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A STGDY OF COLiFGRMS ANY)
EN'E‘EROCDCCS 3N SON.

Thesis for the Degrae of M. S.
MiCHiGAN MATE CQLLEGE
395m"? Cfiauncey Ccopar
E953

“4:515 ‘ ‘ . \ f ,.

«3

This is to certify that the

thesis entitled

has been accepted towards fulfillment
of the requirements for

—r,I »V\

_°‘_'_‘”_ degree in

 

 
   

h jor professor

Date_;‘__~.q_;r_

A STUDY OF COLIFORIS AID ENTSROCOCCI IN SOIL

By

Robert Chauncey Cooper
w

A THESIS

Submitted to the School of Graduate Studies of lichigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

.' "7'1 ‘ "1 3” T"".’""“
141:3...Lln. 01‘ OK) 14;”ng

Department of BaCteriology

1955

lllv. Fitw I 21:. "I
.-

ACKNOWLEDGEMBKT

The writer wishes to express his sincere

appreciation to Dr. J. L. Hallmann by whose able

guidance this thesis was made possible.

{,1 . 733185

TABLE

IHTRODUCTION

XPERIHEETAL PROCSDURE

lTJ

ISCUSSION

U

n'T‘: '7 r . o-
I.) brawl—.111 I

BIBLIOGRAPHY

OF C

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-xJ.

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EJA J...)

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C3

13
29
51

1.).» Alofixf.§‘
V C

I.

II.

III.

IV.

V.

VI.

VII.
VIII.
IX.

The number of coliforns and enterococci in
various treated and untreated soils . . . . .

The number of coliforms and enterococci in
soils treated with peat . . . . . . . . . . .

The number of coliforms and enterococci in
soils treated with sewage sludge . . . . . .

The number of coliforms and streptococci in
soils treated with straw manure . . . . . . .

The number of coliforms and enterococci found
in eight samples of "virgin soil" . . . . . .

The number of coliforms and enterococci found
in control plots . . . . . . . . . . . . . .

Differential reactions among coliforms isolated
Types of enterococci isolated from soil . . . .

Results of standard methods coliform tests on
3011 3317113183 0 o o o o o o o o o o o o o o o

Page

14

16

17

18

19

19
24
26

28

INTRODUCTION

A perennial problem for investigators has been the sani-
tary significance of members of the coliform group of organisms

when found in water and soils; and, in particular, he Aerobactor

 

aerogenes types which are usually thought of as soil or grain

 

organisms. It is thought that the presence of the enterococ-
cus group might aid in determining the significance of the
coliform organisms that are present. The purpose of this in-
vestigation is to determine the possible occurrence of the
enterococci in various soils and to establish their relation-
ships to the coliform organisms occurring in the same soil
samples.

The presence of streptococci in sewage was originally
reported by Laws and Andrews (1894) when they isolated them
from.hospital sewage in England. Winslow and Hunnewell (1902
gave the first report of sewage streptococci in the Un ted
States. They had isolated these organisms from the hands of

school children in conjunction with Escherichia coli and found

 

them to be similar to those found in Boston sewage. One of

the first reports on the sanitary significance of these organ-
isms was that of Houston (1898), who concluded that the strepto-
cocci in water are of sanitary significance and that they in-
dicate more recent pollution than do the coliforms. Due to

this report the organisms received the name "sewage strepto-

cocci of Houston". Savage and Read (1917) demonstrated that

no streptococci were present in polluted waters and are of
undoubted value as evidence of excretal contamination. Hall-
mann (1928) found that E, 321; tends to increase or multiply
in swimming pools while the streptococci do not, demonstrating
that the streptococci are better indicators of pollution in
unsafe swimming pools. Winslow 33 El (1947) stated "although
he significance of the streptococci as sewage organisms is
not established with the definiteness which marks our know-
ledge of the coliform group, these bacteria have been isolated
so frequently from.polluted sources and so rarely from normal
waters that it seems reasonable to regard their presence as
indicators of pollution."

The intestinal streptococci are quite widespread in nature
and usually in conjunction with the intestinal discharges of
animals. Winslow and Palmer (1910) isolated the enterococci
from the intestines of horses, cows, and man, making their
differentiations by using acid production in sugar as a cri-
terion for identification of the enterococcus. Orcutt (1926)

isolated enterococci from. he normal digestive tract of calves
and demonstrated that these streptococci were not a homogeneous

group. Steinhouse (1941) isolated Streptococcus fecalis from

 

a number of insects including seven orders of the class Hexa-
poda. Sylvester and Benedict (1941) isolated 65 true entero-
cocci and 15 related forms.from.the viscera of the fox and

mink explaining their presence in the viscera as being due to

migration from.the intestine to the gut after death. Winter

and Sandholzer (1946) found enterococci in sewage, water,

sea water, and the feces of man, domestic and wild animals.
Ostrolenk and Hunter (1946) made a study of the distribution

of enteric streptococci and found the organism present in

the feces of humans, cats, mice, guinea pigs, dogs and other
animals and insects. These workers found soil to be nega-
tive; however, they only tested two soil samples. Hallmann

and Litsky (1951), in a study on the survival of various enter-
ic organisms in soil, stated that there were no streptococci
present in untreated soil.

It can thus be seen from the work done on the distribu-
tion of the enterococci that they have been isolated mainly
.from animal excreta. Of all the citations made in the litera-
ture, few have been made concerning the presence or absence
<Jf these organisms in the soil; in those cases where soil was
included in the study no streptococci were found.

To be a good indicator of pollution an organism should
neither be able to persist for great lengths of time outside
of its normal habitat nor to multiply outside of its own sur-
roundings. The enteric streptococci seem to fit this classi—
fication somewhat better than do the coliform organisms. Savage
and Wood (1918) studied both coliforms and enterococci in tanks
0f water with small amounts of organic matter present, and
found that the streptococci die out in about two weeks, while
the coliform persisted longer and in some cases multiplied.

They concluded that the presence of streptococci in water may

3

km a better indication of pollution than the coliform group.
Bhllmann (1940) demonstrated that in sewage there are instances
when the coliform organisms will multiply while such is not
the case with members of the enterococcus group. This again
indicates that the streptococci would be better indicators

of contamination. Hallmann and Litsky (1951), in a study on
the survival of enteric organisms in various types of soil,
inoculated lysimeters with material (soil) containing sewage
in.various forms. They found that the enterococci died out
rapidly in soil while coliforms were found to persist for

long periods of time. Young and Greenfield (1925) found that
under certain natural conditions the coliform.group will mul-
tiply and thereby fail to be an indicator of pollution. In
sterile soil they found coliforms surviving for as long as

17 months; in unsterilized soil they found EH1 increased num-
ber of coliforms. Skinner and Murray (1926) inoculated soil
with coliforms and found that the organisms lasted longer than
122 days; while Kulp (1952) reported that at aerogenes and

E. coli lasted almost four years in the soil, demonstrating

 

what he called "the protective influence of the soil on the
coliform organisms."

The literature on the significance of the coliform organ-
isms in soil is quite confusing. The many workers draw diver-
gent conclusions, particularly concerning the intermediate

strains that lie somewhere between the true 2, coli and the

true A, aerogenes. Koser (1924 and 1926) found that there were

 

colon organisms resembling those of intestinal origin in
relation to the methyl red-Vegues Proskauer test in appar-
ently unpolluted soil. He felt, further, that the intestinal
Eh 2212 may be separated from.the soil 3, Eglé_by using his
citrate medium; a positive citrate being evidence of non-
intestinal g, 9213, In contrast, Vaughn and Levine (1942)
gave evidence showing that citrate utilizing organisms are
present in feces, soil and water. They felt the statement,
"intermediate groups of coliforms are necessarily non-fecal
in origin" to be unjustified. Parr (1941) found that some-_
strains of coliforms are not stable on Simmons citrate. He
felt that different strains exhibit varying grades of stabil-
ity. Some citrate positive organisms will reproduce both
citrate positive and negative colonies. Carpenter and Fulton
(1957) reported that 49.8 percent of the fecal samplesthey
examined contained citrate positive strains and 15.5 percent
demonstrated intermediate groups. They, therefore, concluded

that the citrate positive group may have sanitary significance.

Gray (1952) found A, aerogenes universally present in human

 

stools and predominating over E, coli in soil.

 

EXPER RENTAL PROCEDURE

To enhance the value of the study it was deemed necessary
to sample soils of known history in order that their sanitary
conditions might be known. This would show the incidence of
coliforms and enterococci, if any, in soil known to be of both
good and poor sanitary quality.

Soil samples were taken from experimental plots, records
of which have been kept for a number of years. These his-
tories include what has been placed in the soil by way of
treatment and also included when the treatment was carried
out. The soil plots are maintained by the Soils Department
of Michigan State College. Their physical measurements are
112 feet wide and 150 feet deep. They are each divided into
strips 14 feet wide making eight strips, each receiving a dif-
ferent treatment. There are 12 of these plots in the section
used for soil samples, arranged three deep and four across
(see map). The center section (D) was used in taking the
samples because section (E) is on a road and Section (C) is
adjacent to a different set of soil plots. This center sec-
tion appeared to be the least susceptible of the three to
foreign contamination. »

The soils samples had been treated with sewage sludge,
straw manure and peat; the material being placed on the soil

and subsequently plowed under. Control plots, used as a

Sec.

0

 

 

 

 

 

 

 

 

 

 

EXPERIMENTAL SOIL PLOTS

Sec. D

 

 

 

 

 

 

 

 

 

*=———————150

Sec. E

 

 

 

 

 

 

 

14'

 

 

 

i4 . o.- ., 135...?» iii

positive control when testing the effect of the above mater-
ials (in soil) on various creps, were also sampled.‘ The con-
trol plots have had no treatment whatsoever. he "virgin soil"
was taken from a heavily wooded area in the vicinity of the
experimental plots. (The term "virgin soil" is used to indi-
cate an area that has never been cultivated.)

To take the actual soil sample a simple instrument was

required: one which would easily penetrate the soil and

which would retain about ten grams of earth when removed.
Ordinary cheese samplers - which are hollow cylinders with a
sharp edge and perpendicular handle - were selected. In taking
the sample the area to be tested was scraped clear of the first
few centimeters of soil with a sterile tongue blade. The ster-
ile cheese sampler was then driven into the soil to a depth of
about four inches and the sample removed. The specimen was
then placed in a sterile,-wide—mouthed water sampling bottle
with a ground glass stopper and transported to the laboratory
for testing.

Random sampling was employed in taking the soil specimens,
that is, there was no pre-determined distance between sampling
points along the length of the test plot. The number of samples
taken per plot was approximately ten; five in the spring and
five in the fall.

In the laboratory, the sample was transferred to a sterile
mortar and ground with a pestle to insure an even mixture of

all depths represented. Two grams of the sample was weighed

out and placed in 100 m1. of sterile, distilled water. Using
this procedure the coliform and streptococcus count of the 100
m1. will equal the total count present in the two grams of
soil. The counts were made using most probable numbers based
on three portions each of three dilutions. Dilutions of 1.0,
0.1 and 0.01 ml. for the coliforms, and 10.0, 1.0 and 0.1 ml.
for the streptococci proved to be the most satisfactory.

The coliform tests were run according to standard methods
procedures (194C) and were carried out through the completed
test. Lauryl tryptose broth (Difco) was used for the presump-
tive test because, as was reported by Mallmann and Darby (1941),
this medium will inhibit spore formers and shorten the lag
phase of growth. Since soil is predominantly inhabited by
spore forming organisms, many of which are capable of ferment-
ing lactose with the production of gas, it was felt that the
above medium would eliminate a large number of false positive
presumptive tests. Confirmatery tests were carried out in
brilliant green bile lactose broth. The completed test was
performed using nutrient agar slants and lauryl sulfate tryptose
broth. In running the indole, methyl red and citrate tests
for the coliform identifmajion, eosin methylene blue plates
were streaked with ineculum from positive presumptive tubes
and isolated colonies were picked and transferred to the various
media required for the tests.

Lhtheds for determining he presence of the enterococci

have been significantly improved in recent years. Through

advances in technique, moreover, the enterococci have been
shown.te be much more numerous in polluted sources than was
previously thought. Prescott and Baker (1904) gave a me hod
for the detection of enteric streptococci which allowed the
sample to incubate for four to five days in lactose broth;
by the end of this time period the coliforms died out and

he streptococci predominated. The resulting growth was
then observed micrOSCOpically for ceccus forms. This is the
method upon Which much of the work te_date has been based.

In 1940 Hallmann developed a medium for the enterococci
which included sodium azide as an inhibitor of gram negative
organisms. This medium is selective for enterococci and gave
better counts than any previous methods. Hajna and Perry (1945)
independently devised a medium quite similar to Mallmann's,
the only difference being that Hajna and Perry's medium re-
quires an incubation temperature of 45 degrees centigrade.
hallmann and Seligmann (1950) made a study of various media
for the detection of the enterococci. Their data indicated
that azide dextrose broth is the best medium of the group test-
ed which included standard lactose broth, sodium azide broth
(Mallmann, 1940), and Hajna'and Perry's S. F. Broth. This me-
dium, dextrose azide, was chosen as the enrichment medium to
be used in testing for the enterococci in soil. The azide
dextrose broth could not be used as a direct presumptive test
because the sedimentation of the soil in the sample confused

the readings as to whether growth was present or absent. All

10

l.bnilr 5.. o to. Ark-Hunfiflr‘

tubes of the aforementioned medium were assumed to be positive,
and transfers were made from these into ethyl violet azide
broth. Litsky, Hallmann and Fifield (1955) reported on the
use of ethyl violet azide broth as a confirmatory medium for
he enterococci. They found this medium to be highly selec-
tive and to give higher counts than any other medium yet used.

Isolation of the streptococci, for identification, was
done by the pour plate method. Transfers were made from posi-
tive ethyl violet azide tubes to brain heart infusion broth
and allowed to incubate at 55° C for 24 hours. Serial dilu-
tions of these broth cultures were carried out and pour
plates, using brain heart infusion agar, were made. After
24 hours incubation isolated colonies were picked and placed
in brain heart infusion broth again, and the above procedure
was repeated. In this_way two pour plate dilutions were made
before a colony was considered pure. The ethyl violet is
bacteriostatic against gram positive spore formers, and when
enrichments are made from media containing this substance the
gram positive rods will grow; one must, therefore, be careful
when trying to establish a pure culture. Kicroscopic checks
were made to insure that the isolated organism was not a gram
positive rod.

In making identifications of the enterococci two distinct
steps must be carried out; first, one must ascertain whether
the isolated colony fits within the group referred to as the

enterococci; second, it must be identified as a single species.

11

In this work Bergey's manual (1948) was used as the authority.
The writer is aware that this method is not very accurate
as has been shown by Sherman (1937), Sherman, Stark and
Yawger (1938), and Sherman (1958), but some standard of
reference was required for the sake of clarity.

To justify placing the isolated streptococcus into the
enterococcus group it must grow at 45° C and in broth con-
taining 6.5 percent sodium chloride. These are the most im-
portant requirements. Also included in the identification
were reduction of litmus milk, growth in ethyl violet azide
broth and a microsc0pic examination which indicated the
presence or absence of gram positive streptococci. To de-
termine to which of the four species included in the group
enterococcus the organism belonged, the following schema was

used (Bergey's manual):

I. Not Beta Hemolytic
a) Gelatin not liquefied. ‘g. fecalis

b) Gelatin liquefied. S, liguefaciens

 

II. Beta Bemolytic

a) Kannitol and Sorbitol fermented. S, zymogenes

 

b) Mannitol and Sorbitol not fermented. S. durans

12

DISCUSSION

Tables I through VI show the results of the laboratory
tests on the soil samples taken from the various experimental
fields. In Table I both the logarithmic and the numerical
averages of the number of enterococci and coliforms per gram
of soil are given. The logarithmic average was determined
by taking the sum of the logarithms of the number of organ-
isms per gram of soil in each sample and dividing this by
the total number of samples taken in that particular series.
The antilog of this number is called the logarithmic average.
of the number of organisms present in the samples. It should
be noted that the logarithm of one equals zero; therefore, a
negative sample may be considered to have from zero to one
organism per gram. The logarithmic average is the most rep-
resentative, as it adjusts for wide variations in the number
of organisms present; it gives a less misleading average when
there are a number of negative or low count samples with one
or two high count specimens which, when using the whimerical
average, will make a higher average per sample than actually
is the case. In most cases the 10garithmic average will be
lower than the numerical except when the latter is less than
one.

Fields treated with peat in 1950 and 1951 gave entero-
_coccus counts of zero with the exception of one series (Table

II) from a field treated in 1950 in which one sample gave a

13

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l4

count of two organisms per gram, or a logarithmic average
for the field of one organism per gram. The number of
coliform organisms remained relatively constant for both
years represented, he number being quite low, ranging from
two to five organisms per gram of soil. A field treated
with peat was considered non-polluted, which consideration
was supported by the very low coliform count and illustrated
even more strikingly by an essentially negative count for
the streptococci.

Fields treated with sewage sludge gave much the sam
picture as (was obtained from) the peat-treated soils. The
coliform count was somewhat higher with a logarithmic aver~
age of 59 organisms per gram in one series; the number,
however, still would not be considered of sanitary signi-
fiCance. The enterococcus count was completely negative
in all samples taken, again giving striking evidence of no
pollution. It is of interest to note that the coliform count
was higher in those plots which were treated in 1951 than
those treated in 1955. This could possibly be due to the
achievement of a natural balance in the former plots with a
low rate of reproduction, while the latter has not yet
reached such a stage.

The soil plots that had been treated with straw manure
gave results that were somewhat different from the aforemen-
tioned two plots. In this case there were a number of samples

which gave positive results for he enterococci as well as

15

TABLE II
TYE NUKEZR OF COLIFORIS AID EITEROCOCC

N SOILS TREATED WITH 2A

 

Date of samnle Date of Organisms per Gram of Soil
A LL: * Ya q , +-
Application No. Sampling 922:3223é strepiocoqqi

 

V V

Fall 1950 6/2/55 0 o

H 75

(0

N O
I! 18

(O CO \1 0‘: U1 rh (fl [0 l-’
H
(n O O O O

H
H O
(n
\
H I:
(D
\
01
C»!
O 0

Fall 1951

10
gs
01

9/17/53 0

" 75

OOOOOOOOOOOOOOOOO

(OCDQOWUIrfiCR

O
I! O
O

I!

 

16

TABLE I I I

THE 1111175333 OF COLIFORLB AITD EEIT‘zI’LOCOCCI
I}? SOILS TREE; ED ‘."JITH 83.17.5133 SLEDGE

 

 

 

Date of Sample Date of Organisms per Gram of Soil
Application No. Sampling Califorms Streptococci

Fall 1951 1 5/19/55 15 o

2 " 80 o

3 " 45 o

4 " 45 o

5 9/25/55 0 o

5 " 155 o

7 " o o

8 " O 0

Spring 1955 1 5/27/55 0 o

2 " 55 o

:5 " O o

4 " 45 o

5 " O o

5 9/25/55 0 o

7 " O o

8 " O o

9 " 56 o

10 " o o

 

l7

TABLE IV

 

 

 

 

TIE NUHBBR or COLIFORHS Ann 593293000001
IN SOILS TREATS WITH STRAW wirtRS

Date of Sammle Date of Organisms per Gram of Soil
Application No. Sampling COlifOPmS Streptococci

Fall 1951 1 5/22/55 0 o

' 2 " 215 0

5 " 55 15

4 " O o

5 " 15 450

5 9/20/55 0 0

7 " 0 0

5 " 15 0

9 " O o

10 " o 0

Spring 1955 1 5/25/55 0 12

2 " 75 5

5 " 450 450

4 " 45 12

5 " 215 57

5 9/20/55 0 0

I7 I! O O

8 " O O

9 " 1450 12

10 " 0 0

 

18

TABLE V
THE NUKBJR OF COLIFORKS AID ENTEROCOCCI

FOUND IK EIGHT SAKPLES OF "VIRGIN SOIL"

 

 

 

 

 

 

Sample No. ' Date of Sampling Organisms per Gram of Soil

Coliforms Streptococci
1 7/17/55 45 1
2 " 55 O
5 " 75 0
4 " 570 12
5 " 1100 22
6 " 195 O
7 " 4500 450
8 " 215 2

TABLE VI

THE L'HBER OF COLIFORHS AKD EK'EROCOCCI FOUND IN
CONTROL PLOTS These soils have had no treatment)

 

Sample No. Date of Sampling Organisms per Gram 0f 8011

 

 

 

Coliforms Streptococci
1 7/7/55 1450 450
2 " 15 ' 8
5 " 18 O
4 " 45 12
5 " 45 O
6 " O O
7 " O O
8 " O O
9 " 18 O

 

19

the coliform organisms. Again the number of coliforms was
low. The plot treated in the fall of 1951 gave an average
(logarithmic) count of 11 coliform organisms per gram when
sampled in the early summer while the plot treated in he
spring of 1955 gave an average count of 51 per gram when
sampled at the same time. This difference in count is to
be expected, as one was treated two years previous to sampling
while the other was treated in the same year. Both revealed,
as did all the plots except those treated with peat, a de-
cline in he number of coliforms during the summer. The
enterococci counts were much lower than that of the coliforms
in these plots, and fluctuated with the coliform count, a
lowering in the number of coliforms being accompanied by a
lowering in the number of enterococcus which dropped to zero
in the case of the plot treated in 1951.

he significance of the streptococci in low numbers in
the straw manure-treated plots is difficult to evaluate.
Their presence L y be due to contamination by small animals
or to the cow dung present in the manure. There is a large
population of small animals on these fields, and many workers
have shown that these animals harbor enterococcus organisms
in their intestines. The larger number present (26 per gram)
in the plot treated in 1955 is possibly due to both contami-
nation from the straw manure itself and from small animals.
The fact remains, however, hat only one out of 57 samples

taken from the peat and sewage sludge-treated fields was positive

20

for the enterococci. Animal contamination, therefore, may

not be as significant as we believe. These plots, sewage-
sludge and peat treated, are adjacent to those treated with
straw manure and yet have a negative incidence of enterococci.
Such evidence seems to indicate that the incidence of intestinal
streptococci in straw manure is due to the presence of cow dung
rather than animal contamination as all the plots would be
frequented by the same animals. A very low incidence of en-
terococci was found in the control plots and in "virgin soil".
In these cases the soil is supposedly unpolluted: the con-
trol plots having had no treatment and the "virgin soil" having
been taken from a heavily wooded area in which the soil had

not been tilled. The enterococci count was low and relatively
stable in both soils, three to four organisms per gram. The
best explanation for their presence seems to be contamination
by small animals present in the area. The same problem, how-
ever, arises: some of the fields exposed to animal contamina-
tion have negative enterococci counts.

It is interesting to note that the enterococci count
remained fairly constant in the "virgin" and untreated soil
while the coliform count varied from 15 per gram in non-
treated soil to 248 per gram in the "virgin soil".

To test soil from a source known to be polluted two sam-
ples were taken from.the bank of the Red Cedar river, the water

of which is known to be contaminated. Here the logarithmic

21

.II, .E I?» . K‘tbi..fl.+ia1§.r‘

averages of the coliforms and streptococci were quite high
in comparison to samples taken from the test plots, (see
Table I) 306 per gram for the coliforms an‘ 198 per gram

for the enterococci, thus showin that in polluted samples

there was an increase in the numbers of coliforms and a
very marked increase in the streptococci present. The
average (logarithmic) coliform count for virgin soil was
248 per gram, and in the river bank sample, 306 per gram.
The small difference makes an evaluation between the two
very difficult in terms of coliforms alone, but in conjunc-
tion with the enterococci content there was qlite a large
difference between the polluted and non-polluted samples.

An examination of Table VII shows the types of coliforms
isolated from the soils tested. All of them were proved to
be coliforms by the completed standard methods test. The
majority, 72 percent, was of the Aerobacter aerogenes type,
which would be expected in most soils. Twenty-two percent
were aerobacter intermediates that produce acid in peptone
broth (methyl red positive). Three percent were E, 29;;
intermediates utilizing citrate. All of the coliform organ-
isms isolated from these soils were able to utilize citrate.
When streaked on eosin methylene blue agar none of these organ-
isms gave a metallic sheen which is typical of E}.Eflli° In
comparison of these, two samples were taken from the banks of
the Red Cedar river and run through the same process as used

for all the samples. When streaked on eosin methylene blue

22

agar typical E, ggli_colonies develOped and strains of E,
ggli positive for indole and methyl red and negative for
citrate were obtained. This demonstrates that typical E,
coli can be shown to be present by the methods used in
handling these samples.

From the above we have not much choice other than to
assume that E, Egli_was not present in any of the samples
tested with the exception of those taken on the river bank.
If we use g, 221; alone as the standard of pollution, hen
none of the plots can be considered even slightly contaminated.,
This would assume that the intermediates are of no consequence.
many workers have stated, however, that the intermediates of
the coliform group, including those that utilize citrate, do
have some significance. If this is the case, then possibly

he presence of members of the enterococcus group may aid

in evaluating their significance. For example, the absence

of E, ggli_in the fields treated with sewage sludge was a good
indication that there was no contamination present. This was
supported by the fact that the enterococcus count was negative.
However, in the fields treated with straw manure no 2, ggli_
was isolated, and the coliform count was roughly the same as

in those fields treated with sewage sludge, but in this case
enterococci may indicate that there is a small amount of fecal
contamination present and therefore indicates that the coli-
forms present in these particular samples may have been derived

from a similar source of contamination. If this is true then

25

TABLE VII
DIFFEREHTIAL REACT 033 AKOUG COLIFORIS ISOLATED

 

30° 0* Indole Methyl Red Citrate Percent

 

Isolations
2s - - / 72
7 - / / 22
1 / - K 3
1 x ,1 x a
Total 52 100

All organisms tested gave positive completed tests

None of these organisms produced a sheen on E.K.B.

 

24

the enterococci would be of great aid in evaluating the presence
of coliform intermediates in regard to sanitation.

It should be noted that no soil plot showed any signi-
ficant predominance of one intermediate over another; all of
the fields gave similar results.

Table VIII gives the results obtained in identifying
the types of enterococci present in the soil samples. The

predominant type was Streptococcus liqpefaciens with 72 per-

 

cent of the isolates belonging to this group. Seventeen

percent of the enterococci were S. gymogenes and 11 percent

 

S, fecalis. No member of the S. durans species was isolated.
The results of these identifications can not be relied upon
completely because of the variations that may occur among
these organisms in the various biochemical tests prescribed
by Bergey's manual. As was stated previously, however, some

standard reference was needed and at present this classifica-

tion is the most acceptable.

In testing soil for coliforms a great number of false
positive presumptives occurs due to the tremendous load of
spore forming organisms that have the ability to ferment lactose
with gas production. In view of such a condition it was neces-
sary to perform completed coliform tests on all samples which
gave positive presumptive results. Table IX shows graphically
and tabularly the number of false positive presumptive and
confirmatory tests. Eighty seven and six-tenths percent of

all the samples taken gave positive presumptive tests, while

25

TABLE VIII
TVPES OF EN EROCOCCI ISOLATED FROM SOIL

 

 

Species Number Percent
S. durans O 0
S. fecalis 2 ll
S. liquefaciens 13 72
S. zymogenes 3 17
Total IQ” ICU

 

26

only 45 of these or 55.2 percent of all the samples gave
positive confirmed tests. Of these 45 samples only 57 were
shown to be coliform organisms by the completed test, that
is, of all the samples tested only 45.6 percent were coliforms.
The analysis of the soils for enterococci using ethyl
violet azide broth as the selective medium gave no false
positive results. Positive tubes were checked microscopically
and all contained gram positive cocci in chains. At no time
were any other organisms observed to be present. All the iso-
lations made for typing the organisms were taken from the
ethyl violet azide medium and in every case enterococci were
isolated. The number of false positives in the dextrose
azide broth, which would be used as a presumptive medium if
water or like samples were used, could not be determined be-
cause the amount of sedimented soil which was present made
reading the tubes very difficult. It is most probable that
the number of positive azide dextrose tubes that do not con-
tain enterococci would be great, because this medium.will not
inhibit gram positive rods of which there are many in the soil.
It is not known how many false negatives occurred with the two

organisms as no tests were made for this purpose.

27

TABLE IX
RESULTS OF STAEDAR IETIODS

COLIFORK TESTS ON SOIL SAIPLES

 

=7
- ¢
Completed [ _I

 

Confirmed

 

 

 

 

.+

+

Presumptive 1‘

.O 10 20 5O 4O 50 60 70 80
Number of Samples Tested

 

 

Test Number of Number of Percent of
Samples Positives All Samples
Presumptive 81 71 87.6
Confirmed 71 45 55.2
Completed 45 57 45.6

 

SUMILARY

Coliform organisms were found to be present in all
fields tested, and, in most instances, in low numbers. No
typical E, coli was isolated from any of the samples. All

the coliforms found were A. aerogenes or intermediates and

 

all were able to utilize citrate. The sanitary significance
of these coliforms is questionable as they were found in simi-
lar numbers in both polluted and unpolluted soils.

The enterococci were found to be present in some of the
soils that would not be considered contaminated, namely
"virgin soil" and untreated soil; however, they were present
in very low and relatively constant numbers. They were marked-
ly absent from sewage sludge and peat treated soils. In con-
trast, they were present in much higher numbers in polluted
soil taken from the bank of the Red Cedar river. The entero-
cocci present in the soils sampled were predominantly g.

‘7 "i

liquefaciens followed by §, zymogenes and g. fecalis. no 23

 

 

durans was isolated.

The enterococci were always present in lower numbers than
the coliform organisms but no definite ratio between them was
observed. At no time was there a sample positive for the
enterococci and negative for the coliforms, but the reverse of
this happened quite often. There was an effect on both the
coliform and enterococci content in the test soils during the

summer months: both dropped in number during this period.

29

,i.s‘hv.5l'urfiltgi‘
, . .

The presence of the enteric streptococci in a soil may

aid in evaluating the significance of £3 aerogenes and inter-

 

mediate coliform organisms that are present.

50

|4€~gIl.I.IIIJ.IJ
, . .. u . .. r

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55

 

     

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