punctiform,

greyish, fluorescent by T/L.

convex,
At two days

colonies are 2.5 mm with cup-shaped liquefaction advanced,
rtt three days liquefaction is complete and by U/L the lique­
fied and non-1iquefied portions of the medium fluoresce
silvery grey..
Agar stroke:

Abundant,

Nutrient broth:

filiform,

Pellicle,

greyish,

strong clouding,

glistening.
scanty

sediment from one to seven days with slight odor of putre­
faction at seven days.
Gelatin stab:

Abundant granular sediment at 14 days.

Rapid infundibuliform liquefaction com­

plete at seven days with flocculent,

greyish sediment.

-105Ivlotility medium:

After eight days upper half of medium

is moderately or slightly yellowed at 30° and 37° respectively.
No pigmentation of surface
Litmus milk:

growth.

Acid at one day, neutral at two days

with proteolysis occuring at two days and completed at 10
days.
Nitrate:

Reduced to nitrite at one day.

nt

31 days

but not at seven days, nitrogen gas present but nitrate and
nitrite absent.
Indole present at one day.

Hydrogen sulfide negative.

Starch hydrolyzed in two days.
Considerable acid in dextrose,
sucrose, maltose,

fructose,

galactose,

inulin, mannitol, salicin and aesculin.

Slight acid in arabinose, xylose, mannose, lactose, raffinose,
inulin, and dulcitol.

No gas produced.

Growth in citrate broth.
Culture No. 103
Vegetative cells:
day at 30° and 37©.

0.3 by 0.4 to 1.0 u, motile in one

Two undulate polar flagella.

Gram-

ne gative•
Agar colonies:
greyish,

Circular,

smooth, entire,

convex,

strongly fluorescent by T/L increasing in size to

5.0 to 6.0 mm and containing wedge-shaped areas cf. culture
No. 2 at three days.

-106Gelatin colonies:

Circular,

convex,

entire,

smooth,

with cup-shaped liquefaction advanced considerably at two
days.
Agar stroke:

Abundant, echinulate,

glistening,

greyish,

from one to seven days.
Nutrient broth:

Strong clouding,

scanty sediment,

developing a slightly putrefactive odor at two days, a
slight ring and an abundant sediment by seven days.
Gelatin stab:

Filiform growth with infundibuliform

liquefaction complete at seven days.
Motility medium:
Surface

Greyish sediment.

No water soluble pigment produced.

growth at 37° is slightly pink.

Litmus milk:
Potato:
Nitrate:
Indole:

Acid at one day but no curd at 58 days.

Scanty,

glistening,

creamy growth.

Reduced to nitrite by one day.
Present at one day.

Hydrogen sulfide:

Negative.

Starch not hydrolyzed.
No growth in citrate broth.
Acid from dextrose,
maltose,

raffinose,

fructose,

galactose, lactose, sucrose,

dextrin, mannitol,

Slight acid production from dulcitol:
xylose, m a n n o s e , inulin.

No gas.

salicin, aesculin.
none from arabinose,

-107Culture No. 104
Vegetative
to six days.

cells:

0.2 to 0.3 by 1.0 to 1.5 u, from one

Non-motile in motility medium at 30° and 37°.

Flagellated, most commonly with a single polar flagellum on
one or both ends,

three flagella on each end, rarely with

two flagella on one end or two flagella on one end and two
originating on one side.
Agar colonies:
translucent,

Circular,

smooth to rough, greyish,

convex to raised, with thin, effuse marginal

zone which is e r o s e , l o b a t e , or very ragged.
colonies are S.O to 15.0 mm and
Gelatin colonies:
convex,

entire,

Agar

At seven days

(X43) are coarsely granular.

At seven days are circular, 1.0 mm,

smooth ana translucent.

stroke:

Abundant,

filiform,

glistening,

greyish,

becoming somewhat echinulate and spreading by three days.
Fluorescent by T/L.
Nutrient broth:

Moderate clouding and scanty sediment

at one day with pellicle,

strong clouding and slight putre­

factive odor at seven days.
Gelatin stab:

Filiform growth with feathery puffs from

line of stab by 14 days.
Motility medium:
days.

Slight yellowing of medium at seven

No pigmentation of surface growth.
Litmus milk:

Slightly alkaline at two weeks, markedly

alkaline at three weeks.

-108Nitrate:

Reduced to nitrite in one day.

Indole ana hydrogen sulfide negative.
Starch not hydrolyzed and glucose not fermented.
Slow growth in citrate broth.

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