This is to certify that the
thesis entitled

Studies of the hteric Bacterial Flora. of
Raw Savage at East Lansing, lichigsn

presented by

u
- -v— _———.—.—F_—.—.--——’—-—-.
.
.

Irving Dahljeh

has been accepted towards fulfillment
of the requirements for

"mu—1'3 degree 1112;922:9198?

“‘n’k

Maj professor

Date-M

 

 

SWSOTMWICMCTERMWOIMNM

L'l'
HST WING. MICHIGAN

by
Irving Leno: 3113.11

A THESIS

Submitted to the School of Graduate Studies of Michigan
Stete College of Agriculture end Applied Science
in pertiel fulfillment of the requirements
for the degree of

mm 01' some:

Depot-ant of Bacteriology end Public neelth

4

195°

THESIS

“WW

the writer wishes to eeknowledge the assistance end ed-
wice of m. I. L. Hellman 1n the work end the 1:.in suggestions
offered from time to tine by m. I. Olitsky with reference to the
psrsoolobeetrun group of organisms. Acknowledgment 1s else node
of the courtesy extended to the writer by the nichtgsn Department
of Health labor-stories 1n the becteriophsge typing of organisms
suspected of hetng pethogens.

23395383

mm or com

Imowcmxou......... ....... ...... 1
nsmxcumm.... ............ . ....... 2
METHODS or Isonnon.......... ............ 5
minimum smnsmnu... ........... 10
mscussxon.............. .................... . 17
oorcLusIons................... ....... 21
mu ..... 22

mmsWHm... .................. .. 25

INTRODUCTION

m isolation of enteric pathogens from sewage has been re-
ported in the literature on a number of occasions in recent years.
lost. if not all. of the pathogens isolated were obtained from sewage
originating in areas where the occurrence of enteric infections was
either endemic or epidenic. furthermore, the methods used in the
identification of the organisms isolated were often incomplete and
render the results reported of questionable value.

A review of the literature revealed that no study has been
reported on attespts to isolate enteric pathogens from sewage origi-
nating in an area where the occurrence of enteric infections my be

considered to be rare or non-existent.

Because of the present tendency to use sewage and sewage
solids for the purpose of soil conditioning, it appeared advisable
tonaheastudyofrawsewage fronsuchasourceusingwarious nedia
which are currently advocated as being emerior for the isolation of

enteric pathogens. his thesis presents the results of this study.

HISTORIGAL m

'31s isolation of Salmonella typhosa free water was recorded
in the literature as early as 1895. Iillson (l) in 1905 summrised
the instances in mch g. tnhosa had been isolated from contaminated
drinking water up to that date. According to hia. banner reported
that up to 1895 there were 65 cases in which it was claimed that _s_.
tnhosa had been isolated from water. lillson states that. 'It is
possible that in some of these the bacillus was really that of typhoid,
but the evidence of identity is now known to be insufficient and none
of these cases can be accepted as conclusive." The 1895 isolation was
from the Berlin water supply. he isolated organisms exhibited all
the then-known characters of g. typhosa and were afterwards confirmed

as such by Pfeiffer's reaction.

hon 1895 until 1905. various workers have reported the iso-
lation of the typhoid organisn free 11 water supply sources. Since
1905 (2), there are 13 instances in which _8_. ms; or related enteric

pathogens are reported to have been isolated from water or sewage.

Special nention should be made of the work of the London
letrepolitaa tter Board Laboratories which is. perhaps. the most com-
plete and carefully controlled. Immated by the late Sir Alexander
newton. the 1913 report and each report from 1927 to 1933 (5 to 15)
carries sons reference to isolations of enteric pathogens. In the
19133 report, 23,353 non-lactose fermenting colonies developing on
plates planted naialy with eagles ef London (wring ) or Bendon sew-
age and also individual samples froa Dublin. Belfast. Edinburgh

and Aberdeen were examined. He was unsuccessful in isolating a single
g. mhosa. and concluded that the typhoid organism is not uniformly
present in 0.00066 ml. of crude sewage, and that this amount of sew-
age contains 70” excretal bacteria per ml. Benton. in calling

attention to the difficulties of the task, writes:

'Sewage contains such an enornous number of
bacteria that it is quite impracticable to examine
nore than a very small amount of this material.
for exanple. to examine one cubic centimeter of
crude sewage for typhoid bacilli by the direct
plating method would nean making at least 1,000
mcial plate cultures and at least 20,000 primary
subcultures, not to speak of the secondary and
other cultures for the purpose of differentiation.
It is, however, quite practicable to work with as
much as 0.01 c.cm. of sewage spread over from 10
to 20 special plates, and therefore to nabs 200
to 1100 pri-ry ennui-«J

lilson (16) states. ”Ion-lactose fermenting organisms are
so cemon in sewage that many thousands or hundreds of thousands of
such colonies would require to be examined in order to isolate a
single typhoid bacillus. ror example, in Belfast sewage I find that
on an average l100.000 to 500.000 organisms develop from 1 c.cn. on
lacOonhy lactose bile salt agar plates. and that of these more than
one-half are non-lactose fermenters. In such sewage I have found

about one typhoid bacillus in each cubic centinetre, so that. using

-3-

and Aberdeen were examined. He was unsuccessful in isolating a single
g. tnhosa. and concluded that the typhoid organism is not uniformly
present in 0.00066 1111. of crude sewage, and that this amount of sew-
age contains 70“ excretal bacteria per ml. Houston. in calling
attention to the difficulties of the task, writes:

'Sewage contains such an enormous number of
bacteria that it is quite impracticable to examine
more than a very small amount of this material.
for example. to examine one cubic centineter of
crude sewage for typhoid bacilli by the direct
plating method would mean making at least 1,000
mcial plate cultures and at least 20,000 priury
subcultures. not to speak of the secondary and
other cultures for the purpose of differentiation.
It is. however. quite practicable to work with as
much as 0.01 c.cm. of sewage spread over fron 10
to 20 special plates, and therefore to make 200
to ‘100 primary cultures.'

Iilson (16) states, “Ion-lactose fermenting organisms are
so cemnon in sewage that many thousands or hundreds of thousands of
such colonies would require to be examined in order to isolate a
single typhoid bacillus. for example, in Belfast sewage I find that
on an average l000.000 to 500,000 organisms develop from 1 c.cm. on
hacOonhey lactose bile salt ear plates. and that of these more than
one-half are non-lactose fermenters. In such sewage I have found

about one typhoid bacillus in each cubic centimetre, so that, using

-3-

and Aberdeen were examined. He was unsuccessful in isolating a single
g. typhosa. and concluded that the typhoid organism is not uniformly
present in 0.00066 ml. of crude sewage, and that this amount of sew-
age contains 70” excretal bacteria per ml. Houston, in calling

attention to the difficulties of the task, writes:

“Sewage contains such an enormous number of
bacteria that it is quite impracticable to examine
more than a very small amount of this material.
for example. to examine one cubic centimeter of
crude sewage for typhoid bacilli by the direct
plating method would mean making at least 1,000
spgcial plate cultures and at least 20,000 primary
subcultures, not to speak of the secondary and
other cultures for the purpose of differentiation.
It is, however, quite practicable to work with as
much as 0.01 c.cm. of sewage spread over from 10
to 20 special plates, and therefore to make 200
to 1600 primary cultures.'

Iilson (16) states. Won-lactose fermenting orpnisms are
so cannon in sewage that many thousands or hundreds of thousands of
such colonies would require to be examined in order to isolate a
single typhoid bacillus. ror exasple, in Belfast sewage I find that
on an average l{00,000 to 500.000 organins develop from 1 c.cm. on
lacOonkey lactose bile salt agar plates, and that of these more than
one—half are non-lactose fermenters. In such sewage I have found

about one typhoid bacillus in each cubic centimetre, so that, using

-3-

the meonkey medium, there would have been a chance of isolating the
bacillus if 250,000 non-lactose fermenting colonies had been tested.
In all probability, even after such a Herculean effort, failure would
have resulted, as the chances are that the typhoid bacillus would not
have a clear space on the plate to develop, and its growth would have
been obscured and inhibited by the coliform colonies. The addition
of brilliant green to the medium would doubtless render the isolation
of _B_. tugsus (_s_. typhosa) from sewage not quite so difficult, but
even so the chances against a non-lactose fermenting colony being
composed of typhoid bacilli would be many thousands to one.‘'

Kehr and Butterfield (11) in 1918 presented a review of the
results of some recent attempts to isolate pathogens from sewage and
an interesting discussion of the indicated relations among the g. 31-
2132 density of sewage and water, the coliferm density and the
typhoid fever morbidity in the co-unity.

Dunlop (18) reported on the isolation of typhoid, dysentery
and salmonella organisms in the final effluent of the Denver sewage
disposal plant. lbre recently. Dunlop (19) has reported on the quan-
titative isolation of pathogens from sewage. his work, however,
cannot be accepted as conclusive because the evidence of identity is
insufficient. For example, he assumes that turbidity in an enrichment
medium is presumptive evidence of the presence of g. mhosa, and the
appearance of non-lactose fermenters on solid differential media and
characteristic reactions of the Salmonella or Shigella in Kligler's

iron agar results in a completed test.

gthods of W“ and Related Pathogens from _!ater or 39.92

 

 

 

Various methods have been used or suggested for the isola-
tion of pathogens from water or sewage. All of the methods fall into
three main groups: (a) Physical concentration produced by agglutina-
tion, chemical precipitation, centrifugation or filtration followed by
isolation on differential solid media with or without enrichment:

(b) isolation on differential solid media after preliminary cultiva-
tion in selective enrichment medium: (c) direct isolation on differ-
ential solid media.

{the various early differential and enrichment media have
been adequately reviewed and discussed by Prescott (2) and lilson
(16). h good purpose could be served by enumerating and discussing
these media here. Mfice it to say that the media more recently
developed have proved to be far superior but are not all that is to
be desired.

Laine (20) in her work reviews the development of more re-
cent media and discusses the author's reasons for race-ending each

media.

Sons of the media used in this study have presented problems
not ordinarily encountered when the media were used for the isolation
of enteric organisms from other sources, such as feces or urine. I'or
this reason, the media used will be considered in some detail, even
at the risk of presenting material adequately covered elsewhere. as

specific problems encountered will be considered in the discussion.

hodifications of some media were attempted. a discussion
of these is not included as it could serve no purpose other than add
to already existing confusion.

for the purposes of enrichment the udia most favored today
are sodiu tetrathicnate broth and selenite broth. Sodium tetrathi-
cnate broth was first found to be useful as an enrichment medium by
meller (21) and reported by him in 1923. be usefulness of the
media is based on the regulation and inhibition of the physiological
activities of contaminating organises by sodims tetrathionate. this
is formed by the reaction. of sodium thicsulfate and iedine. Iodine
is inhibitory to gram-positive organisms and the bile salts contained
in the medics inhibit the non-intestinal types. Pretecse peptone aids
as a buffer against too great a chanp in the reaction of the ndiu
and serves as a ready source of energy for the bacteria. Calcium
carbonate serves to maintain an alkaline reaction.

Idefson (22) in 1936 described selenite I' broth. me devel-
opment of this medium was based on the observations of M1 and
meodoraecu who, according to Oath (23), observed that Echerichia
931; was such more susceptible to the toxicity of sodiu selenite
than was 5. m. Idefson showed that the selenite broth was not
sufficiently toxic to inhibit fecal coli and enterococci coqletely.
mwever, he found that the colon bacilli were refined in numbers
during the first 8-12 hours and thereafter increased rapidly. he
typhoid bacilli multiplied fairly rapidly from the start. Proteus
and pseudomonas were not inhibited, but dysentery and alcaligenes
were inhibited.

‘Pruary plating media are used for initial isolation of
orpnisms. the composition ef each medium is usually such that it
will readily differentiate groups of organisms, or be selective for

a particular group of oranisms.

hedonhy's ear (23) described in 1905 continues to enJoy
popularity as a differential medium. his present modification not
only serves to differentiate strains of g. m from members of the
coliform group, but has the added advantage of supporting excellent
growth of all Shigella and Salmonella strains. the differential ac-
tion of this -diu is clear and distinct. Isolated colonies of coli-
ferm bacteria are described as being brick red in color and may be
surrounded by a sons of precipitated bile. fie reaction is due to the
action of acids, produced by fermentation of lactose. upon bile salts
and the subsequent absorption of neutral red. uphoid, paratypheid
and dysentery bacilli have little effect on the appearance of the
medium. dram-positive organisms are inhibited by the selective
bacteriostatic action of crystal violet.

8. 8. (Mlle-Salmonella) ear, developed by Difco Imb-
oratories, was devised to provide differentiation of lactose fermenters
from lactose non-fermenters. and to give maxim. inhibition of coli-
form ermisms without restriction of the growth of pathogenic gram-
negative bacilli. Shigella, Salmonella and other organisms not
fermenting lactose form opaque, transparent and translucent uncolored
colonies which generally are smooth. Lactose fermenting organism
which my not be inhibited are generally recognised by the formation
of a red color in the colon. some coliform colonies do not show a

-7-

definite red color, being pink or nearly colorless with a pink center.
dole Proteus and Salmonella types produce black-centered colonies; a
characteristic large white or cream-colored opaque and mcoid colony
my be developed by some types of aerogenes.

Bismth sulfite agar as mdified by Iilson and Blair (23)
is a selective medium used by almost all investigators in recent years.
the cognition of this media is based on the fact that the Salmonella
areable to reduce sulfites to sulfides. In the presence of a far--
mentable carbohydrate and metallic salts, this reduction results in
the media surrounding the colonies becoming blacloened. he acids
produced by the organisms from the carbohvdrate, which serves as an
indispensable source of energy, facilitate the characteristic changes
by bringing the metallic salts into solution. mini-at green pre-
motes the blackening of the Salmonella colonies and aids in the inhi-
bition of the colon group. bis-1th in ccmbimtion with sodium sulfite
causes a suppression of lecherichia 29;; without reparably suppressing
the development of the Salmonella colonies. sodium phesphate is used
as a buffer to absorb excess acids produced.

Iaine (20) used Boyle's brilliant green acid fuchsia ear
which was recomended by cruickshank (25). She confirmed the finding
that the medium was particularly inhibitory to Proteus. he mediu
had the advantage of eisplicity in preparation, and good colonial
differentiation was obtained. Sodium taurocholate enters into the
compositien of this Indium and seems to stimulate the growth of en-
teric pathogens when present. At the same time, it is inhibitory to
non-intestinal organisms. Hilliant men serves to inhibit the

growth of gram-positive organism, while the decolorised acid fuchsin
indicates the production of acid resulting from the fermentation of
lactose.

Jeter and lynne (26) developed and described acid fuchsin
methylene blue agar. his is a medium which was formulated to provide
a mre satisfactory differential medium essentially free from objec-
tions described as conon to other media of primarily differential
nature. are objections listed for ether media are: (1) Generally,
only colonies ef lactose fermenters are colored, whereas the color-
less colonies of non-fermenters tend to be naked by the diffusion
of dye from fermenting colonies. (2) the medium may be too toxic
for the growth of delicate enteric species. (3) Deterioration may
occur on exposure to light. (it) weak fermenters my not be differ-
entiated from non-fermenters. Acid fuchsin methylene blue agar con-
tains, in addition to peptone, lactose and agar, a coflination of
acid fuchsin and methylene blue buffered at an optima concentration
of 0.3 per cent. Colonies of lactose fermenters take on the red
color of the acid dye, and colonies of non-fermenters take on the
blue color of the basic dye. m. is possible because the dyes do
not combine chemically. The optimum buffer concentration of 0.3 per
cent prevents the making of non-fermenting colonies by these pro.
ducing acid which occurs at lower concentrations, while higher con-
centrations tend to prevent ready differentiation of weak fermenters.
he recs-ended pH is 6.6. since at appreciably himr values the
increase in adsorption, and therefore in toxicity, of methylene blue
results in dye-sensitive species, such as g. gmnteriae ship and
.i_. faecalis failing to develop.

-9-

WW S‘NDIES

a study of the organisms occurring in raw sewage. with par-
ticular reference to the incidence of enteric pathogens, should be a

useful contribution to knowledge.

considering the conclusions drawn by Houston (3) as a result
of his failure to isolate 5;. whose, and the statement of Wilson (16)
regarding the incidence of non-lactose fermenting organisms in sewage,
it at once becomes apparent that it would be desirable to concentrate
the bacteria present in the sewage into a small volume in order to in-
crease the probability of isolating any enteric pathogens which might
be present. It would also be desirable, if not absolutely necessary,
to use some form of selective enrichment media to increase the number
of pathogenic organisms and, at the same time, inhibit the growth of
non-pathogens. Various methods of concentration were tried. is a
result of these trials, it was concluded that precipitation of the
sewage using alum would best serve the purposes of this investigation.
Other workers usually add a constant volume of a 10 per cent solution
of elusinnm sulfate to each liter of water or sewage. Iilson (27),
for instance, added 2.5 ml. of a 10 per cent solution of aluminum
sulfate to each liter of water and then adjusted the pH to about 7.
In the present investigation, this method did not give satisfactory

results.

Aluminum hydroxide is amphoteric in nature; the varying
kinds and concentrations of mineral salts present in water or sewage

definitely affect the solubility of the precipitate. hperience in

-10..

water treatment has demonstrated that each water has a definite opti-
mum pH value at which the aluminum hydroxide precipitate is least
soluble, and that the optimum pH value for a given water may vary

from day to day, or hour to hour.

the composition of sewage varies considerably, and it was
decided to carry out all precipitation at the optima pH as deter-

mined by jar tests.

Growth and the production of ensymes by bacteria are closely
associated with or dependent upon the pH of the medium in which the
bacteria are seeded. no subject enteric pathogens, already in an
enviromnent not conducive to their growth, to additional adversity
is not likely to enhance the chances of recovering such organisms.
Porter (28) gives the minimum, optimum and maxismm pH for the growth
of several bacteria. the minimm pH values for all of the enteric
forms listed are above MO. lhen aluminum sulfate was used as the
precipitating agent, the cptismm pH value was often found to be as
low as 3.5. in. effect of such a low pH on the organism, which may
have been adversely affected from the conditions of its environment.
is likely to be death. Aluminum ammnium sulfate was found to give
optimma pH values ranging from lt.6 to 6.6. on last lensing sewage,
and for this reason was selected as the precipitating agent to be

used.

A. number of enrichment media was tried; and in addition,
experimental enrichent broths were devised. None, however, gave

entirely satisfactory results. 01 the enrichment media used,

-11-

Bacto-‘l'etrathionate Broth and Bacto-Belenite Broth showed the greatest
inhibition of the coliform organisms and were selected for enrichment

purposes as representing the most satisfactory of the media available.

For primary plating purposes, a large number of media was
used. Goliform organisms grow abundantly in all media. 011 Dosoxychol-
ate Agar there was a tendency for the coliforms to overgrow or mask
the non-lactose fermenters. It was found that best colonial differ-
entiation could be obtained on Bacto-Bishluth Sulfite Agar. Date-88
Agar. Bacto-IacOOnkey Agar. Bnlliant Green Acid fuchsin Agar and Acid
Fuchsia Methylene Blue Agar. Brilliant Green Acid fuchsin Agar was
particularly useful in suppressing the growth of Proteus, although a

few strains were found to grow on this medium.

:l'or prinry differential screening. Iligler's Iron Agar,
Triple Sugar Iron Agar and urea Broth were used. xliglor's Iron Agar
gave the best differentiation as far as the Paracolobactrum was con-

cerned. urea Broth was ideal for the differentiation of protons.

Secondary differentiation was accomplished using dextrose,
lactose, saccharose, maltose, mannite and xylose in Bacto-Purple
Broth Base. he Mic reactions were also determined. than it was
considered desirable, motility tests were also used.

For final identification of suspected pathogens, agglutina-
tion tests, using specific antisora. and bacteriophage typing were

used.

'Ehe general treatment of the sewage samples is shown in

outline form as hble I.

IFares 2-liter portions of sewage were collected at the sow-
age plant in sterile flasks for each sample. Ono portion was further
divided into ten 200 ml. portions in 1100 ml. bankers, and varying
amounts of aluminum amaonium sulfate solution were added. After thor-
ough rapid stirring, the bankers were allowed to stand and observations
were nde to determine in which beaker the precipitate formed first,
and settled not rapidly, leaving the clearest supernatant fluid above
the precipitate. 'Ihat portion was chosen having the optimam condi-
tions for concentration of the organisms, and the pH value of the
supernatant fluid was determined. this pH value was designated as

the optimal.

no proper amount of alminum amoni'om sulfate solution was
then measured into the remaining 2-litor portions. the flasks were
thoroughly shaken and the precipitate was allowed to settle by gravity
for a period of four hours. The supernatant fluid was decanted until
the remaining volume was about 200 ml. The entire amount of concen-
trate was planted. me procedure generally followed was to plant 1 ml.
of the precipitated sewage directly onto each of the various primary
isolation media without prior enrichment. The remaining precipitated
sewage was planted into the various enrichment broths in ten-fold

serial dilutions ranging from 10 ml. in the first tube to 10-.9 m1.

o
in the eleventh tube. After incubation at 37 0. for 8-12 hours in
the case of Solenito Broth, and 12-21} hours in the case of retrathi-
onato Broth, material from each tube was streaked onto the various

primary isolation media regardless of whether the tube showed visible

.1}.

TABLE!

 

General Outline ShowLng Treatment of Raw Sewage

I. Concentration
Prec p tation using amenium aluminum
sulfate at the optimum pH value for
flocculation
a) Centrifugation of precipitate
and sediment
b) Gravity settling of precipitate
and sediment

II. lurichment
15 Sodium Betrathionate Broth
2) Selenite Broth
a) Primary Isolation
l. Bismuth Sulfite Agar
2. 8.8. Agar
a. laoOonbey's Apr
. Brnliant Green Acid
Zl'uohsin Apr
5. Acid ruchsin Methylene
Blue seer

III. Primary Isolation as in II-a without enrichment

IV. Pr Differential greening
l Iligler's Iron Agar
2) Triple Sugar Iron Agar
3) Urea Broth

Y. Seoo Differentiation
7W
2) Lactose Broth
a; Saccharose Broth
Maltese Broth
5) Nannitol Broth
6) blue Broth
7) IIYid Reaction

VI. Identification
15 nglutination in specific antisera

2) Bacteriophage typing

.1)...

evidence of growth. After incubation at 37° 0.. all plates were ex-
amined at the end of 18, 21} and lle'houro. Only those plates showing
isolated colonies were retained for further study.

An effort was made to secure cultures of all different
types of organisms growing on the plates, although colonies which
showed the typical appearance described for E. coli and A. aeroggnes

were not tahen.

After purification, the mltures were planted in Iligler's
Iron Apr slants. Triple Sugar Iron Agar slants. Urea Broth and Car-
bonrate Broths. me nmc reactions were also determined.

Broth cultures of g. M and g. schottmuelleri were
added to raw sewage and used as controls. The results of this study

are given in 1Ib1e II.

-15..

run 11

Cultures Isolated from Baw Sewage
at last Lansing, Michigg

 

Mber of sewage samples examined.

lumber of colonies studied
"her of Pseudoamnae. . .
lumber of Proteus. . . . .
tuber of Paraoolobactrum.
lumber of Aloaligenes . .
merofg.go_li. . . . .
lumber of ;. aeroggne . .
“er of g. M . . .
Bomber of other Salmonella
Mer of Bhigella . . . .

-15-

.656

323
no

103

DISCUSSIOI

m greatest concentration of organisms in water or sewage,
when using alum as the precipitating agent. was found to occur when
the precipitation took place at the optimm pH value. In the present
investiation. it was found that the optimm pH value when using alu-
minum anonims sulfate was higher than when aluminum sulfate was used
as the precipitating agent. then a constant amount of the precipitat-
ing agent was added to a given volume of sewage, poor settling took
place and the supernatant fluid was turbid, having the appearance of
colloidal material in suspension. Gravity settling for a four-hour
period. when precipitation was accomplished at the optimm pH value.
was superior to centrifugation at 3,300 r.p.m. (3.600 x G.) for a
period of one-half hour.

lhen precipitated sewage was planted in enrichsent mdia,
there was little inhibition of the coliform organisms. IJ'Ehis my have
been due to the massive plantings of coliforms. It is possible that
the great numbers of coliforms were able to overcome the inhibitory
effect of the medium, thus allowing more rapid growth. In the em-
ination of sewage. there is a definite need for an enrichent medias
which will be capable of greater coliform inhibition and at the same
time allow rapid development of enteric pathogens which might be

present.

0f the plating media. Bismuth Bulfite Agar showed the

greatest inhibition of coliforms.

The development of numerous black colonies during July.

-11-

August and September by organisms having the ability to reduce sul-
fites confirmed the observations of lilson and Blair (16). the chief
characters of this organism to which Wilson gave the name B. effluviei
are as follows: A gram-negative actively motile bacillus with growth
on agar resembling g. 923-33 it liquefies gelatin. is medal red nega-
tive and gives a positive Yoges-Proshauer test; it resembles B.
clcacae. from which it differs in its reduction of sulfites and in
being a non-lactose fermenter; it grows in Ioser's citrate solution:
it ferments glucose, maltose. mannite, saccharose (and starch) with
the production of acid and as, and has no action on lactose, dulcite
and salicin. and it forms indol and does not decomcse urea.

Acid ruchsin Brilliant Green Agar was excellent for elim—
inating the troublesome Proteus organisms. It did. however. allow
the more-cr-less unrestricted development of coliforms. IBhe plates
were usually overcrowded with lactose fermenters. making the recog-
nition of lactose non-fermenters difficult. It should be mentioned
that difficulty may be expected in using this medium if the somemt
alkaline pH value of 7.“ is not strictly observed in its preparation.

Salmonellas have a tendency to be inhibited at lower pH values.

Acid luchsin Hethylene Blue Agar had no advantage over
the other plate media in the examination of sewage.

d

Ilotile and non-motile, indol-positive cultures were iso-
lated which produced no pay from carbohydrates when originally iso-
lated. lien: such cultures could easily be classed as Bberthella or
Shigella. but after varying periods of laboratory cultivation. many

-15..

of the strains produced as. Stuart. Wheeler, Rustigian and liner--
man (29) include these in the Paracolobactrum group as anaerogenic
variants of coliform. mey found from serolcgical and physiological
properties that the Paracolobactrum group is intermediate between
normal coliforms and Salmnella. mere appears to be no sharp dis-
tinction between the groups, and a more-or-less continuous series of
types exist. Ii'his group of organisms was the cause of considerable
concern as they were often mistaken for Salmonellas on original

isolation.

During the early part of the study. Hollenbach and DehlJelm
(30) isolated an organism on Bismuth Sulfite Agar which gave the typ-
ical colonial appearance of g. typhosa. When first cultured for
biochemical reactions, the results in lactose and sucrose were atyp-
ical in that acid was produced. continued incubation resulted in
these two media becoming alkaline and thus giving the typical reac-
tions expected of this organism. Subcultures from the original
isolation all gave biochemical reactions typical of those described
for g. mhosa. Agglutination tests were positive in a titre of
1-320, and a culture sent to the Michigan Department of Health
Salmonella Dping Station was reported as having the somatic anti-
gens II and III and the flagellar antigen d. The organism was
sluggishly motile and the presence of ti antigen could not be

demonstrated.

Agglutination tests using the control _8_. ghosa organism
were positive in a titre of 1-5.120 using the same antisera as that
used for the organism isolated from sewage. Iilson and Blair (27)

-19..

when discussing the identification of organisms isolated from sewage
state that. "me identification was completed by seeing whether the
organism was agglutinated to full titre by a typhoid agglutinating

serum. '

-20-

 

CONCLUSIONS

'i'he results of this study indicate that enrichment media,
which are quite satisfactory for fecal samples and urine. will not
adequately suppress the growth of coliforms found in precipitated

raw sewage.

Interic pathogens. if present in raw sewage at last Lans—
ing. Iichigan. were not in numbers sufficiently large to permit
their ready isolation by the methods used in this investigation.

Irhe use of aluminum ammnium sulfate for precipitation
of sewage at the optimum pH value is more satisfactory than the
use of almninum sulfate. A greater concentration of the organisms

was obtained with aluminum ammonia sulfate.

me Baracolobactrum group of organisms often give bio-
chemical and serological reactions similar to the" Salmonellas upon
first being isolated from sewage. mic is especially true of the

anaerogenic strains.

-21-

1.

3.

APPENDIX

Aluminum amonium sulfate solution

Aluminumassnonium sulfate...... ............... .....
m.t111.d nurOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOO.

3K2 grass
1000.0 I1.

One ml. of this solution. when added to a 200 ml. sanple equals

one grain per gallon.

retrathionate Broth (Bacto)

nth base

Proteose-peptone lo. 2 (Difco)..... .......... ......
mm.b11° "lt'eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
calcium carbonate..................................
Sodium thiosulfate........................... ..... .
Distilled water............ ......... . ............ ..

Iodine Solution

Ioum ca‘t‘1.0000eeeeee eeeeee eee eeeeeeeeeeeee eeOO
Pom.iu iouueeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
m.t111.d nt.rseeeeeeeeeeeeeeeeeeeeeeeeee eeeeee as.

5.0 gram
1.0 m
10.0 grams
30.0 grams
1000.0 ml.

6.
5.
0

fit

2

1b prepare 1000 ml. of media. to 1000 ml. 8f broth base which
has been boiled and then cooled to below ‘15 o.. add 20 ml. of
the iodine solution. Shake well to mix and dispense in 10 ml.
quantities in test tubes. taking care to obtain an even distri-
bution of the insoluble material. ‘lhe medium was found to give
better results when freshly prepared and used on the same day.

Selenite Broth (acts)

ww‘mumeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
wt0-mt°..eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo
Disodium phosphate.
8011‘“ “1d “I‘m“eeeeeeeeeeeeeeeeeeeeeeeeeeoeeee

'i'o prepare the medium. 23 grams of the dehydrated medium are
suspended in 1000 ml. of distilled star and heated to boiling.
It is then dispensed into sterile culture tubes to give a depth
of medium of at least 2 inches. hcessive heating is to be
avoided. W autoclave. me final reac-

tion of the medium will be pH 7.0.

-22..

h.

5.

6.

”non, .m‘“000000000000000.0....00000.00.00.00.

lacconkey's Acu- (recto)
(DeMdrated - Difco)

...-

wtO‘PQPtoneeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeee
nonalo-Paptom...ooo...... eeeeeee eseeee eeeeeeee ee
w“-mt0'°eeeeeeeeeeeeeeeeeeeeeeeeeeeeeee eeeee ee
Bacto Bile Salts 30. 3.............................
Sodium chloride....................................
ww-w.eoeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeo
mm‘mtr‘l "doeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseem
mm‘cmt‘l “Olit (DeGe‘a)eeeeeeeeeeeeeoeone.oee e
Distilled water... 1000.0 ml.

né’é’fiifi

o

o.- F
8° Taxonwpu-e
I-‘U

1b prepare the medium. 53.5 gram of the dehydrated medium are
suspended in 1000 ml. distilled water. Boil for one or two
minutes to dissolve the medium. Sterilise for 20 minutes at

15 pounds pressure.

Bismuth Sulfite Agar (Bacto)
(Dehydrated - Difco)

mmmooooceeoooonOOOQOOOeeeeeeeeeeeeeeeeeeeeeeoo
B.“ “untoooeeeeeeeeeeeeeeeee eeeeeeeeeeeeeeeeee a

“It”.........OOOOOOOOOOCOCOOO0.000000000000000.0.

m.°d1mphO'phuseeooooooweeeeeeeeeeeeeeeeeeeeee.

Bi.mth.n1£1t. 1m1c‘wr0000000.0.0....00000......
w...CCOOCOOOOOOOOOOO0.00.00.00.00.0.00.00.00.00.
Brillmt y’enCOCCOOO......OOOOOOOOO......OOOOOOO.
m.t111.d nt'rCOOOOOO......OOOOOOOO00.00.00.000... 10m.0n1.

N H
OMOWWO

mooQoooo
assess
iit55533

.°
8

Suspend all dry ingredients in the distilled inter and heat to
boil as rapidly as possible; then allow to sinner for a minute.
Into sterile Petri plates pour 15 to 20 ml. of the medium.

This medium should not be autoclaved as rolonged heating
destroys its selectivity. rinal pH 7.6 -.

Salmonella and Shigella Medina (Bacto)
(Dehydrated - Difco)

Beef extract............................. ....... ...
mm..'pept°meeeeeeeeeeeeeeeon... ...... eeeoeeooe
mtoao.0......00.000000000000000.00.0.00000000000.
Bile salts.........................................
“um citr‘“000000000O00.00000000000000000000000.
Sodium.thicsulfate.................................
Jerric citrate.....................................
meeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
mill-mt m’neeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
mtm redOOOOOOOOOOOO0.00000000000000000000.0000. 0e
m'tillod ”“rOOOOOOOOOOOO00.0.0.0...O0.0.0.000... 1m.

0 O

...-
“NGOU'IW

OH
Out-i
O.

ofium ox'numa o o o
F???§3???3§

-23-

7.

Dissolve ingredients in distilled water. Steam for 15 to 20
minutes or bring to the boiling point. Do not allow to boil
or do not sterilise. Pour about 20 ml. medium in each sterile
Petri plate. Allow to dry with covers partially removed.

Brilliant Green Acid ruchsin Apr

_Btase medium

mooeoeeeeeeeeee oooooooooo eeeeeeeoeeeeeeeeeeeeeee

MmeOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO000.000...

0
0
Sodium taurocholate....................... ....... .. 5
Sodium chloride.................................... 5.
Distilled water.................................... 1000

Autoclave at 10 pounds pressure for 20 minutes. adJust pH to

7.13 and filter. Add 10 game lactose. mix and distribute in

200 m1. quantities in flasks and sterilise at 12 pounds pres-
sure for 15 minutes.

To prepare plates. to 200 ml. of base medium, melted and cooled
to 50° 0.. add 2 m1. of uni-ow. indicator and mix. Add 0.8 ml.

of freshly prepared brilliant green (1 per cent) solution. Mix
and pour plates.

Acid fuchsin Methylene Blue Agar

Difco peptone. 10.0 grams
lactose........... ...... ...... . 10.0grams
Dipotassium phosphate.............................. 3.0 gram
Acid fuchsin................ ..... . 0.5 gram
lethylene blue.. 0. 5 grams
Agar....... 15.0 grass
Distilled water.................................... 1000.0 m1.

Dissolve ingredients in distilled water by heating. Adjust
pH to 6.6. Sterilise at 15 pounds pressure for 20 minutes.

-2h-

 

 

 

7.

Dissolve ingredients in distilled water. Steam for 15 to 20
minutes or bring to the boiling point. Do not allow to boil
or do not sterilise. Pour about 20 ml. medium in each sterile
Pstri plate. Allow to dry with covers partially removed.

Brilliant Green Acid l'uchsin Agar
48‘ medium

mOOOCOOOOOOOOOOO 0000000000 OOOOOOOOOOOOOOOOOOOOQO

mtomOOCOCOOCOOOOOOO......OOOOOOOOOOOOOOOOOO0....

Sodium taurochclate.................... .......... ..
an“ cmorid‘Oeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeee.
m.t1n°d nt‘reeeeeoeoeeeeeeeeeeeeeeeeeeeeeOOeeeeO

Autoclave at 10 pounds pressure for 20 mimites, adjust pH to
7.1; and filter. Add 10 grams lactose. m and distribute in
200 ml. quantities in flasks and sterilise at 12 pounds pres-

sure for 15 minutes.

To prepare plates, to 200 ml. of base medium, melted and cooled
to 50° 6.. add 2 ml. of Andrade's indicator and mix. Add 0.8 m1.
of freshly prepared brilliant green (1 per cent) solution. Mix

and pour plates.
Acid Iuchsin Methylene Blue Apr

Difco peptone............ ..... .
Lactose........... ....... ................... .......
Dipotassium phosphate..............................
‘cid m111...............s eeeeee eeeeeeeeeeeeeeee'e
lethylene blue.....................................

Wooooeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee.

m'tillod nt'rOCOOOOO0.0.00.0.00...00.000.00.00...

Dissolve ingredients in distilled water by heating.

Adjust

pH to 6.6. Sterilise at 15 pounds pressure for 20 minutes.

-2h-

 

1. I‘illson, H. 8. 1905. The isolation of 3. mm susfrom in-
fected water, with notes on a new process. Jour. an. 5: 1I29.

2. Prescott. 8. 0.. Winslow. Owl. i.. and McCrady. It. ISM-5.
fiter Bacterioloy. 6th Dd. John liley and Sons. Inc..
New York, N. Y.

3. muston. A. c. 191% Tenth Report on Research work to Metro-
politan Water Board. Inndon.

h. undon. Metropolitan tater Board: Twenty-second annual report
on the results of chemical and bacteriological examination of
the London waters for the 12 months ended December 31, 1927.

5. ---------- Twenty-third annual report on the results of chemical
and bacteriological examination of the London waters for the
12 months ended December 31, 1928.

6. -------- - ‘fsenty-fourth annual report on the results of chemical
and bacteriological examination of the London waters for the
12 mnths ended December 31. 1929.

7. ---------- floaty-fifth annual report on the results of chemical
and bacteriological examination of the London waters for the
12 months ended December 31,1930.

8. --------- twenty-sixth annual report on the results of chemical
and bacteriological examination of the London waters for the
12 months ended December 31, 1931.

9. - ------- Twenty-seventh annual report on the results of chemical
and bacteriological examination of the london uters for the
12 mnths ended December 31, 1932.

10. ------- - Twenty-eich annual report on the results of chemical
and bacteriological examination of the London waters for the
12 months ended December 31, 1933.

ll. --------- Twenty-ninth annual report on the results of chemical
and bacteriological examination of the Inndon eaters for the
12 months ended December 31, 1931»

12. ~--------- Thirtieth annual report on the results of chemical
and bacteriological examination of the Iendon waters for the
12 months ended December 31, 1935.

13. ------ -- Thirty-first annual report on the results of chemical

and bacteriolodcal examirmtion of the Inndon waters for the
12 mnths ended December 31, 1936.

-25-

 

 

1’4.

15s

16.

17.

V 18.

19.

20.

21.

23.

21+.

25.

26.

 

-'1'hirty-second annual report on the results of chemical
and bacteriological examination of the London nters for the
12 mnths ended December 31. 1937.

 

Thirty-third annual report on the results of chemical
and bacteriological examination of the London uters for the
12 lanths ended Decoder 31. 1938.

Iilson. I. J. 1928. Isolation of _n_. t beans from sewage and
shellfish. Rrit. lied. Jour. 1: 1.061.

10hr. R. I. and c. T. Butterfield. 191+}. Notes on the relation
between coliform and enteric pathogens. Public Health Reports.

58: 589.

Dunlap. S. G. 1991. '1!» occurrence of typhoid. dysentery and
salmonella organisms in the final effluent of the Denver sewage
disposal plant.” latter of Science 'niesis. University of Colo-
rado. Boulder. Colorado.

Dunlap. S. G. 19149. Bacterial studies of soils and washings of
irrigated fruits and vegetables. Presented at a smosium on
Recent Research in Hilk and Food Sanitation held under the aus-
pices of the Sanitation Study Section. Division of Research
Grants and Fellowships. National Institute of Health. 0. 8.
Public Health Service at Washington. D. O.

Laine. S. L. 19148. "in evaluation of media for the isolation
of Salmonella from feces.' Hester of Science Thesis. Michigan
State college. hit Lansing. Kichigan.

Mueller. 1.. 1923. Un nouveau milieu d'enrichissemsnt pour la
recherche du bacille typhique et des paratyphiques. Oomptes
renthis de la Societe de Biologie. S9: 1&3".

Liefson. I. 1936. New selenite enrichment media for the iso-
lation of typhoid and ratyphoid (Salmonella) bacilli.
“1'. Java m‘e 2“: 3e

liacOonlney. A. 1905. Lactose-fermenting bacteria in faeces.
Jour. mg. 5: 333.

Iilson. I. J.. and Blair. I. ll. Rev. 1927. Use of a glucose
bismth sulfite iron medium for the isolation of g. mhosus
and 3. protons. Jour. Eye. 26: 27“.

Oruickshank. J. 0. 19h}. A brilliant green acid fuchsin medium
for the isolation of Salmonella. Dull. 53's. 18; 505.

Jeter. I. 8.. and lynne. D. S. 19149. Acid fuchsin methylene

blue agar: A new differential medium for enteric bacteria.
Jour. Ract. 58: "29.

-26-

27.

28.

29.

Iilson. I. J.. Blair. 1:. M. McV. 1931. Further experience
of the bismuth sulfite media in the isolation of Bacillus

typhosus and R. arat hosus D from faeces. sewage and
water. Jour. Hyg. 31: 1?.
Porter. J. B. 1946. Bacterial Chemistry and Physiolog.
let M. John Iiley and Sons. Inc.. New York. N. Y.
Stuart. 0. 1... Wheeler. 1. u.. Rustigian. R. and Zimmerman. A.
19%. Biochemical and antigenic relationships of the para-
colon bactetia. Jour. Beet. ‘#5: 101.

Bollenbach. s. 1.. and DahlJelm. l. L. lane. Unpublished
data.

-27-

 

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