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QTUDIES ON PRIMARY AND
SELECTIVE MEDIA
FOR COLIFORM ORGANISMS

Thesis Ior the Degree of M. S.
MICHIGAN STATE COLLEGE

Charles Willis Darby
I943

 

 

 

This is to certify that the

thesis entitled

Studies on Primary and
Selective Media
Ibr Coliform Organisms

presented by

Charles Willis Derby

has been accepted towards fulfilment
of the requirements for

M. 3, degree inmogy

   

Major profe

Date Eggs“; 9. 1933

 

 

 

 

 

 

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STUDIES ON PRIMARY A33 SELECTIVE

MEDIA EC? CCLIFCRM ORGANISES

By
Charles Willie Darby
P

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied

Science in partial fulfilment of the I

requirements for the degree of .

I

MASTER OF SCIENCE

 

Department of Bacteriology and Hygiene

19 LL3

 

THESIS

PREFACE

Because the academic requirements for the Master of Science
Degree were fulfilled on a part-time basis this work was extended
over a period of four years. After the completion of the first
part of the problem, Professor W. L. Mallmann suggested that it
would.be highly desirable to have this work published. Permission
was obtained from the Graduate Council for the publication of
Studies 9}; _l_al_e_d_i_a £9; _C_<_>_liform Organisms. which was published in
the Journal g£_the American water flg§5§_Association, April, 1939.

Studies here then started to devise an improved selective
medium for the bacteriological examination of water. The laborer
tory and field results of these investigations were presented as
a second paper, Eggs 9;; 3.3. Lauryl Sulfate Tryptose M for; as
Detection 9f,Coliform Organisms. This paper was published in
the Americag,Journ§l‘g£_Public'ggalgh, February, l9hl.

A study of these papers will show the necessity for early
publication so that other workers would.have the Opportunity
to check the new media and methods under practical field condi—
tions.

The Graduate Council kindly consented to allow the published

papers to be included, in reprint form, as a portion of this thesis.

ifs}.
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TABLE OF CONTESTS
Page
Introduction

Part I Studies on a Basic Medium for Coliform Organisms ...... ..l

Appended,reprint-~Studies on Media for Coliform Organisms.......

Part II Studies on Bacteriostatic Agents ................... ....h
Experiment 1 ...................... .. ................ ..9
Experiment 2. ..................................... ...12
Experiment 3. ...................................... ..lu
experiment h ...................................... ...17
Experimcn 5. ........................................ 20

Part III Studies on Selective Media for Coliform Organisms....21

A Comparison of Brilliant Green and Ethyl Purple

in a. 3119 Liedium. o 0000000 o ooooooooooooooo o o o o oooooo ?3
Ethyl Purple and Brilliant Green Bile in Tryptose
Broth... .................................. .........2M
Growth Curve Studies of Selective Media for water
Analysis ........... . ............... ...... ...... ....26
Acknowledgment.. ..................... . ........... ... ........... 28

Second appended reprint~-Uses of a Lauryl Sulfate Tryptose
Broth for the Ibtection of Coliform Organisms. ........ . ......

Literature Cited. ........ .. .................................. ..29

 

IEI.ODUCTIOK

A study designed to develop selective media for cultivating
a specific group of bacteria naturally divides itself into three
phases.

The first phase is to develop a.practical basic medium that
will supply an ideal environment for growth. The experimental
work covering this phase of the problem is presented in Part I,
Studies gghg.§g§ig Medium for Coliform Organisms.

Finding an ideal selective agent to retard or eliminate
undesirable organisms is the second.major phase. The experi-
mental work dealing with this phase of the problem is presented
as Part II, Studies gg Bacteriostatic égents.

The third and final phase is to combine the basic medium
and.the selective agent into a selective medium. This medium
must then be subjected to laboratory and field trials for a
final evaluation. These studies are presented in Part III,

Studies 2; Selective Media for Coliform Organisms.

Part I

Smudies on a Basic Medium for Coliform
Organisms

Introduction

Soon after the introduction of quantitative laboratory tech—
nice, bacteriologists began to compare the various media then
available for the growth of different groups of bacteria. From
these studies evolved numerous media, especially a great variety
for the more fastidious organisms.

Because the coliform (1) group of bacteria grow in a wide
variety of the more simple media. relatively little attention
has been given towards developing more suitable media to support
their growth.

Winslow (2), in 1905, demonstrated that the type of medium
and the period of incubation would make radical differences in
bacteria counts from water and sewage.

The first report on investigations concerning media and
technics to be used for water analysis was published in 1898
(3). A permanent Committee on Standard.thhods was formed in
1901 (5). From 1901 to 1925 numerous changes were made in
standard methods, both in the content and.preparation of the
media used and in the methods of executing these tests in the I
laboratory. The latest major changes were made in the report

of the American Committee on Standard Methods (1925).

As early as 1891, Reinsch (6) demonstrated that the

reaction of the medium would cause a marked difference in

bacteria counts from the same water samples. In 1922 Bunker
and Schuber (7) demonstrated the value of haVing a definite
standard hydrogen-ion concentration for all media.used in
water analysis. They recommended a,pH of 6.8-7.2 for ordinary
solid and liquid media.

In 190M Gage and.Adams (8) demonstrated that marked dif-
ferences in counts resulted.from using different peptones in
the basic medium. The same authors demonstrated the importance
of using distilled water in the preparation of standard media.

A definite step toward.uniformity was made when the time-
consuming and inconstant meat infusions were replaced.by
Liebig's or any comparable meat extract (4).

The pioneers in water analysis first relied on agar or
gelatin plates for the primary isolation of coliform organisms.
As early as 1892, Mathews (9) used litmus-lactose-agar plates
for this purpose.

In 1893 Smith (10) demonstrated the value of a primary
enrichment medium of dextrose broth. By seeding definite
amounts of water into a series of tubes a quantitative test
was deve10ped.using only a.broth medium. That this method

was qualitatively superior to the direct plating method was 1
demonstrated by Irons in 1901 (11).
Soon after the introduction of dextrose broth as a pre-
sumptive medium, numerous workers found that only 60 to 70 per

cent of the tubes showing gas in this medium actually contained

coliform organisms. Although these reports were published and

a great deal of discussion resulted. it was not until the 1923
edition of Standagd Methggg that lactose was substituted for
dextrose in the presumptive test.

Although various enrichment media have been devised in
recent years, standard lactose broth has remained unchanged
since 1925. This medium contains the following per liter:
beef extract, 3 grams; peptone, 5 grams; and lactose, 5 grams.
The final hydrogen-ion concentration should be between 6.8 and
7.0.

In practically all of the papers that have appeared in the
literature comparing media used in water analysis. the same
.standards have been used for comparison. They have taken total
counts after one, two or more days' incubation or gas production
in 2h to M8 hours or longer as a measure of efficiency.

The first appended publication, Smudies‘gn‘ggdlanfgp 991;:
form Organisms, presents the data obtained in an effort to de-

vise a more efficient basic medium to be used in the presumptive

test for water analysis.

 

 

 

 

 

 

  

Reprinted from JOURNAL or rm: AMERICAN WATER Wonxs Assocurxon
Vol. 31, No. 4, April, 1939

 

 

 

Studies on Media for Coliform Organisms
By C. W. Darby and W. L. Mallmann

HERE has been considerable doubt in the minds of the writers
as to the value of standard lactose broth as an enrichment me-
dium for culturing coliform organisms from water and other sources
when the organisms have been attenuated or when they appeared
in minimal numbers. The fact that coliform organisms frequently
appear in fermentation tubes after incubation periods longer than 48
hours or in tubes incubated at temperatures below 37°C. would seem
to indicate that the prolonged lag phase of growth might be caused
by an unfavorable environment in the nutrient medium. Little
effort has been made to check the efficiency of this medium by the
development of better enrichment media for comparative test-s.
More frequently this medium has been used as a standard to measure
the value of new selective media in an attempt to produce a new
medium with selected properties for the coliform organism with
enrichment value equal to that of the present standard lactose broth.
The appearance on the market of new peptones which are being
used extensively in. the growth of such pathogenic bacteria as the
diphtheria bacillus and gonococcus offers an opportunity for the
application of these nutrients to the growth of other organisms
such as the coliform group. In this laboratory Bacto—tryptose, a
peptone, which is being used extensively for the cultivation of or-
ganisms that grow scantily on Bacto-peptone media, seems particu-
larly adaptable to this application.
In most studies on the elaboration of a nutrient medium it has
been customary to measure the response of the organisms to the
medium by using a loopful of organisms to each tube of material

A record of research contributed by C. W. Darby, B.A., and W. L. Mall-
mann, Ph.D., Michigan Engineering Experiment Station, East Lansing,
Michigan. This study was made by Mr. Darby in partial fulfillment of the
requirements for the master’s degree.

689

 

 

690 c. w. DARBY AND w. L. MALLMANN [.1. A. w. w. A.

tested and then measuring the maximum growth obtained at the
end of 24 to 48 hours incubation. The medium giving the greatest
amount of growth at this time has been selected as the best. In
most instances, perhaps, this procedure has obtained its end, par-
ticularly if the investigator desires large quantities of organisms.
It is the authors’ contention, however, that the medium giving "the
greatest number of organisms in 24 or 48 hours incubation is not
necessarily the best diagnostic medium. It would seem that thr-
best diagnostic medium is one that will allow the development of
the desired organism when minimal numbers are present in the

material to be examined. In waters of questionable sanitary qual-

ity it is conceivable that the use of standard lactose broth may not
obtain such results.
TABLE 1

Viability of Esch- Cali in Different Media Before and After Lethal Exposure
to Irradiation

 

 

MEDIA UNTREATED IRRADIATED

 

 

 

 

HEATED
Nutrient agar ........................ 570,000 20 27
Nutrient agar/blood .................. l 570,000 65 l 102
Nutrient agar/glucose ................ 600.000 4.5 105
Nutrient agar/yeast .................. l 610.000 l 25 l 27
Infusion agar ......................... l 610,000 38 l 180
Tomato juice milk powder agar ....... 5 540,000 I 69 i 237

 

 

 

It is not within the province of this paper to show the practical
significance of the increasing numbers obtained but it is our thesis
that a diagnostic medium should produce a maximum number of
the organisms present from the material examined whether it be the
diphtheria bacillus from a throat swab or coliform organisms from a
water supply. The value of a diagnostic medium lies in those of its
properties which promote a rapid growth of the dormant viable cells
in the material examined. There should be a continuance of rapid
reproduction until the cells appear in such abundance that. they
can be recognized by their physiological action on the medium or so
that they can be transplanted to differential media.

Many media do not present a suitable environment for the viable
cells to reach the reproductive stages and thus fail to give positive
cultures. Many viable cells of Escherichia. coli fail to reproduce on
certain media as shown by Curran and Evans (2) in table 1, It will

 

 

 

 

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VOL. 31, NO. 4] NEW MEDIUM FOR COLIFORM 691

be observed from this table that on the tomato juice milk powder
agar 237 cells survived heat treatment; whereas on the nutrient agar
only 27 cells survived. That the latter medium is best suited for
demonstrating surviving organisms is quite apparent.

It is a well established fact that when organisms are transferred
from one environment to another, particularly if the new environ-
ment difiers radically from the previous one, many viable cells fail
to reproduce due to their inability to adjust themselves to the new
conditions or to adjust their immediate surroundings to their needs.
Because of this it is customary in transferring many organisms to
use mass seedings, hoping that a sufficient number of viable cells
will reproduce and thus assure a positive culture.

In single-cell isolation' work Wright and Hendrickson (20) found
that by incubating the single cell in a drop of broth in a moist cham-
ber, rather than in a tube of nutrient medium, they were able to
grow as high as 90 per cent of the cells isolated. It would appear
from this work that the cell must adjust its environment to its own
needs and that by the use of a minimum rather than a large volume
of broth the cell is able to adjust itself more rapidly and more surely.
A number of years ago, the junior author of this paper spent several
months trying to single-cell several strains of Salmonella pullorum
using many types of enrichment media without success. Then one
night in a demonstration of single-cell culture, five out. of six cells
grew in plain nutrient broth. Following this discovery, using the
same batch of medium, single-cell cultures were prepared for all
the desired strains of Sal. p'ullorum with a very high percentage of
the single-cells growing. No explanation was found as to why
this particular batch of medium gave results when previous batches
and other media failed. But it appears quite obvious that environ-
mental conditions were such that each viable single ccll found its
surroundings conducive to immediate growth without considerable
adjustment prior to reproduction.

Reproduction begins after a cell has overcome its stationary phase
and has taken in sufficient food material. As soon as the cell has
undergone fission the resulting two young cells begin growth and
ultimately reproduction. A new critical period in the life of the

.organism begins with the completion of fission. The work of

Salter (11), Schultz and Ritz (12), Reichenbach (10), Sherman and
Albus (13, 1,4), Stark and Stark (17), Heiberg (4), and Sherman and
Cameron (15) shows that young cells are much more susceptible to

 

 

 

692 C. w. DARBY AND w. L. MALLMANN [J. A. w. w. A.

adverse conditions such as heat, 2 per cent sodium chloride, 0.5 per
cent phenol, and dilute crystal violet, than cells found in the lag
stages of a logarithmic growth phase. Huntington and Winslow
(6) show that during the lag stage and the early logarithmic stage of
growth the young cells exhibit all the characteristics of physiological
youth comparable to those exhibited in multi-cellular organisms.
This means that a good medium must produce an environment
such that no inimical conditions exist during the critical period of
lag and early logarithmic stages of growth. Otherwise the mortality
of the cells may be such that no growth results.

It is our thesis in the light of past experience and the studies cited
that a culture medium for diagnostic use should be measured not by
gross growth obtained at the completion of the logarithmic stage

TABLE 2
Influence of Bactmpcplonc in Various C(mccnfratirms1 in the Base illcdimn‘ on
Lag Phase of Growth of Esch. (.‘oli N 0161

 

 

 

 

 

 

 

 

 

 

 

 

Co'ggiNggx- i NUMBER 01' BACTERIA 111511 UL.
13:32: i Initmrs '— 6 hours mw—Hmiours — i i 24 tours —T— 48—;urs—fl‘
percent 1 i t
0.5 15 i 800 i 2,000 52.(Kll,),000 ' 413,000,0(7Xl 408,000,000
1.0 ’ 20 i 1.200 4.700 30 “000 000 [I 704, 000, 000 731, 000, 000
2.0 18 1,200 4 400 32 000 000 300 000 000L 872, 000. 000
3 0 i 16 J 770 . 1 750 t 8 3110. one i 1 9411:11114100’1 244 (11111111

 

 

 

 

 

 

* Base medium consists ole521ctt-1 beef exttat t t) —3 per tent, lat tosc 0.51m

cent.

but by the behaxior of the ortgmism during the lag and early 105121-
rithmic stages of growth. Therefore in the studies presented 1111
have worked with minimal numbers of organisms that have been
attenuated by age and we have studied the behavior of these or-
ganisms primarily during the lag and 1arly logarithmic growth
stages.

Procedure

Growth rates were determined in broth media containing varinlr
concentrations of the ingredients that enter into a nutrient medium:
to ascertain the amounts giving the maximum rate of reproduction
during the early stages of the logarithmic growth phase of the. or.
ganism. For these studies a laboratory strain of Esch. coli (No. 1611

 

 

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VOL. 31, NO. 4] NEW MEDIUM FOR COLIFORM 693

was selected. This culture was kept on an agar slant and, after the
full development of the culture, it was kept in the refrigerator.
Storage in the refrigerator over a period of months tended to at-
tenuate the culture sufficiently to increase the stationary phase of
the organism and thus give a better picture of the value of the
medium studied in the recovery of the viable cells present. In all
experiments organisms were taken directly from these slant cultures
and introduced directly into the medium under investigation.

Small seedings were used to intensify the difficulties of the or-
ganisms to overcome unsuitable environments and thus increase
the marginal differences in growth rates of the different types of
media used.

Considerable care was exercised in the preparation of the media
and in the technics of growing the cultures and the determinations
of the numbers present in order to eliminate as far as possible all
variables other than the one variable under study.

Experimental Background

Peptonc. In an earlier study of bacteriostatic action of dyes, it
was observed (table 2) that a concentration of 3 per cent Bacto-
peptone in a medium caused a decreased growth in the lag phase of
growth of Esch. coli. The fact that this concentration acted as an
inhibitory agent seemed to indicate that a concentration of 0.5 to
1 per cent was best, not because the organisms grew best at these
concentrations, but that. in more dilute amounts it. was less toxic.
It would appear that its use in an enrichment. medium such as lac-
tose peptone broth certainly would not favor the development of
isolated cells or groups of cells that were partially attenuated by
chlorination. It appears quite evident that a more satisfactory
sourceof nitrogenous food should be found.

A comparison of Bacto-tryptose and Baeto-peptone showed that
much more rapid growth occurred with the former peptone (table 3).
The two media are not exactly comparable because the constituents
and their concentrations differ; however, the medium containing
the Bacto—tryptose was far superior at all stages of growth through a
12-hour incubation-period. The data are presented to show the
marked difference between the two media.

The most effective concentration of tryptose was determined by
varying the amounts of tryptose from 1 to 3 per cent in the same
base medium. In table 4 data are presented for growth rates of

 

 

 

 

604 C. w. DARBY AND w.'1.. MALLMANN [J. A. w. w. .1.

E's-eh. coli in varying concentrations of tryptose in a base medium
of 2 per cent bile brilliant green lactose broth while for the growth
rates in table 5 a phosphate, buffered medium was used. In the bile
brilliant green medium, 3 per cent. tryptose gave the most rapid
growth while in the buffered medium there was little choice be-
tween 2 and 3 per cent tryptose, although the 2 per cent medium
showed slightly higher rates of growth. In similar experiments 2
and 3 per cent. tryptose showed about the same effect on growth
rates, although in general the 3 per cent concentration appeared to
be slightly the better medium. In concentrations up to 4 per cent,
the highest concentration tested, Bacto—tryptose showed no iu~
hibitory effect such as that observed with Bacto-peptone.

TABLE 3

Comparison of Earle-peptone and Bade-tryptose ilfctlia by Means of Growth
Rates of Each. Cali, No. 161

 

 

 

 

{ NUMBER OF BACTERIA PER ML.
MEDIUM I ‘ _ __ ___#._ ,, __ .__—~_

 

 

 

 

’ Initial l3 hourst 6 hours l ”ti-hours 12 hours
Lactose 111-01111 ........ l 86 t 1,560{ 573.000 ) 31,300,000 232,000,101
Tryptose brotht.......l 07 t 5,300 12,700,000 , 585,000,000; 1,050,101,111)

 

 

 

"‘ Lactose broth: dehydrated Difco lactose broth.
l Tryptosc broth: liacto-tryptose 3.0 per cent, lactose 0.5 per cent, NaCl
0.5 per cent.

The concentration of 2 per cent was selected as optimum as the
increased activity induced by 3 per cent tryptose did not appear
to warrant the additional expense involved in practical use of the
medium. It is possible that in practical applications, a one per cent
concentration may be satisfactory.

Hydrogen ion concentration. Although the pH range for the
growth of Each. col'i appears to be quite wide, it was felt that the
influence of pH should be carefully checked to determine the effect
on early growth rates. Media consisting of 3 per cent Bacto—trytr
tose and 0.5 per cent lactose were adjusted to pH values of 6.8, 7.0.
7.2, 7.4, 7.6, and 7.8 and tested as in previous experiments. N0 pH
values below 6.8 were tested as acid will cause a hydrolysis of the
lactose during sterilization. The results are presented in table 6.
It should be observed that at the end of 6 hours incubation at pH
6.8 the number of organisms was nearly double the numberfit .

 

 

 

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TABLE 5
Influence of Bade—tum in Various Concentrations in the Base Mediiun“ on i - i.-
- , ‘ 1 Lag Phase of Growth of Each. 0011', No. 161 ‘
m o “m l mun or RAM I'll Ila.
limo. ' Inithl 8 hours 6 houn 12 hour-
pcrouu '
m. 1 84 3,800 333,000 1,330,010,000
1 2 80 3,300 397,000 995,000,000
not - 3 88 2,700 402,000 1,150,000,000
will , Average 80 3,300 377,000 1,158,000,000
wt
1 102 4,500 1,712,000 1,700,000,000
h, 2 2 90 3,000 040,000 1,450,000,000
0 3 80 3,400 1,144,000 1,510,000,000
all“; Average 94 3,800 1,168,000 1,587,000,000
we
BM 1 78 2,000 1,202,000 1,950,000,000
«till 3 2 90 2,900 994,000 2,000,000,000
3 n 3 79 3,700 1,400,000 1,780,000,000
its.t Average. 84 2,900 1,218,000 1,953,000,000
1|!is '

 

 

 

 

 

 

 

 

 

 

"' Base medium consists of: lactose 0.5 per cent; K2HP04 0-4 per cent;
KHJ’O; 0.15 per cent; NaCl 0.5 per cent; adjusted to pH 6.8 before steriliza-

.tiqn.

 

 

696 c. w. MALLMANN

DARBY AND w. 1..
pH 7.0 and that at increasing pH values the amount of growth
decreased until at pH 7 .8 the growth was only approximately one-
fourth that obtained at pH 6.8.

In a number of other similar experiments, the maximum growth
appeared at pH 6.8 during the early periods of the lag phase of
growth. It will also be observed from these data that with the
higher pH values, greater amounts of growth appeared at 24 and 48
hours incubation. This would be expected as the limiting acid con-
centrations caused by the fermentation of the lactose would be
- approached sooner in the growth cycle of the organisms in the
media which were adjusted to lower pH values. A reaction of pH
6.8 was selected as the optimum value for the growth of Esch. (011'.

TABLE 6

Influence of (he [13/11111ch1 Ion (’1111C1ntrali1m of [(11(omeclo-tryplose Broth."
on Lag Phase of Growth of Escl1.(.'o11, 1V1) 161

 

 

 

NUMBER OF BACTERIA PER ML.

(.1. A. w. w. A.

 

 

 

 

I
pH i____ . _ _ .__ .___ ..-, -___-___.__ _.
I Initi1l I 6 hours I 12 ‘ 1011111 I 24 hours 48 hours
0 8 I 38 I 31,800 I 182,000,000 I 9211 .01111111111 880,000,000
711 I 35 I 17,800 I 177,000,000 I 1,150,000,000 1,070,000,000
7 2 1 41 17,21111 229,101,100 1,218,000,000 1,040,000,000
7 4 I 30 , 11.11111 I 185,000,000 1,250,000,000 1,428,001,000
7. 0 31 I 8,800 I 240 0110 11011 1.003.000.1100 2,510,010,000
7.8 I 35 I 7 11110 281 0110 0110 I 2,015,01111,11110 2,630,011,011)

 

 

" The base medium consists of: Bacto-tryptose 3 per cent and lactose 0.5
per cent.

\Valker and Winslow (19), Mallmann and Gallo (7), Slanetz and
Rettger (16), and others have demonstrated that the addition of a
phosphate buffer to a medium caused an increase in the total num-
ber of bacteria produced as compared to a similar medium without
buffer. The use of buffered media for fermentation experiment:
was suggested by Bronfenbrenner and Schlesinger (1). The i11-
creased number‘of bacteria was believed by Mallmann and Gallu
(7) to be due not. only to the buffering effect on the acid pr0duc1'1i
by the organisms, but also to the need of phosphate in the mctah
11lism of the organisms. The use of a buffered lactose peptone b10111
as a presumptive medium for the coliform organisms was recom-
mended by Thompson (18).

 

 

        
 
 

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ngam- e: mam Medium an the Log Phase of aroma of
, wt; . . Each. €091,170. m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I! ' ' unusual
min-1i ._ , an: , Numberofbactetiaperml.
1511181 11110qu --12hours 2111mm 101mm
None. ., 7.0 35 17,800 177,000,000 1,150,000,000 1,670,000,000
Pmenit'. . . '. . . 7.0 82 81,111) 578,000,000 1,850,000,000 2,790,000,000
slam no. 3
Initial 8 hours 6 hour: 9 hours 12 hours
None ......... 6.8 19 725 l,217,(l)0 470,000,000 997,500,000
Premnt ....... 6.8 16 890 3,362,000 840,000,000 1,555,000,000
. sums no. 3
Initial 2 hours 4 hours 6 hours 8 hours
11M None ......... 0.8 95 350 28,700 2,050,000 88,200,000
11 Present ....... 6.8 108 430 40,700 2,570,000 139,700,000
W. ‘ Bufl‘er mixture: K3HPO4 0.4 per cent; KH1P04 0.15 per cent.
,. l Series N o. 1, base medium: Bacto-tryptose 3.0 per cent, lactose 0.5 per cent.
W. Series No. 2, base medium: Bacto-tryptose 2.0 per cent, lactose 0.5 per cent,
M! NaCl 0.5 per cent.
um“ Series N o. 3, base medium: same as No. 2.

l- '1': served that in all instances the growth during the early stages of
1 ”do reproduction and the later stages of the logarithmic phase, was much
idF" greater for the buffered medium. In media prepared by the Difco
the” Laboratories, there was little choice between the bufl‘ered and non-
Wt buffered media during the early growth period but in most instances
was” in the late logarithmic phase the buffered medium was superior. In

. general, the data showed that the addition of a buffer to the medium,

. '1 ‘ ‘4‘ ...__‘D t..-

 

 
   
    
      
  

   
 
 
  
 

 
   

i ‘o. ‘s 7‘"
‘0
.. i
this. ‘
’ o '. ‘ 3
'83-‘0. ‘r
.‘ ° ' .\.o
I
.. J 7“ N )

 

 

 

698 e. w. DARBY AND w. 1.. MALLMANN [J. A. w. w. A.

although not always causing an increase in reproduction during the
early stages, did not hinder the rate of growth and in the late stages
of the logarithmic phase the buffer caused a marked increase in the
number of organisms.

TABLE 8

I nfl ucnce of Sodium Chloride in a Base M cdiu 111’ on (he Lag Phase of Growth of
Esch. Coli. No. 16'!

 

 

 

BUFFERED Mzmou‘

 

 

 

 

 

 

1
(‘ON(EN- .' f~—r -
TRATION f Number of but teria per ml
01 Nat‘l _ -__ _ *_fi_ w_# ._
l I 1111111 I 6 hours i 12 hours 24 hours 48 hours
1cm! “ ' l
None 32 8l . l00| 578, 000. 000 l.850,000,000 2. 790. 000 0le
0.5 , 35 . 106.0001“— 1 (168, 000. 000 2,51Xl.000.0001.970.000 000
: NON-BUFl-‘ERED urznruut
None l 35 ' 17,800 177.000.1111 1 1 1.50 111111 111111 . 1. 670 000 11111
0.5 l 39 ' 67,000; 535,000,000 I l ,755. 000 000 1,610,(XX).000

 

 

‘ Base medium consists of: Baeto-tryptose_ 3 per cent, lactose 0.5 per cent.
KgHP04 0.4 per cent; KHzPOt 0.15 per cent.
1‘ Base medium consists of: Bacto—tryptosc 3 per cent, lactose 0.5 per cent.

TABLE 9

Influence ofSodium (.‘hloridt 1'11 the Base llcdium“ on the Lag Phase of Growth of
Esch.CoI.1',N.o 161

__ _ _ _ _ ;__,>__ , _ __ _, __

 

CONCENTRA‘ ' NUMBER 0!" BA('I‘ERIA PER ML.

 

 

 

T1011: ‘ pH {.___ - -. _ _ ________ _ _ ___, _*_ __ e.
01- NaCl 1 1 Initial 1 2 hours i 4 hours !' 6 hours i 8 hours
percent t l l 1' ‘

0.5 ‘ 6.8 l 1118 1 .1311 l 411.7011 2,570,000 1 139,701,000

1 11 6.8 | 00 ’ 540 53.1011 2 111111 1100 153,211,110

2 11 l 6.8 1 100 , 2511 l 20 000 , 650 0110 1 42, 000 (100

 

 

 

" Base 111edium consists of: Ban to- trx ptose. 2 per Lent, lat tose 0. 5 per cent
K,HPO. 0. 4 per cent, Kll,P04 015 per cent.

Sodium C hlurz'dc. Dunham (3) reports that the addition of sodium
chloride to eosin-methylcne blue medium causes a marked increase
in the number of positive coliform organisms isolated from lactose
presumptive tubes showing gas. Mooney and Winslow (8) obsen'f‘d
that Salmonella pullorum would not grow in peptone—glucose broth
medium when aerated but when 0.5 per cent. NaCl was added, growth
was rapid. For these reasons the influence of NaCl was tested for

 

 

 

  
   
  

. ' hit: am
i. - 3.045111915110114“ Mhbeth a
‘ - * 3, £54; ”I ‘ fie Salim ‘ 'of N80! chimed a marked
". : ~9~ ."'”‘ (3* 2% If. infiheeufyetages algmwth and min

J- In); 154434.le .. . . ‘phflhe In We 9 data showing the
.. . .. ; >1. firm eeiit Na‘Ci-in a medium are presented.

‘ IMIIW bemoan 0.5 and 1.0 per cent but 2.0
W Inhibitory efie'ct on the rate of What

no” of; c w; .11 r l

  

flit"
.g.

 
   
  

  
 

  
  

   

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 10
1M aim tn the Basic He'dium‘ on Lag Phase of Growth of Each. Cali,
No. 161
Wh’d— 3".f" mmorummnsn.
mm W _
m Initial 2 hours } 4hours 61mm 8 hours
pram;— .
- ' 1 86 240 8.2% 626,000 78,0(X),(l)0
0.25 . . 2 97. 240 7,200 804,000 52,500,000
Average . 91 240 7,710 715, 000 65,250,000
M ' f 1 95 280 7,000 558,000 70,000,000
"3 0.5 2 108 250 9,400 470,000 66,600,000
J Average 102 240 8,200 514,000 68,300,000
:0! - 1 92 220 9,400 640,000 53,800,000
/ 1.0 2 100 280 9,100 710,000 82,0(X),0(X)
Average 96 250 9,250 675,000 67,900,000
HT; 1 86 250 9,200 404,000 30,500,000
/ 2.0 2 97 240 8,150 506,000 36,200,000
39”! Average 91 245 8,675 455,000 33,350,000
l 1
In,” "' Basic medium consists of: Bacto-tryptose 2 per cent, NaCl 0.5 per cent,

6.” KHJ’O. 0.15 per cent, K,HPO. 0.4 per cent; pH 6.8 before sterilization.
/

1 all stages of the logarithmic phase up to 8 hours incubation. In
1 the light of these data, the addition of 0. 5 per cent NaCl seemed
an“ advisable.
ted” Lactose. Hershey and Bronfenbrenner (5) found that a concen-
wy tration of 3.0 per cent lactose in a synthetic medium encouraged
(3) a“ greater production of carbon dioxide and growth of slow lactose-
I009. fermenting organism than did a concentration of 0. 5 per cent. To
I M} determine the influence of the concentration of lactose an experi-
” w“ ment was set up using 0.25, 0.5, 1.0, and 2. 0 per cent lactose. The

 

    

 

  
   
   
  

\

v

4
6

 

700 c. W. DARBY AND w. 1.. MALLMANN [.1. A. w. w. A.

results are presented in table 10. No difference was observed in the
rate of growth for concentrations of 0.25, 0.5 and 1.0 per cent lactose.
With 2.0 per cent lactose a decrease in growth was evident after 4
hours incubation.

Mallmann and Gallo (7) found that Esch. coli even in a bufiered
medium seldom utilizes more than 0.5 per cent glucose and seldom
more than 0.3 per cent in an unbuffered peptone broth. Inasmuch
as no difference was found in concentrations varying from 0.25 to
1.0 per cent and because actual utilization of lactose is generally
not more than 0.5 per cent, there appeared no reason for a change
from the present accepted concentration.

TABLE 11
Comparative. Growth Rates of Strains of Escherichia coli and A erobactc-r
aerogcnes on Tryptosc lactose Broth“

 

l NUMBER OF BACTERIA PER ML.

 

 

 

 

 

CULTURE . ___, “fl ______A _. ._f_
l.) Initial i 4 hours | 8 hours 12 hours

Esch. coli-- 161 ................. l 72 44,200 02,000,000? 1,400,000,010
Esch. ooh—165 ................. l 211 16,500 37,000,000 990,010,011
Each. colt—466 ................. ‘ 207 58,000 28,000,000 1,430,000,000
Esch. colt-e171 ................. 1 253 41,000 74,000,000 1,100,011.101
Aer. aerogcncsd140 ............. 225 24.400 20,160,000 740,000.00“
Aer. aerogencs—l3l. ........... 215 1 40,500 46.2(')0,000 1,100,000.10»
Esch. coli~—l67 ....... . ......... 182 l 37,500 103,000,000 1,530,010,111)

 

 

 

 

 

"' The medium consists of: Bacto—tryptose 3 per cent, lactose 0.5 per cent.
KQHP04 0.4 per cent, KH2P04 0.15 per cent. NaCl 0.5 per cent; medium ad-
justed so pH 6.7 after sterilization was obtained.

These results do not in any way fail to confirm the work of Hershey
and Bronfcnbrenner (5) since their obser 'ations were made at the
end of two days incubation and the above-mentioned observatitms
were limited to 8 hours incubation.

For comparative purposes and uniformity of data, only one
strain of the coliform group was used, namely Each. coli, No. 161.
To check the efi'ectivcness of the tryptose lactose medium in growing
the coliform organisms from their stationary phases, five strains of
Escherichia coli and two strains of Aerobactcr aerogcncs were tested.
The, results are presented in table 11. No cases of marked retarda‘
tion were observed although there was a difference in the rates of
reproduction during the early growth phases.

 

   

  

  

 

jéf'd :7-

--------

 
 
 
 

Emmottheathertests.whemm

   
 
  
  

1 ' . ' ,the tryptose lactose medium was . ”'3, .1.

is Wmnebmthatausmgesofgmwth ' . '. .35

I. M” “flmmmdlacphasesweremwh 1 . _;. '4.-
TABLE 12 73.? A a,

     

.-_ Wet Lam” Broth and Lactose Tryptose 3917" 370‘" fl”
-" MofWhofEach coh’, Na 161

     

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

..0 gran... i' ' . O , “33.301 [0 a"; .6
- l- -— *_ .° ._4 o v- .
_ m ,1!!! Numbcofbocteu’aparml. ‘ . I’f firfigi '
‘ . lam-l Shouts Ohoun 911mm l labours Ll .' i 1 'f :11 .
.. . f. '. 7* \. 1- ‘ ,‘f 113.-
Standard". ..... 0.55 51, 960 79,000 7, 750, 000 229,500,0m ‘ '
Tryptooe‘l’. 0.8 64 5,885 1,440,000 970, 000, 000 2,205,010,000 “
unnamed
Standard ....... 6.8 29 49 l 2,300 62,000 360,000,000 :
Tryptose ..... 6.8 49 42) 80,300 115,000,000 2,070,000,000
Insured
Initiall Shouts 4 hours 6 hours 8 hours
Standard ....... 6. 8 25 41 455 25 , 000 1 , 235,000
Tryptose ....... 6.8 27 72 1,785 170,000 13,600,000

 

 

 

,- “ Standard: Bacto-lactose broth, A.P.H.A.
id“ 1 Tryptose, medium consists of: Bacto-tryptose 2 per cent, lactose 0.5
Mil per cent, KJIPO. 0.4 per cent, KHsPO. 0.15 per cent, N aCl 0.5 per cent.

1 shorter in the tryptose lactose broth, indicating very clearly that
may! this medium allowed the rapid development of the viable cells by
11', NJ the fact that it offers a much better environment for the early
1M" reproduction of the organisms. This, in turn, means that more
‘ viable cells will reproduce so that the colon index will be higher in
M“ this medium than in standard lactose peptone broth particularly if
red“. the coliform organisms present have been attenuated.
the 14', The physiological by-products of metabolism parallel roughly

1 the rate of reproduction of the organisms. In tables 13 and 14 data

 

 

702 C. w. DARBY AND w. L. MALLMANN [J. A. w. W. A.

are presented to show the time of appearance of gas. Three media
are presented, the proposed tryptose lactose broth, the same medium
minus buffer, and standard lactose peptone broth. An examination
of the data shows that gas appeared sooner in the tryptose media
than in the standard lactose peptone broth and that the quantity

TABLE 13

Influence of the Medium on the Rate of Gas Production of Esch. Cali, No. 161
(Series No. 1)

 

 

i TIME OF APPEARANCE 0F GAS AND GROW'TH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

usmuu "33:5“, _9_-5:0:rs_v‘I 10.5 hours -- 11.5 hours i 12.5 hours —l3.5 hO—llE
l H 2.53:2: ‘, 23:22:: .... 2:21:25 222:5: .... 1:22:32:

H 1 \ + + + 10 + 40 + 60 + '90

ll 2 + + + 15 + 40 + 60 + 80

1 3 I + + + - 10 + 40 + so + 80
i 4 i + + + 15 + 40 + 70 + 80

| 5 + + + 20 + 50 + 70 + 90

‘ 1 + + + 2 + 20 + 40 + 60

i 2 + + + 20 + 60 + 70 + | 70

2 3 + + + 40 + 75 + 80 + _ 90
“ 4 + + + 25 + 60 + 70 + $ 81)

. 5 + 10 + 50 + 75 + 80 + ’90

i 1 2t — + — + — + 5 + .20

2 i — + — + + + 5 + 1 10

3 l 3 i — + — + . — + 10 + 15
1| 4 :t — + — + ‘ — + 5 + '10

L 5 , i - + -— l + g + + m + ‘20

 

 

__ —.. --....

l
|

1

Medium 1 consists of: Bacto-Tryptose 2 per cent, lactose 0.5 per (‘9nt-
NaCl 0.5 per cent, KgHPO. 0.4 per cent, KHQPO. 0,15 per cent.

Medium 2 consists of: Bacto-Tryptose 2 per cent, lactose 0.5 per cent, Natl
0.5 per cent.

Medium 3: Bacto-lactose-peptone broth.
Inoculum: 580 organismo per tube.

of gas was far greater in the former media. The non-buffered tryp
tose broth showed more gas than the buffered medium at the. Sam
early periods due to the fact that the fall in pH was more rapid
in the non-buffered medium; hence there would be less conversion
of the carbon dioxide to carbonates. As would be expected from 31-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘J— {.Lt' _9\. Jai’ll “Jurl$ .
Q...- ‘ ‘ akin-1 . 1‘: Hi: . . - _.. ’ ”1.: ,r ”a
' ' ‘30?!de Cali, 'Ncc 161 . ' , ' j.’ .""_".-~§!_
.: V‘Hs . “F%’) ...- . w_ . -. ‘ - '3751'.' ‘o'l‘
1 t ' ' “W".fimoflmwoumoaom ‘. i f ’ . ’4. .kfl‘" ; v“
j" "' . f7 3’3“ um " mm- mm ‘-‘ ' '5- - . " 5 3': “-1143
' 1* .... m. w... e : s.» . ..
In!“ ‘3” comb-G“ m C" m G“ l ' : E's-imp?
’+‘.—'— + - + 10 + 90 ... _"".,' -
‘+ - + - + 10 + 90 a. , -.
2r . ‘1‘ - + - + + + 100 if urifl". ' ‘
- + - + 5% +' 5 + 90 y ° '13“
+ - + 5% + 5 + 90 -. .- _
+ — + 5 . + 20 + 70 '
+ ‘ - + +1" + 5 + 70
'+ - + 10 + 5 + 60 .
1 + - + + + 10 + no
N + - + + + 20 + 70
{I
.| l 1 - —- a; — a: — + — + 10
1 2 -— - :1: —- a: - + — + 2c
47‘ 3 1 8 - - :l: - :t — + - + 20
+ 4 - - :9: - :1: - + - + 10
4.. _ 5 - - :l: — :l: - + — + 10
Li! ‘ Medium 1 consists of: Bacto-tryptose 2. 0 per cent, lactose 0. 5 per cent,
”a NaClO5percent, KzHPO¢04 percent, KH,PO¢0.15 percent.
Medium 2 consists of: Bacto-tryptose 2. 0 per cent, lactose 0. 5 per cent,

vii NaCl 0. 5 per cent.
9" Medium 3: Bacto-lactose-peptone broth.
1. 1' Bubble of gas is indicated by +.
Inoculum per tube: 40 organisms.

the medium of pH 6.8 gave a count of 31,800 in 6 hours incubation
1 while the medium at pH 7.8 gave a count of 7,900. At the end of
3:3 48 hours incubation the medium at pH 7.8 showed a. count of 2,630-

Mfifl table 6, in measuring the significance of pH in the growth of Esch. 0012',

000,000; whereas the medium at pH 6.8 showed only 880,000,000

 

 

initial number of viable cells that reproduce, the medium at pH

704, c. w. DARBY AND w. L. MALLMANN [.1. A. w. w. A.

organisms. If the two media were measured by total growth, the
medium at pH 7.8 is superior but if the media were measured by the

6.8 is far superior. It is quite evident that the medium at pH 7.8
does not offer a favorable environment for the development of the
viable cells present and hence such a medium would be an unsatisn
factory diagnostic or enrichment medium.

Another example of the value of this procedure is shown in tableS
in the influence of sodium chloride in the medium on growth rates.
The amount of growth in both‘ the salt and non-salt media is approxi-
mately the same at the end of 48 hours incubation whereas at the
end of 6 hours incubation the salt media show a much higher count.
If the media were measured at the end of 48 hours incubation, it
would appear that salt had no appreciable value in the medium;
whereas at the end of 6 and 12 hours incubation, it is quite evident
that salt increases growth rates materially. Repeatedly during this
study similar experiences have occurred, demonstrating the value
of the growth rate method for the development of a culture medium
designed for diagnostic purposes.

It is realized that the medium presented in this paper differs rad-
ically from the present standard lactose peptone medium, but it is
the opinion of the writers that an enrichment medium used as a
presumptive medium for the detection of the coliform organism
should allow the development of all viable cells present in the ma-
terial to be examined. This medium based on studies made with
minimal numbers of viable. cells shows that a much greater rate of
reproduction occurs during the lag and early logarithmic phases of
growth with a materially shorter stationary phase than standard
lactose peptone broth would show. That it does allow the develop-
ment of more viable cells than standard lactose peptone broth we:
demonstrated this past summer on swimming pool samples.

The use of this medium on routine water analysis has not as 3’01
been tested on a wide scale. At the present writing it is being used
in seven water purification plant laboratories in parallel with stand-
ard lactose peptone broth for comparative purposes. The result
of this study will be presented at a later date.

The medium is being presented at this time to give water analyst
an opportunity to make comparative tests so that it will be possihh
to determine whether or not its use would be worthwhile for routim
use in place of the standard lactose peptone broth.

 

1! ~11"?
$1112"
1111'. .ii‘
:11 {d
um i'

1111 1.1::

1 11:13

111'11 .".

am 31‘
ll 11??"
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VOL. 31, N0. 4] NEW MEDIUM FOR COLIFORM 705

Summary

1. Bacto-tryptose was found to be superior to Bacto-peptone in a
base medium for all growth phases Of Esch. coli.

2. A concentration Of 2 per cent Bacto-tryptose in the medium
was found to give the greatest rate of reproduction during the lag
and early logarithmic growth phases.

3. The addition of phosphate buffer to the medium caused a much
greater growth in the late logarithmic phase and a slightly greater
increase during the lag phase than the non-buffered medium.

4. The rate of growth during the lag phase was greatest. at a pH
of 6.8.

5. The concentration of lactose appeared to have no influence on
the rate of growth during the lag and early logarithmic phases.

6. The addition of 0.5 per cent sodium chloride to the medium
caused a marked increase in the rate of reproduction during the lag
and early growth phases.

7. A 2 per cent Bacto-tryptose lactose buffered broth was far
superior to standard lactose peptone broth for the growth of coliform
organisms during the lag and early logarithmic phases. The in-
crease in numbers persisted into the late. logarithmic and decreasing
logarithmic phases of growth.

8. The medium is suggested as an enrichment medium for the
growth Of coliform organisms. The formula follows:

BactO—tryptose .......................... 2 per cent
Lactose ................................ 0.5 per cent
K2HPO4 ................................ 0.4 per cent
KH2P04 ................................ 0.15 per cent
NaCl .................................. 0.5 per cent

pH before sterilization: 6.8

In a subsequent paper the results showing the use of this medium
as a presumptive medium for the detection of the coliform organisms
in water supplies will be presented.

EDITOR’S NOTE. This paper has been published at the request
of the Executive Committee Of the Water Purification Division in
the hope that the media therein suggested will be extensively tested
elsewhere so that experiences may be summarized in a discussion
before the Water Purification Division.

706 C. w. DARBY AND w. L. MALLMANN [J. A. w. w. A.

1-1.
15.
16.

17.

19.

‘20.

. BRONFENBRENNEB, J ., AND SCRLEsINGER, M. J. Carbohydrate Fermenta-
. CURRAN, H. R., AND EVANS, F. R. The Importance of Enrichments in the

. DUNHAM, H. G. Personal communication.
. HEIBERG, B. Die Thermoresiste'nz bei jungen und alten Bakterien und

. HERSHEY, A. L., AND BRONFENBRENNER, J. The Influence of the Compo-
. HUNTINGTON, EVELYN, AND WINsLow, C.-E. A. Cell Size and Metabolic
. MALLMANN, W. L., AND GALLO, FRANK. The Influence of Phosphates on
. MOONEY, 0., AND WINsLow, (‘..-E. A. The Metabolic Activity of Various

. PERRY, C. A., AND HAJNA, A. A. A Modified Eiijkmann Medium. J.

. REICRENBACH, H. Die Absterbcordnung der Bakterien und ihre Bedeu-

. SALTER, R. C. Observations on the Rate of Growth of Bacillus (‘011

. SCHULTZ, J. H., AND Ri'rz, H. Die Thermoresistenz junger und alter Celi-

. SHERMAN, J. M., AND ALBUs, W. R.

. THOMPSON, R. E. Irregularities in the Test for B. Coli. in Water. 1-

References

tion by Bacteria as Influenced by the Composition of the Media.
Proc. Soc. Exp. Biol. and Med. 16: 44 (1918).

Cultivation of Bacterial Spores Previously Exposed to Lethal Agen-
cies. J. Bact. 34: 179 (1937).

“jungen” and “alten” Bakteriophagen. Zeitschr. Hyg. u. Infek-
tionskr. 114: 425 (1932).

sition of the Medium on the Metabolism of Some Slow Lactose-
Fermenting Bacteria of Intestinal Origin. J. Bact. 32: 519 (1936).

Activity Of Various Phases of the Bacteria Culture Cycle. J. Bact.
33: 123 (1927).

the Metabolism of Bacteria. Mich. Acad. Sci. Arts, and Letters 14:
617 (1931).

Colon Group Organisms at DitTerent Phases of the Culture Cycle.
J. Bact. 30: 427 (1935).

Bact. 26: 419 (1933).

tung fur Theorie und Praxis der Ifksinfektion. Zeitschr. f. Hyg.
69: 171 (1911).

Jour. Infect. Dis. 24: 260 (1919).

Bacillen. Centralb. f. Bakt. I Abt., Orig. 54: 283 (1910).
Physiological Youth in Bacteria
J. Bact. 8: 127 (1923).
Ibid. The Function Of Lag in Bacterial Cultures. J. Bact. 9: 303 (19211
SHERMAN, J. M., AND CAMERON, G. M. Lethal Environmental Factor.-
within Natural Range of Growth. J. Bact. 27: 341 (1934).
SLANETz, C. A., AND RETTGER. L. F. The Influence of Phosphate Butir.’
in Carbohydrate-free and in Glucose. Containing Media. J. Bact.
15: 297 (1928).
STARK, C. N., AND STARR, P. The Relative Thermal Death Ratesct
Young and Mature Bacterial Cells. J. Bact. 18: 333 (1929).

Bact. 13: 209 (1927).
WALKER, H. H., AND WINsLow, C.-lfi_ A, Metabolic Activity 01th:-
Bactcrial Call at Various Phases Of the Population Cycle. .1.
Bact. 24: 209 (1932).
WRIGHT, W. H., AND HENDRIcRsON, A. A. Studies on the Progeny ui

Single-cell Isolations from the Hairy-root and Crown-gall Organisms
Jour. Agri. Res. 41: 54] (1930).

 

 

 

 

 

 

PmfiII

Studies on Bacteriostatic Agents

It is not within the province of this thesis to review com-
pletely the work that has been done in the field of bacteriostasis
The major problem was to investigate the bacteriostets under con-
trolled laboratory tests with the hOpe that some of the newer dyes
or compounds at our disposal might be more effective than those
in current use. The experimental data are presented, not as a
completed stuiy of any one series of dyes or other bacteriostatic
agents, but merely as experimental data that may be of practical
use in future investigations. The practical possibilities of
each set of data will be discussed with each experiment.

In 1926 Churchman (27) gave the following definition for
the term ”bacteriostasisP "At least four phases of bacterial
inhibition have been observed in the case of bacteria exposed
to the action of gentian violet and related dyes: cessation of
motility; inhibition of reproduction; mspension of animation;
and.inhibition of sporulation. Anilin dyes may show these four
types of inhibition without killing. The difficulty of distin-
guishing between death and inhibition of growth has led the
writer to use the term 'bacteriostasis.' In the cases of tripheny
methane dyes, gram-positive bacteria are. as a rule, much more
susceptible than the grampnegative."

Although bacteriologists were using bacteriostatic agents

as early as 1902 (23), Ghurchman (29), in 1912. was the first

“9“

worker to present a comprehensive paper on this subject. He studied
the action of gentian violet on a large collection of bacterial
species. He divided these organisms into violet negative and
violet positive groups. and demonstrated the practical importance
of this work. In the same year Churchman presented a second.paper
(25). in which he demonstrated that by the use of gentian violet
it was possible to differentiate between closely related bacterial
species. In this paper he made the following prophetic statement.
"---that a substance will be found.possessing a similar selective
affinity between such bacteriologically troublesome organisms as
Bacillus tvphosus and Bacillus 0011 does not seem to be out of
the question."

In 1923 Churchman (26) demonstrated that this inhibition
of gram-positive bacteria by gentian violet could be accomplished
by adding the dye to the medium (extrinsic bacteriostasis) or
the organisms could be stained with it before being planted on
plain agar (intrinsic bacteriostasis). He also demonstrated
that selective bacteriostasis is not necessarily associated with
selective penetrability. Organisms that are heavily stained
may grow, and others. apparently unstained.by the dye, were in-
hibited. At this time he pointed.out that the bacteriostatic
action may be due to changes effected by the dye at the surface
of the organisms.

Stearn and Stearn (28), in 192h, offer an elaborate chemi-
cal explanation for the mechanism of bacteriostasis. Their paper

includes bacteriostatic titers obtained by other workers in this

in this field. Any investigator interested in bacteriostasis
should review the material presented in this paper.

Dubos (29), in 1929, and Ingraham (30). in 1933, point out
that the bacteriostatic action of gentian violet and related
compounds is closely associated.with the ability of these com-
pounds to poise the oxidationpreduction potential of the medi-
um. Ingraham definitely shows that during the lag phase of
growth neither the dye nor the organisms are altered.

The possibility of inhibiting gram-negative bacteria and
allowing gram-positive bacteria to grow was first recognized
by Churchman in 1923 (26, 31). He demonstrated that in suit-
able concentrations acid fuchsin would exhibit the reverse
action of gentian violet on gramppositive and gram-negative
organisms. This phenomenon has been called ”reverse bacterio-
stasis.” In 1939 Bryan, Ibvereux, Hirschey and Corbett (32)
demonstrated the value of sodium azide as an inhibitory agent
for gramnnegative bacteria. Mallmann (M6), in 19h0, demon-
strated the value of sodium azide for the isolation of strep-
tococci from sewage. In 19uo Snyder and Lichstein (33) and
in l9hl Lichstein and Snyder (3h) presented the value of sodi-
um azide in inhibiting the growth of gramrnegative bacteria
when.culturing feces for the isolation of streptococci. In
l9hl Mallmann, Botwright and Churchill (35) published a paper
on the selective bacteriostatic effect of slow oxidizing agents.
In this study they demonstrated that both potassium dichromate

and sodium azide exhibited the property of ”reverse bacteriostasis."

In 1913 Browning, Gilmour and.hhckie (23) presented a
technic for the isolation of typhoid bacilli from feces. They
demonstrated that brilliant green had the ability to inhibit
Escherichia coli but allow Eberthella typhosa to grow. They
stated that Conradi and Drigalski, Loffler, and Savage were
using this selective action of brilliant green as early as
1908. Savage was using it in his studies for the isolation
of the Gartner's bacillus from fecal material. In 1927
Bakieten and.Rettger (36) used brilliant green in a buffered
broth as an enrichment medium for typhoid and paratyphoid or-
ganisms. Nbllmann, Thorp, and Semmes (37), in 1928, demon-
strated the value of a brilliant green liver infusion medium
for the isolation of Salmonella pullorum from infected chicks.
The currently pepular selective media, bismuth sulfite agar
and Bacto—S. 8. medium depend. to a large extent, upon the

selective action of brilliant green. Currently, the use of

 

brilliant green as an enrichment broth has been supplanted by

tetrathionate broth (HM).

 

 

..

For a Complete discussion of the chemical composition and
physical properties of the dyes studied in this paper the reader
is referred to Conn (M5).

In any series of experiments designed to determine the
bacteriostatic preperties of a compound there are several fac-
tors that must be carefully controlled and recorded if the data
are to be of any value for future work. The importance of stat-

ing the source, serial number, certification number and any

-0.

other available data concerning the particular bacteriostat
under study has been emphasized by Conn (38). The value of
stating definite pH values has been shown by Stearn and Stearn
(39, no), McCalla and Clark (hi) and Reed and Genung (he).
The importance of the age of the culture or cultures used.was
stressed by Churchman in 1926 (M3). That the constituents of
the medium may vary the bacteriostatic titers of dyes was
shown by Reed and Genung (he) and other workers in this field.
Stearn and Stearn (NO) point out the importance of the pre-
vious environment of the organisms under study. Other impor-
tant factors that should.be carefully recorded are (1) time
and temperature of incubation; (2) concentration of the bac-
teriostat; (3) time and temperature of sterilization (if
bacteriostat is subjected to heat); (h) additional organic
compounds added to the medium (serum or blood, for example);
(5) whether the medium is broth or agar in nature; (6) size
or numbers of inoculum; (7) possible antibiotic or symbiotic
effects of mixtures of bacteria; (8) the possibility of oxi-
dation or reduction of the bacteriostat by the particular
microorganisms under study; (9) the particular growth require—
ments of the bacterium under study; (10) the exact technic used
in carrying out any series of experiments and (11) the stabil-
ity of the medium under study.

The results of the experimental work carried out on various
bacteriostats and mixtures of bacteriostats are recorded in the

following series of five experiments.

Experiment 1

[£[g_. The following dyes were used in this experiment:

(1) Ethyl purple 63. No. 673k. National Aniline and Chemi-
cal 00., Inc. NBg-h.

(2) Brilliant green. Schultz H . M99. 0. I. N0. 662. Total
dye content 9hfi. national Aniline and Chemical 00., Inc.

(3) Acid violet. Schultz Ho. 530. Lot No. 8393. National
Aniline and Chemical Co., Inc.

Basic Medium. In this experiment the following basic medi-
um was used: Bacto—beef extract, 0.3%; Bacto~peptone, 0.5%;
sodium chloride, 0.5% and agar, 2.0%. The pH was adjusted to
7.2 and sterilization effected by autoclaving for 20 minutes
at 15 pounds pressure. All dyes were prepared aseptically and
added to the basic medium after autoclaving. Twenty m1. amounts
were poured into standard Petri dishes (63.5 sq. cm.) and the
plates incubated at 37°C. for 2h hours to insure sterility.

Technic. The plates were marked on the bottom so that
four equal areas were obtained for seeding. The inoculum was
placed upon the agar surface as a single streak using one 100p-
ful from a standard h mm. platinum loop. All plates, including
controls were run in duplicate. After seeding, the plates were
incubated for 30 hours at 37°C.

Edlutions of Eyes. The following dilutions were made of

 

each dye: 1 in 25,000; 50,000; 75,000; 100,000; 150,000;

200,000; 500,000; and l in 1,000,000.

-10-

Cultures. Twenty-four hour broth cultures of the following
organisms were used in this study: Salmonellajpaggtyphi é,
Salmonella pullorum, Eberthella typhoia, Singing gallinarum.
Stflglococcus m, Escherichia 991;, and Bacillus cer_eu__g.

The results, showing the bacteriostatic titers obtained
in experiment I, are tabulated in table I. If the growth com-
pared favorably with that obtained on the control plates, it
was recorded as 1L. A growth of approximately one-half that of
the control was recorded as 2, and only a few scattered colonies

as l.

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-11..

Summary of
Experiment 1
'1. Acid violet. The dilutions used in this experiment
were too high to demonstrate if this dye has any practical value.
In the dilutions of l in 25,000 and l in 50,000 it was demonstrated
that this dye produced some bacteriostatic effect on the gram-
positive bacteria.

2. Ethyl purple. This experiment has shown that selected

 

members of the genera Salmonella and Eberthella grew in all di-

 

lutions. S. pullorum was completely inhibited at a dilution of
l in 25,000, and very little growth was obtained at the l in 50,000

dilution. Ԥ. gallinarum and E. coli grew in all dilutions of

 

ethyl purple. The gram-positive representatives were inhibited
in high dilutions.

3. Agpilliant_green. The Salmonella organisms grew in all
dilutions of this dye. Ԥ. gallinarum was inhibited completely
at l in 200,000. E. 2313 and Staph. aureus were completely in-
hibited at l in 150,000 and §.'g§£§g§ at 1 in 1,000,000 dilution.

Practical Applications

1. More experimental work should be done with ethyl purple,
especially on its toxicity for coliform organisms. The inhibition
of §. gallinarum by brilliant green (1 in 75,000) and the excellent
growth of this organism on ethyl purple (l in 75,000) compared
with the complete inhibition of §. pullorum on ethyl purple
(1 in 25,000) might be another method of differentiation of
these closely related organisms. This work should include the

study of numerous strains of each gemus.

-12...

Experiment 2

Purpose. Experiment 2 is designed to determine the bac-
teriostatic titers of the dyes studied in experiment 1 in a
broth medium.

‘Q[g_. The following dyes were used (1) brilliant green,
(2) gentian violet, (3) ethyl violet and (h) ethyl purple 6B.
The gentian violet used had the following identification:
National aniline and Chemical Company; 81% dye content; 0. I.
No. 681; Certification No. N. C. 6. The other dyes were from
the same lots as used in experiment 1.

Cultures. Twenty-four hour broth cultures of the following
bacteria were used: _E_. typhosa, E. _c_9_]_._i_.,, _S_. naratyphi g, _B_. w
and Staph. aureus.

Basic Medium. The dyes were added to the following'basic
medium: Bacto-beef extract, 0.3%; Bacto-peptone, 0.5% and
sodium chloride, 0.5%. The pH was adjusted to 7.2. The dyes
were now added to make the following dilutions: l in 25,000,
50,000, 75,000, 100,000, 150,000, 200,000, 500,000 and l in
1,000,000. Ten ml. amounts were dispensed into test tubes,
plugged, and autoclaved for twenty minutes at 15 pounds pressure.

Technic. All dilutions were run in duplicate. The broth
tubes were seeded with a standard h mm. 100p. These broth
tubes were then incubated for MS hours at 37°C. The results
of this experiment are tabulated in Tables IIuA and B.

Conclusions. The bacteriostatic titers of the dyes

studied are apparently higher in broth than in an agar medium.

-13.

Note. It will be noted that in this experiment both ethyl
violet and ethyl purple 63 were used. Conn (#5) states that

ethyl purple 6B is a synonym for ethyl violet.

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-lh~

Experiment 3

Pugpose. This experiment was carried out to determine the
bacteriostatic titers of a selected group of dyes in a Bacto-
tryptose agar medium.

‘Q[g_. The following dyes were studied: (1) Brilliant gaggn.
Schultz No. bag. 0. I. No. nee. Total dye content 93%. Cert. No.
N 3g 7. National Aniline and Chemical Co. (2).§£hzl 133133.
Schultz do. 518. Lot Ho. 3330. Hational Aniline and Chemical Co.

(3) Iodine green. The Coleman and Bell Co. (h) Trypan Blue.

 

Vital stain. No. 3&17. National Aniline and Chemical Co. (5)
dethyl violet gg. Schultz No. 515. C. I. No. 680. Total dye
content 81%. Cert. No. N. MV-h. Rational Aniline and Chemical
Co. (8) Basic fuchsig. Total dye content 83%. Cert. No. R. F.
30. National Aniline and Chemical Co. (7) Eyronine Q. Schultz
No. 568. Lot. me. 2179. (8) Fast Green. F. C. F. The Coleman
and.Be11 Co.

Basic Medium. The dyes were added in suitable amounts to
the following basic medium: Bacto-tryptose, 2.0%; NaCl, 0.5%;
KeaPoI. o.n%; KHzPoI, 0.15%; dextrose, 0.1% and agar, 2.0%.
The pH of this medium was 8.8 before sterilization. The medium
containing the appropriate dye was sterilized by autoclaving for
20 minutes at 15 pounds. Twenty ml. portions were dispensed into
Petri dishes, allowed to cool and incubated to check for steril-
ity. The following dilutions of the various dyes were used:

1 in 10,000; 1 in 50,000; 1 in 150,000; and 1 in 500,000.

-15-

Technic. The method of seeding the plates and interpreta—
tion of the results were the same as those used in experiment 1.
Incubation was carried out at 37°C. for 30 hours to be compares
ble with experiment 1.

Cultures. The following cultures were used in this experi-
ment: .§. enteritidis,|§. ggdlinarum,‘§. gallinarum (10H0),‘§.
paratmhi 13., _S_. typhimurium, §. paratyp. hié, _§. pullorum,

.§. pullorum (Coburn), Shigella dysenteriae,‘§. typhosa (Hopkins),
1§.'ggli (lbl), Pasteurella avicida, Aerobacter aerogenes (131),
.§- cereus and.§1caligenes fecalis.

Results. The following dyes exhibited very little bacteri—
ostatic action: trypan blue, fast green, F. C. F., pyronine G,
and.iodine green. Trypan blue had no effect on the growth of
the organisms tested in a dilution of l in 10,000 in the basic
medium. Fast green exhibited a very slight action (a No. 3
growth) on all the organisms in the l in 10,000 dilution. Pyronine
G completely inhibited E. cereus at a dilution of l in 50,000
and.partially (Ho. 2 reading) at 1 in 150,000. The gram-negative
organisms all grew in the 1 in 10,000 dilution, bud there was
some evidence of toxicity (a No. 3 growth was recorded for the
gram-negative group). Iodine green had the same action on the
gram—negative group as was exhibited by pyronine G. It inhibited
‘g. cereus completely at the 1 in 10,000 dilution and.partially
(a ho. 1 reading) at 1 in 50,000. The bacteriostatic titers of
the other dyes tested are presented in tables III A, B, C and D.

_Qiscussion. In general the bacteriostatic titers obtained

in a tryptose base were lower than those obtained in a Bacto-

~16-

peptone base. One reason for this was the increased amount of
peptone in the tryptose medium. The second factor of importance
is the composition of the tryptose. Also, in this experiment,
the effects of a greater amount of dye were studied (a dilution
of l in 10,000 as compared to a dilution of l in 25,000 in ex-
periment 1. Brilliant green inhibited §. pullorum in a dilup
of 1 in 25,000 and almost completely at l in 50,000 (a No. 1
reading) in the nutrient agar base. .§. pullorum.grew in the
tryptose base at a dilution of 1 in 10,000. Brilliant green
inhibited.§t ggli at a dilution of l in 150,000 in a.nutrient
agar base, whereas in the tryptose base a concentration of
1 in 50,000 was required. It is particularly important to
note that in the tryptose medium p. _c_c_>_l_i_ and 1.1. tzghosa were
inhibited at the same concentration of brilliant green. Neither
a.peptone or a tryptose base with brilliant green, would be
suitable for the isolation of §. gallinarum or|§.,dysenteriae.
Brilliant green still retains a high degree of selectivity for
granhpositive bacteria. 3
With ethyl purple, the other dye used in both experiments 1

and 3, there was a.uniform reduction in bacteriostatic titers when

the tryptose base was used.

Table III

The Bacteriostatic Titers of Selected.Ives
in a Tryptose Agar Medium

But-{ethyl Violet 213

__J

" ilution of dye l in l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cultures D
10T 50T 150T 530:4
S. enteritidis AH=3 3 3 M
S. gallinarum 3 3 3 ’4
S. gallinarum No. lOMOfl 3 3 3 3
s. paratyphi B E 3 3 it 1+
S. typhimurium HA3 3 M h
s. paratyphi A 3 3 LI ’4
S. pullorum H 3 3 3 M
S. pullorum - Coburn fl 3 3 3 U
S. dysenteriae fl 2 3 3 M
E. typhosa 1 3 3 ’4
E. col;_ % 1 2 3 h
P. avicida 2 3 3 n
A. eerogenes 3 3 3 h
Al. fecalis M h M M
B. cereus - - - ’
thousand

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growth 1/2 control

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no growth

 

-17-

Experiment h

Pugpose. This experiment was carried out to determine
the bacteriostatic titers of a selected group of compounds
in a Bacto—tryptose broth medium.

Dyes agd Compounds. The following selected group of
compounds was used in this experiment. (1) Brilliant green.
Schultz No. #99, C. I. 662. Total dye content 93%. National
Aniline and Chemical 00.. (2) Methyl violet g3. Schultz No.
515, C. I. No. 680. Total dye content 81%. National Aniline
and Chemical Co. (3) _.e._g_i_g fuchsin c. I. No. 692. Total dye
content 60%. National Aniline and Chemical 00. N. R.-10.

(h) Thionin Schultz No. 3kg, 2nd Ed. 0. I. No. 920, NT—Z.
National Aniline and Chemical Co. (5) Ethyl purple ég,
National Aniline and Chemical Co. no. 673M. (6) Rosalie
ggid. (7) Sodium azide, Pract. (8) eroninn—Schultz No. U69,
hth Ed. National Aniline and Chemical Co. Cert. 'o. N. P. 3.

(9) Erythrosin-—Fisher Scientific Co. (10) Nigrosine (Water

 

Soluble). (ll) Acriflavine hydrochloride. (12) Elggp-
No. 36092. (13) Toluidin Blue Cert. No. HU—l. Fisher Sci-
entific 00.

Basic Medium. The dyes and.other compounds tested were
added to the following basic medium: Bacto—tryptose. 2.0%;
dextrose, 0.1%; NaCl, 0.5%; mm, 0.15% and harm, 0.1%
The recorded concentrations of the compounds were added to

the basic medium and dispensed, in 10 ml. amounts, into test

tubes. Sterilization was effected.by autoclaving for 20 min-
utes at 15 pounds pressure. The pH of the medium, after ster-
ilization, was 6.8. All tubes were checked for sterility by
incubating them at 37°C. for ME hours.

Technic. All tests were run in duplicate. The tubes
were seeded with a 2h-hour culture of the apprOpriate organ-
ism, each tube being seeded with one loopful, using a h mm.
platinum 100p. Growth was determined by gross observable tur-
bidity, using unseeded tubes of the same dye-dilution as con-
trols. The readings were made after MS hours incubation at
37°C-

0u1tures. The following cultures were used in this ex-
periment: ,g. coli, Staph. aureus,‘§.‘ggg§ug, §, pullorum,

g. paratzphi 5, _§. typhimurium (230), §. typhinnlrium (226),
,§. gallinarum,'§. tzphosa, and'S. dysenteriae.

Results. The following compounds showed very little
bacteriostatic action and these data are presented.below:

(1) gig fuchsin-u-all cultures grew in a dilution of
1 in 5,000.

(2) Thioninp-B, cereus and E. txphosa were inhibited in
a dilution of l in 10,000. All of the other cultures grew at
this dilution.

(3) Rosalie Epigrg§£gph..gggg3§ was inhibited in the
1 in 10,000 dilution but grew in the 1 in 50,000 dilution.

_B. g_e_r_e_u_s was inhibited in both the 1 in 10,000 and 1 in

50,000 dilutions, but grew in the l in 100,000 dilution. All

-19-

of the other cultures grew in the l in 10,000 dilution.

(h) .Erythrosin——All cultures grew in the l in 10,000 di-
lution.

(5) Nigrosine--All cultures grew in the l in 10,000 di-
lution.

(6) Elong-B, cereus was inhibited in a 1 in 5,000 dilu-
tion, all other cultures grew in this dilution.

(7) Toluidine b1ue--§, cereus and §. dysenteriae were in-

 

hibited in a dilution of 1 in 10,000. All other cultures grew
in this dilution.

The experimental data obtained from the other compounds
tested are shown in the table IV series.

Discussion. In a tryptose broth medium brilliant green

 

loses much of its selective action for gram-positive bacteria
and all of its selective action for g. 291i. Using gentian
violet or ethyl violet in a tryptose medium does not markedly
effect the bacteriostatic properties of these dyes. In a
tryptose broth base, sodium azide exhibits "reverse bacterio-
stasis" but the differential titers are too close for practical

use.

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920-

Experiment V

Eggpggg. This is a continuation of experiment IV to show
the bacteriostatic action of a mixture of bacteriostatic agents.

In this work only five cultures were used. The results are

presented in the table V series. The sodium lauryl sulfate

was Duponal W. A. paste. 3.33 grams in 100 ml. were used as
a 1% solution.

Discussig_. The addition of sodium lauryl sulfate to
brilliant green in a tryptose base had very little action. The
bacteriostatic action of the sodium lauryl sulfate was not
affected by the brilliant green. (Table V—A)

The addition of sodium lauryl sulfate to methyl violet 23
reduced, to some extent, the toxicity of methyl violet for
gram-negative bacteria, but had no apparent effect on the
gramepositive organisms. (Table V-B)

Sodium lauryl sulfate, when added to acriflavine hydrochloride.
reduced the toxicity of this compound for gram—negative bacteria.
The mixture of acriflavine and ethyl purple gives a greater toxicity,
than either compound acting independently for both gram~positive

and gram-negative bacteria. This did not occur when brilliant green

and methyl violet were mixed. (Table V-E)

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Part III

.§tudies_on Selective Met'a for_Coliform Organisms

Any selective medium designed to be used in water analysis
must be based on a thorough knowledge of the bacterial flora of
various types of water. The basis of any method of water analy-
sis is to determine the presence or absence of coliform organisms.
The whole technic is based on the thesis that when fecal contami-
nation occurs, coliform bacteria will be present in the water.
Elaborate technics have been devised to perfect a medium that
will eliminate any organism, or group of organisms, other than
the coliform group, that might possibly give a positive test by
the technic employed.

An exhaustive study has been made by Greer and Greer and
associates of the various types of organisms, other than coli-
form, found in water supplies. (12, 13, in, 15, 16)

A second very complete discussion of both coliform and non-
coliform lactose fermenting bacteria was published.by Levine (17).
A comprehensive review of the coliform bacteria was published by
Parr in 1939 (1).

From a review of the cited references it was found that num-
erous non-coliform organisms have been isolated which have the

ability to ferment lactose. Species of the genus Clostridium

 

have probably been incriminated more than any other in false
positive presumptive tests. Bergey (18) lists 18 species of
this genus that have the ability to ferment lactose with acid

and gas production. A few spore-forming and non-spore-forming

gram-positive, aerobic rods have been described which have the

ability to ferment lactose. Organisms such as §._a§rosporus

 

(an aerobic spore—former) and Eouston's "leather" bacillus,
described by Greer (12) have the ability to produce gas from
lactose. Greer also refers to many undescribed species that
are capable of lactose fermentation. Many streptococci and
staphylococci have the ability to ferment lactose without the
production of gas. This action.nmkes possible a synergistic
fermentation of lactose. Members of the genus Proteus, for
example, have the ability to produce acid and gas from dex-
trose, but not lactose. In a mixed bacterial population gross
ing in lactose broth2it is possible to have the lactose hydro-
lyzed'by one species and the resulting dextrose fermented with
gas production by a second species. This synergistic action
probably causes many false positive presumptive tests.

Other non-coliform lactose-fermenting bacteria are members
of the genera Erwinea and Klebsiellas These organisms, however,
are rarely found in water and are of little significance in
water bacteriology.

With the inception of a definite technic for water analysis,
bacteriologists began to devise methods for the elimination of
these non—coliform organisms. In part II of this thesis the
early work concerning bacteriostatic agents has been reviewed.
Greer (15) has presented a review of the deveIOpment of the
various selective media used in water analysis. The first selec—
tive agent used for this purpose was bile, which was added to

lactose broth to inhibit the growth of the gram—positive bacteria.

-23..

It was found. however, that the concentration of bile necessary
to obtain the desired results was inhibitory to the coliform
bacteria. various media containing gentian violet as the selec—
tive agent were devised for use in water analysis. Crystal
violet lactose broth, formats ricinoleate broth, fuchsin lac-
tose broth, and brilliant green'bile 2% lactose broths are now
recommended.by "Standard Methods of Water Analysis" (19) for
the confirmation of positive presumptive tests.

Of these selective media the brilliant green bile medium
is the least toxic and most efficient in the elimination of
false positive tests. The efficiency of this medium has been
reported by Muer and Harris (20) and Dunham and Schoenlein (21).
Jordan (22) reported that this medium was slightly superior to
plain lactose broth for the determination of coliform organisms
in water.

To warrant consideration in field tests any new selective
medium should be superior totmilliant green bile 2% lactose broth.

The following experimental data are presented as prelimi~
nary work to the development of the selective medium.presented
in reprint form and appended to this thesis.

A Comparison of Brilliant Green and
Ethyl Purple in a Bile Medium

In part II it has been shown that ethyl purple (ethyl violet)
has high bacteriostatic action on gram-positive organisms, with
relatively little toxicity for E. ggli. This study was designed
to determine the effect of 2i‘bile on the selective action of

brilliant green and ethyl purple.

-2h.

The basic medium contained.the following: beef extract, 0.3%;
Bact-peptone. 0.5%; Bact-oxgall, 2.0%; lactose. 1.0%. The pH was
7.0 before sterilization. The dyes were added to obtain the concen-
trations indicated in tables VI and VII. The media were dispensed,
in 10 ml. amounts. into tubes containing fermentation inserts.

The tubes were then autoclaved for 20 minutes at 15 pounds pres-
sure. One N mm. loop of the SM-hour broth culture was seeded
into the broth as indicated in the tabulated data. Incubation
was carried out at 37°C.

An examination of the data presented in tables VI and VII
shows that although the ethyl purple bile medium was slightly
less toxic for the coliform organisms, it was also less effi-
cient in preventing the growth of the gramapositive bacteria.

It was concluded from these studies that an ethyl purple bile
medium would not be as efficient as the brilliant green bile
medium.

Ethyl Eprple and Brilliant Green
Bile in Tryptose Broth

 

 

In the appended reprint. Studies 9n Media for Coliform

 

Organisms, the value of tryptose broth for the growth of coli-
form organisms was definitely demonstrated. Although the bac-
teriostatic titers in Part II indicated that brilliant green
would not be a suitable bacteriostat in a tryptose base, the
combination of brilliant green and bile had not been studied.
The technic used in this study was similar to that used in the
preceding study. Fburteen different media were tested.using
different combinations of organisms that would give true and

false positive presumptive tests in selective and non—

selective types of lactose broth.

 

 

 

 

 

 

 

 

 

 

 

   

 

   

 

 

 

 

 

 

 

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The "heunt Clemens" organism was an.unidentified gramrpositive
rod incriminated in false positive presumptive tests from the
Mount Clemens water plant. This organism had apparently lost
its ability to produce gas from lactose. The results of these
tests are presented in the table VIII series.

The table IX series presents a comparison of brilliant
green bile tryptose. standard.brilliant green bile and the
ethyl purple tryptose medium. In this series a more compre-
hensive group of organisms was used. The data obtained in
these studies are presented in tables IK;A.and.B.

A study of the data presented in the table VIII and IX
series justifies the following conclusions. (1) Ethyl purple
in dilutions of l in 100,000 to l in 200,000 exhibits slightly
less toxicity for coliform organisms than does the standard
brilliant green bile 2% medium. (2) The ethyl purple in a
l in 200,000 dilution in tryptose broth inhibited the clostridia
in all tests in the VIII series. In the IX series this medium
failed to inhibit the clostridia in two instances (culture No.

2 in 116 hours and culture Ho. 13 in #8 hours). (3) The ethyl
purple tryptose medium inhibited all of the aerobic gramnpositive
organisms tested. (M) Brilliant green, even in high concentrap
tions, in a 22% bile-tryptose medium failed to inhibit the clos—
tridia and mirtures of clostridia tested in this series. (5) The
brilliant green 2%‘bileutryptose medium inhibited all of the
aerobic grsnhpositive organisms tested. (o) The results obtained
‘using the standard brilliant green bile medium were about the

same as those obtained.using the ethyl purple tryptose medium.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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This medium failed to inhibit the clostridia in two tubes as
shown in table VIII-D and did not inhibit culture Nb. 2 in
table IXLA. .A mixture of clostridia and Pseudomonas aeruginosa
produced gas in this medium, as demonstrated in table IXLB.

(7) These studies have demonstrated that the ethyl purple tryp-
tose and standanibrilliant green bile media are equally effi—
cient in inhibiting the growth of non-coliform lactose fer-
menting organisms. Neither medium is 100% efficient in its

designed selective action.

Growth Curve Studies of Selective
(edia for Water Analysis

The preceding studies have shown that standard brilliant
green bile and ethyl purple tryptose broth are equally effi-
cient in their selective action. The next step was to deter-
mine the relative toxicity of these media for coliform organ—
isms. The technic used to demonstrate toxicity of selective
media.has been outlined in the appended reprint Studies 9g
ggggg for Coliform Organisms. The test organism for these
studies was 3. 22;; (161).

The tabulated data showing the media studied and the bac—
teria counts after definite time periods are presented in tables
X. XI, XII—A.and XII-B. A study of these data justify the fol-
lowing conclusions. (1) In two of the three growth curves the
ethyl purple medium was superior to the standard brilliant green bile
medium. In the data presented in table XII the standard brilliant
green medium was superior. (2) In testing the selective media now
recommended.by standard methods it was found that in using E. ggli:

Bacto crystal violet lactose broth was most toxic; Bacto fuchsin

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lactose broth second; Bacto formats ricincleate broth third;
and Bacto brilliant green bile 2:35 was least toxic. (3) Ethyl
purple (1 in 200,000) in a basic medium containing 3% tryptose
was superior to basic media containing 2.0, 2.5, or M.O% tryp-
tose. (h) Although ethyl purple, in a dilution of 1 in 200,000,
compares favorably with the other selective media tested, it
does exert a marked toxicity for coliform organisms.

To establishdefinitely the toxicity of ethyl purple in a
tryptose base another series of growth curves was run. As in
the preceding studies, E. coli (161) was used as the test or-
ganism. These data are shown in tables XII-A and B.

In 1938, Cowles (see appended reprint) demonstrated the
value of sodium lauryl sulfate (Drene Shampoo) as a selective
agent incorporated in lactose broth. The toxicity of the lauryl
sulfate was also determined.by means of a growth curve. The
results of this study are presented in table XIII-B.

A study of the data presented in tables XIII-A and B jus-
tifies the following conclusions: (1) Ethyl purple, even in a
dilution of l in 500,000 is toxic for E. ggll; (2) sodium lauryl

sulfate (as Drene Shampoo), in the dilution used, did not inhibit

the growth of E. coli.

 

Because of its non—toxic action on coliform organisms, sodium
lauryl sulfate gave promise of being an ideal selective agent. If
this compound had the ability to suppress non-coliform lactose—

fermenting organisms it would be far superior to any other medium

now being used in water analysis.

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«Game 8. mm. no. on 4% em We mm... %W U“.
B .0 m 3 0.0 90 Me 1 0 mm 11 P1
6 not. 0.... 1.1. 1.... .... em U. .. 00 I...“
mm. 9T. 0 1...... 1...... ET. "HQ. 40 08 3w. .30. OR
1;”... T. pT. TI... LT. 0U. MU 01 .... 89 Ba...
T. nr. 8 an ....n en 39 .8 It. um uo .... 8.3de
90 SS . SS 83 8 ET. U.T. 7:0 0 81. 9
mm m. 1w m m; an
m E B S
8
mothdO

’

 

3&Hsm H.993 538 .Ho .8304 oHpfimofloposm 9a

Run 0."me

 

.. 98...

Table XIV shows the data obtained in a.preliminary bacterio-
static study of sodium lauryl sulfate in a.buffered tryptose»
lactose broth. This preliminary study indicates that this compound
has the ability to suppress the growth of aerobic gramwpositive
bacteria.

The second appended reprint, Uses _3 a Lauri; Sulfate Egyptose

 

Broth for the Dsctsction‘gi Coliform Organisms. completes the
studies of part III of this thesis.
ACKKOWLEDGHSET
The author wishes to express his sincere appreciation to
Dr. W. L. Hallmann for his assistance and active participation

in these studies.

  

I
I I
. _ .
Ir. _ l- .
1 __" ' I
'. i\_ I I
' I
' l
. ~ . I '
:u- .-. mm

_ ” W72 Journal of

_.° ic Health

f g "~5 And T/ae Nation ’5 H641”)

February, 1941 mm. 2

 
 
      

 

 

 

 

9":
I
_r ...

Uses of a Lauryl Sulfate Tryptose
Broth for the Detection of ,
Coliform Organisms

W. L. Mallmann, Ph.D., F.A.P.H.A., and C. W. Darby, D.V.M.

 

 

Published by the

American Public Health Association
1790' Broadway, New York, N. Y.

 

 

f1

 

   
  
 

 

   

 

 

 

.-

———- mm;- M,J.J~- _. as. -

7

Reprinted from AMERlCAN JOURNAL or PUBLIC HEALTH, Vol. 31, No. -, February, 1941

Uses of a Lauryl Sulfate Tryptose
Brorh for the Detection of

Coliform Organisms"

w. L. MALLMANN, PH.D., F.A.P.H.A., AND
c. w. DARBY, D.V.M.

Section of Bacteriology, Michigan Agricultural Experiment Station,
East Lansing, Mich.

AN attempt has been made to
develop improved methods of
procedure for the isolation of the
coliform group from water, both quali-
tatively and quantitatively. These
studies were undertaken primarily be-
cause of the fact that, seemingly in
various water supplies in various parts
of the United States, organisms were
passing through water purification
systems that caused intestinal upsets.
In many instances these water supplies
were meeting all bacteriological re-
quirements of a safe water supply. In
all instances the method of bacterio-
logical analysis was based on the
standard procedure of the American
Public Health Association.

Coupled loosely with these epi-
demics were observations by a number
of workers that longer incubation
periods and special technic demon-
strated the presence of organisms called,
for want of a better term, “ slow lactose
fermenters.”

Darby and Mallmann 1 showed in a
laboratory study that the use of a new
nutrient, tryptose, caused many 50-

‘Journal Article No. 490 n.s. from the Michigan
Agricultural Experiment Station. Read before the
Laboratory Section of the American Public Health
Association at the Sixty-ninth Annual Meeting in
Detroit, Mich... October 10, 1940.

called “slow lactose fermenters ” to
produce gas in greater quantities in a
shorter period of time. This was due
to the fact that this substance with a
few other chemical agents allowed a
more rapid growth of the coliform or-
ganisms and also caused a larger number
of the bacteria initially present to grow.
Thus an enrichment medium is avail-
able that grows a higher percentage of
the bacteria initially present in the
water in a shorter period of time than
the present standard lactose broth.

Many attempts have been made to
introduce into the primary lactose broth
tubes a selective agent which would pre-
vent the growth of Gram-positive bac-
teria and thus make the primary pre-
sumptive tests more significant. In all
cases where dyes have been used, such
as crystal violet, brilliant green, fuchsin,
and others, not only the Gram—positive
bacteria are inhibited but a marked
toxicity is also exhibited for the Gram-
negative organisms. In using the
selective agent in the primary medium
many coliform bacteria are inhibited
and lower colon indices result. The
Standard Method Committee of the
American Public Health Association has
never accepted any of these for
primary use for this reason.

[127]

 

128

AMERICAN JOURNAL OF PUBLIC HEALTH

Feb., 1941

TABLE 1

The Bacteriostatic Titers of Surface Tension Depressants

Dilutions of Compounds
4

 

Compounds Test Organisms " 1-

E. coli
Streptococcus

S. aureus

B. megatherium

E. coli
Streptococcus
S. aureus

B. megathcrium

E. coli
Streptococcus
S. aureus

B. megatherium

E. coli
Streptococcus
S. aureus

B. megatherium

E. coli
Streptococcus

S. aurcus

B. megathcrium

§

Aerosol —— MA.

Nacconol
N.R.S.F.

Duponol W.A.
Paste

Santomerse #1

lgepon T

++i+ ll|+ l|l+ lll+++++

' Incubation 37° C. [or 48 hours

In 1938, Cowls2 demonstrated that
the addition of sodium lauryl sulphate
to lactose broth gave a medium selec-
tive for the coliform group.

Before adopting sodium lauryl sul-
fate in these studies the writers tried
a number of wetting agents with the
object of selecting the best preparation.
In Table 1 are presented a few com-
pounds to show the selective properties
of these preparations. It will be ob-
served that Nacconol N.R.S.F. and
Duponol W.A. Paste are equal in selec-
tivity, both as to organisms and degree
of action. It will also be observed that
two other wetting agents, namely,

1-5T

I—IOT 1—20T 1-—30T 1—40T 1-5

+
+
+
+
+

|+1+++++

fi
01‘
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

++++ |+l+1||+ ||1+ ++++

++++ |+++1+|+ |+l+ ++++

++++ ++++ |+l+ I+|+ ++++
|+|+

++++ ++++ ++++ ++++ ++++

++++ ++++

Aerosol MA. and Igepon T. have no
selective action. In Table 2 are pre-
sented the surface tensions of these
compounds. The data show that the
surface tension depressing action is
about the same for all of these products.
It is apparent that the reduction in sur-
face tension alone is not the cause of
the selective action.

To check the toxic properties, growth
curves were made to determine the
effects of various concentrations of the
wetting agents on the rate of growth
of the coliform organisms. In Table3
are presented the bacterial counts which
were obtained. The wetting agents

TABLE 2

The Sur/ace Tensions of Several Wetting Agents in a Tryptose Broth Base

Surface Tension in Dynes ’ at the Following Dilutions
4

”M

 

 

f 1 set
Compound 1-100 I—IT 1-«5T I-IOT I—ZOT 1—3or 1—401‘ -
Aerosol A.Y. 33.37 36.66 _ __ _ __ __ ..

Nacconol N.R.S.F. 36.19 36.66 37.60 37.14 38.54 40,39 41,37 44.19

Duponol WA. 33.37 39.95 39.01 39.95 39.49 42.30 43.24 47.00
Santomerse #1 36.66 36.66 — _ __ __ ~_ ..
Igepon T 36.66 36.66 —- __ _ __ __ ..
Base Medium 55.94 —— ._ __ __ __ __ _.

’ Taken at 25° C. from autoclaved samples,

______.._——---""""

 

 

H]
I’m
xiii
T.;

 

I

Vol. 31

LAURYL SULFATE TRYPTOSE BROTH 129

TABLE 3

The Eject of Surface Tension Depressants on the Growth of Escherichia Colt
in a Basic Tryptose Medium

 

 

Concentration
of Number of Bacteria per cc.

Compound (Per cent) Initial 3 Hours 6 Hours 10 Hours
Nacconol N.R.S.F.

1-100 1 56 5,700 1,260,000 480,000,000
Duponol W.A. Paste

1—100 1 39 5,900 1,290,000 480,000,000
Base Medium ' — 67 15,700 10,200,000 1,280,000,000
Nacconol N.R.S.F.

1—10,000 0.01 64 24,200 5,860,000 990,000,000
Duponol W.A. Paste

1-10,000 0.01 78 29,500 15,020,000 1,040,000,000
Bu: Medium ' -— 81 37,700 18,360,000 990,000,000
Bacto Formate

Ricinoleate Broth 71 3,000 340,000 59,000,000

' Base medium consists of Bacto-trypth%; lactose—0.5%; NaCl—0.5%; K,HPO.—0.4%;

KH,PO‘—O.15%; pH after sterilization—6.8%

show some toxicity at concentrations of
1:100, but at concentrations of 1:10,000
no such effect is shown. These products
therefore make possible the addition of
a selective agent to a primary medium
without the attending toxic effects on
the organisms desired.

An ideal medium would appear thus
to be the tryptose lactose broth plus
1:10,000 dilutions of Duponol W.A.
Paste (sodium lauryl sulfate) or
Nacconol N .R.F.S.

To check the value of this medium in
laboratory work the Difco Laboratories
kindly supplied the base broth medium.
The following laboratories collaborated
in these studies: Detroit Board of
Health, Water Purification Plants at

Detroit, Highland Park, Wyandotte,
Flint, Saginaw, and Bay City, Mich.

The same medium was used in all
tests.
laboratory for comparative study.

A comparison was made by testing
three media in parallel, namely, stand-
ard lactose broth, lactose tryptose broth,
and lauryl sulfate tryptose lactose
broth. The results on tap waters for
6 plants are presented in Table 4.
Using lactose broth, 238 tubes showed
gas but, upon confirmation with bril-
liant green bile broth, only 4 tubes were
confirmed. Lauryl sulfate tryptose lac-
tose broth, on the other hand, showed
gas in only 3 tubes and all of these
tubes confirmed.

TABLE 4

A Comparison of Standard Lactose Broth, Tryptose Broth and Lauryl Sulfate T ryptose
Broth as Primary Media for Top Water Samples

No. of Tubes Showing Gas
4

No. Tubes Confirmed
L

 

 

 

N 0. Tube: r 4 r fl
Source of Sam ples Tested LB. T.B. L.S.T.B. L.B. T.B. L.S.T.B,
Flint 498 61 4 0 0 l 0
Wyandotte 240 2 5 4 O 0 0 0
Detroit-Springwells 12 6 16 2 0 0 0 0
Detroit W.W.P. 96 34 18 0 0 0 0
Saginaw 204 S9 35 0 0 O 0
Bay City 334 43 s 3 4 3 3

Total 1 ,584 2 3 8 68 3 4 4 3

1LB. denotes lactose broth.
’L.S.T.B. denotes lauryl sulfate tryptose broth.

Data were submitted to our.

 

r-v-v- a.

 

130

AMERICAN JOURNAL OF PUBLIC HEALTH

Feb., 1941

TABLE 5

A Comparison of Standard Lactose Broth and Lauryl Sulfate Tryptose Broth as Primary
Media for Top Water Samples—Highland Park, Mich.

 

No. of Tubes
Period 0/ Examination Tested
Mar. 6, 1939-May 28, 1939 585
May 29, 1939—Aug. 8, 1939 825
Aug. 9, 1939—Feb. 20, 1940 2,100
Total 3,510

1 LB. denotes lactose broth.
2L.S.T.B. denotes lauryl sulfate tryptose broth.

An interesting observation is the fact
that only 68 tubes showed gas in
tryptose broth, although no selective
agent had been added. The writers are
not prepared at the present time to offer
any explanation of this selective action.
In Table 5 are presented the data for
tap water tested at the Highland Park
Filtration Plant over a period of one
year. These data are presented in
groups to show the effect of seasonal
change on the number of tubes showing
gas in lactose broth that do not con-
firm. 'An examination of this table
shows that of 3,510 tubes tested, 107
produced gas in lactose broth but none
confirmed. In lauryl sulphate tryptose
broth 2 tubes showed gas but failed to
confirm. These data are very similar
to the ones presented from the various
laboratories in Table 4.

When comparative tests were made
on polluted raw water the results were
slightly different. For example, in
Table 6, which shows a comparison of
the tests at Flint, Saginaw, and Detroit,

No. of Tubes Showing Gas
r-—-—-——-*-———-—\

No. Tubes C onfimed

%

LB.1 L.S.T.B.2 LB. L518.
59 2 0 0
38 0 0 0
10 0 0 0
107 2 0 0

it will be observed that 454 tubes
showed gas in lactose broth of which
only 32 failed to confirm. On the other
hand, 469 tubes showed gas in lauryl
sulfate tryptose broth and 43 failed
to confirm. As judged by these data,
it would appear that the latter medium
fails to hold back organisms which are
not members of the coliform groups.
In all cases confirmations were made
with brilliant green bile lactose broth.

Because of the failure for confirma-
tion on tubes from raw water our study
with the various plants was discontinued
until further information could be ob-
tained. In order to determine the type
of organisms which were failing to be
inhibited by the lauryl sulfate, Mr.
Dahljelm of the Highland Park Filtra-
tion Plant 'kindly consented to send us
tubes of lauryl sulfate tryptose broth
that failed to confirm on either cosin-
methylene-blue agar or brilliant green
bile broth. As soon as these tubes were
received at our laboratory they were
plated on various media to isolate the

TABLE 6

A Comparison of Standard Lactose Broth, Tryptose Broth and Lauryl Sulfate Tryptose Brillll
as Primary Media for Polluted Raw Water Samples

No. Tubes Showing Gas No. Unconfirmed Tube:
—JL

 

f— —\ >

Source of Sample LB. TB. L.S.T.B. L,B_ T,B_ 1.5.1.5.
Flint 193 211 212 3 16 2°
Saginaw 98 91 91 2 1 3
Detroit'Springwells g3 85 8 S 21 18 17
Detroit-W.W.P. 80 83 31 6 5 3
Total 454 470 469 32 41 ‘3

1L.B. denotes lactose broth.
’ L.S.T.B. denotes lauryl sulfate tryptose broth.

iMitt
[Act '

 

—-- "'A-"".J"~“w'g'§ “.A.’ '_'.'t,.._....' ..z. J- (-

Vol. 31 LAURYL SULFATE TRYPTOSE BROTH 131

organisms present. Various typical Tables 7, 8, 9, and 10, samples which
colonies were picked and transferred to failed to confirm in lauryl sulfate tryp-
suitable media for examination. In all tose broth are reported. It will be ob-
instances where tryptose lactose broth served that in each instance members
was used we were able to isolate from of the coliform group were isolated from
these tubes members of the coliform tubes which failed to confirm on either
group. In the following tables, a num— brilliant green bile broth or E.M.B.

ber of such samples are reported. In agar.

TABLE 7

A Comparison of Standard Lactose Broth and Lauryl Sulfate Tryptose Broth on Raw
Water—Highland Park, Mich., July 5, 1939—9 A.M.

Amounts of Water Tested
__.L

 

 

 

r
10 cc. 10 cc. 10 cc. 10 cc. 10 cc.
_A JL .__—M
. ,— fi fi W
Medium lneub. Time Gas Conf. Gas Conf. Gas Conf. Gas Conf. Gas Conf.
Lact. broth 24 hr. 0 0 0 0 O
48 hr. 0 10 — 0 SO — 0
LS. Tryp. broth 24 hr. 01 0 0 02 0:'1
48 hr. 5 —— 5 + 5 + 10 —— 10 -—
1 Aerobacter
’Aerobacter
3Aerobacter
TABLE 8

A Comparison of Standard Lactose Broth and Lauryl Sulfate Tryptose Broth on Raw 6
Water—Highland Park, July 7, 1939—3 P.M.

Amounts of Water Tested
A

 

r ——W
10 cc. 10 cc. 10 cc. 10 cc. 10 cc. .'
. ,_ L r___1\.._W
Medium lncub. Time Gas Con}. Gas Con]. Gas Cost]. Gas Conf. Gas Conf.
Lact. broth 24 hr. 0 O 0 O O
48 hr. 10 —— 10 -— 0 30 + 0 —-
L.S.’l‘. broth 24 hr. 0 0 1 0 2 0 3 r
48 hr. 0 4 —- 0 10 -— 10 —- '

1 Aerobacter
"’ Not checked
3 Aerobncter .

TABLE 9 1

A Comparison of Standard Lactose Broth and Lauryl Sulfate Tryptose Broth on Raw 1
Water-—Highland Park, July 10, 1939—9 A.M. "

Amounts of Water Tested
A._

 

fi
10 cc. 10 cc. 10 cc. 10 cc. 10 cc.
F—‘A—‘fl FF—‘A—“fi
Medium Incub. Time Gas Con]. Gas Con]. Gas Conf. Gas Con]. Gas Conf.
Lact. broth 24 hr. 0 ' o o o o ‘1
48 hr. 0 1o —— o I
L.S.'l‘. broth 24 hr. 0 1 0 0 9 0 0 i
43 hr. 10 -— 10 + 10 — 10 + 30 +
1 Escherichia "

’ Aerobacter

132

AMERICAN JOURNAL OF PUBLIC HEALTH

TABLE 10

Feb., 1941

A Comparison of Standard Lactose Broth and Lauryl Sulfate Tryptose Broth on Raw

Water——Highland Park, July 7, 1939—3 RM.

 

.1 . ,
, Ei—Jii

Amountflater Tested
fi a .15!
10 cc. 10 cc. 10 cc. 10 cc. 10 cc.
r—Mfi r—A—fi f__’Lfi
Medium lncub. Time Gas Conf. Gas Conf. Gas Con]. Gas Con]. Gas Calif.
Lact. broth 24 hr. 0 0 0 0 0
48 hr. 10 — 10 — 0 30 + 0
L.S.T. broth 24 hr. 0 0 1 O 0 ‘ 0'
48 hr. 0 S — 0 10 - 10 —

1 Aerobacter
’ Not checked
’ Aerobacter

These observations demonstrated that
apparently the confirmatory media
either caused the organisms to lose their
ability to ferment lactose or inhibited
these organisms from growing. It also
demonstrated that in many instances
tryptose broth will allow organisms to
grow and produce gas that otherwise
failed in lactose broth.

In order to follow through more
closely the results which might be ob-
tained from the two methods under
study, a laboratory procedure was set
up for checking the water samples sub-
mitted to our laboratory. Due to the
amount of work necessary in the con-
firmations which we made it was impos-
sible to ask any of the water purification
plants to carry on this type of survey.

TABLE 11

Comparative Confirmation Tests by Parallel Plantings from E.M.B. Plates to Standard is,
Lactose Broth and Tryptose Broth

In each instance the sample of water '
was plated in parallel on standard lac-
tose broth and lauryl sulfate tryptose
broth. At the end of the 48 hour ‘ '
period for gas formation, E.M.B. agar
plates were smeared from all tubes
showing gas on both media. After the
proper incubation of these plates, ob-
servations were made for the type of
colony. If typical colonies were not
obtained, atypical colonies were fished
and planted to lactose broth and tryp-
tose lactose broth for confirmation. ll
gas failed to appear in the lactose broth Vi”-
tubes, transfers were then made to the
tryptose broth. The selected results
are presented in Table 11 for lactose
broth with confirmation on E.M.B. agar
plates, followed by checking the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

M it
i ' . ‘ut oi

Sample (Ilijrti'olnlgacf E.M.B. from Lact. Broth Trypt. Broth 7,:22‘54 it
No. Broth Lact. Broth from E.M.B. from E.M.B. 3",”, t;
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Lilli 65

43 3333—+’+++ ————-— 10202010-++++ 75
44 5333—+-+++ 20——-— 30302015 +++ s:
46 205111nx+++_ 2__ 305__ + a;

65 15535+++++——22—~—1010——-55++ +-

39 33333+++++ ————— lozoloss++++‘i"
95 60 90 40 70 90 A, Cl.“ + + E _ _ 2 ‘0 + + :L‘,
96 40 15 30 10 40 + Cl. C1. C1. C1. —_ __ __ g,
104 20 SO 40 —“ ""+ + + “‘ '—‘ —" 10 4 3 + + + 1,4,]
140 —3335 ++++ -—————- ssss+++'f
187 2 - 10 —— 2 + A _ __ 3 3+ "' ii?
261 50505050—.E+++ —-—-— 332 +++ an
____ ,‘M:_ A~ AM :30

' + indicates atypical colonies. g,

1 E indicates typical Escherichia colonies. it
3.4 indicates typical Aerobacter colonies. 1
'Cl. indicates presence of Clostridia. A
it

 

 

 

 

Vol. 31

5‘
N
V
1‘

LAURYL SULFATE TRYPTOSE BROTH 133

TABLE 12

Comparative Confirmation Tests from Lauryl Sulfate Tryptose Broth by Parallel Planting:
from E.M.B. Plates to Standard Lactose Broth and Tryptose Broth

 

Per cent Gas in Per cent Gas in

 

 

 

 

 

 

 

 

 

 

Salicyple 10 cc. Lact. 10 cc. L.S.T. E.M.B. from Lact. Broth Trypt. Broth
0. B'Oth Broth L.S.T. B705]! ’70"! E.MB. from 851.8.
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 l 2 3 4 5
43 3 3 3 3—5 5 S 5 5+'++++ ——— 31010101010
44 5 3 3 3 — 5 S — 10 5+ + + +10 2 — 230 30 25 25
46 20 S l 1 l 5 5 S 10 S.+ A1 + + + —— l 2 1 2 2 5
as 1 s s 3 $1010 40 40 —'A + A E3 —— s
89 3333355555-l—++++ ————— 5553030
95 60 90 40 70 9040 3 -- -— — + E — 10
96 40 15 30 10 40 —————
104 205040——322——-+++ —-—-3 333
140 —333555553+++++—3310101 7020
187 2—10—2I—ss—— + —— 13
261 50 SO 50 50 SO.-— —- 5 10 — + A — 2
' +. indicates atypical colonies.
1 A indicates typical Aerobacter colonies.
'E Indicates typical Escherichia colonies.
colonies on lactose broth and tryptose gas production was obtained. These

lactose broth. It will be observed from
these data that many instances have
appeared wherein atypical colonies on

E.M.B. agar plates obtained from lac-

, tose broth failed to produce gas in
lactose broth. When transferred to
. tryptose broth, gas was produced in
. considerable quantities.
'3 when negative lactose broth tubes were

'- transferred to tryptose lactose broth,

Furthermore, _

data thus confirmed the observations
made at Highland Park that E.M.B.
and brilliant green bile cannot be used
as confirmatory media if it is desired
to obtain confirmation on all coliform
organisms which appeared in lactose
broth. In Table 12 are presented the
results for parallel planting of the
samples shown in Table 11 on lauryl
sulfate tryptose broth. It will be ob-

TABLE 13

comfiarative Colon Indices Obtained by Parallel Plantings by Standard Methods and
Lauryl Sulfate Tryptose Broth

Stand. Methods
A

L.S.T. Broth Method
_jc

 

 

f‘230

 

Sample r fl r—— N
No. Gas‘lndex Confirmed Index Trypt. Broth Con]. Gas Index Tfypt- 3’0‘,’ C0"!-
43 3 o 3 10 10
44 8 2 8 3 8
45 10 2 6 10 10
49 4 0 0 0 0
65 10 4 8 8 ' 8
75 z 0 z o 0
89 10 0 10 10 10
95 10 4 4 4 4
96 10 0 0 0
103 2 0 2 6 6
104 6 0 6 6 6
140 8 0 3 10 10
187 6 2 4 4 4

200 . 10 0 4 1° 1°

2 2 2 2 2

234 6 2 6 8 8

261 8 2 8 4 4
Average 7_os 1,3 50 5.9 5 9

134

served that the same picture obtained
as in the case of the lactose broth, except
that a higher incidence of coliform
organisms was obtained. It will also
be observed that gas production was
not obtained in the lauryl sulfate tryp-
tose broth when coliform organisms
were not isolated by the confirmation
technic used.

In Table 13 are presented compara-
tive colon indices obtained by parallel
planting by standard methods and
lauryl sulfate tryptose broth. It will
be observed that the gas indices ob-
tained in standard broth are much
higher than the confirmed indices ob-
tained by confirmation through E.M.B.
agar and lactose broth. Where tryp-
tose broth confirmation was made, the
colon indices obtained compare favor-
ably with the gas indices made from
the standard lactose broth. The colon
indices obtained from the parallel
planting in the lauryl sulfate broth by
confirmation in tryptose broth were
the same as the gas indices obtained in
this medium.

These data show that the confirma-

AMERICAN JOURNAL

OF PUBLIC HEALTH Felt, 1941

tory media used at the present time in
standard methods act as suppressing
agents to the coliform organisms and
produce a lower colon index than would
be obtained if more suitable confimsa-
tory media were used.

These data also indicate that in
lauryl sulfate tryptose broth gas pro
duction could serve not only as a pre-
sumptive test but also as a confirma-
tory medium for routine testing. It
has been our observation that, when
gas is produced in lauryl sulfate
tryptose broth, confirmation is always
obtained. These data should be
checked further in routine laboratories,
but until a satisfactory confirmatory
medium for checking gas production in
lauryl sulfate tryptose broth is de-
veloped, the results obtained would not

check with those obtained in this
research.

REFERENCES

l. Darby, C. \V.. and Mallmann. W. L. Studio
on Media for Coliform Organisms. I. Am. Warn
Works A., 3l1689. 1939.

2. Cowls. P. B. A Modified Lactose Broth in!

['se in the Presumptive Test. J. Am. ll'atrr limit
A., 30:979. 1938.

 

 

 

 

 

 

‘.f’ “‘h .__.

 

7 4-1‘9'

 

 

 

 

 

Literature Cited

(1) Parr, L. u.. Coliform Bacteria. Bact. Rev. 3 (1) : 1-hs, 1939.

(2) Winslow, C. E. A. The number of bacteria in sewage and sew-
age effluents determined'by plating upon different media and by
a new method of direct microsc0pic enumeration. Jour. Infect.
Dis. Sup. 1 t 209, 1905.

(3) Committee of Bacteriologists. 1898. Procedures recommended
for the study of Bacteria, with especial Reference to Greater
Uniformity in the Description and Differentiation of Species.

Being the Report of a Committee of Bacteriologists to the

Committee on the Pollution of Water-supplies of the American

Public Health Association. Transactions of the American Public

Health association. 1897 Meeting. 23, 60. (u)

(H) Prescott, S. C. and Winslow, C. E. A. Elements of Water
Bacteriology. 5th Ed. John Wiley and Sons. Inc. Hew York, 1931.

(5) Fuller, G. W. Report of Committee on Standard hethode of
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