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TH '

ST‘JDEEﬁ QM THE EFFECT" 0f
ETHY'L EWRPLE QN CEREMN EACTERM

Thesis {301* the game cf M. 5-.
MﬁCMi’GAN STATE COiLiéGE
Ei‘i’iéi MM jo-liiﬁ‘a
€948

This is to certifg that the
thesis entitled

Studies on the effect of Ethyl Purple
on Certain Bacteria

presented by

Ethel Jolliffe

has been accepted towards fulfillment
of the requirements for

M . S . degree in Bacteriology

Mijor professor - .
Datewg

”-795

       

STUDIES ON THE EFFECT OF ETHYL PURPLE
ON CERTAIN BACTERIA

By

Ethel Mae J olliffe
q”um-u!

A THESIS

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

for the degree of

MASTER OF SCIENCE

Department of Bacteriology

1948

THESIS

ACKNOWLEDGMENT

. I wish to express my
sincere appreciation to Dr.
W. L. Mallmann for his able

advice and guidance.

204076

STUDIES ON THE EFFECT OF ETHYL PURPLE ON CERTAIN BACTERIA

Much of our knowledge concerning differential and selective
media for the isolation and identification of organisms, particularly
those of the colon-typhoid -paratyphoid group, is based on the discovery
by Churchman (1), in 1912, of the selective action of gentian violet on
Gram positive organisms. This action on the Gram positive organisms
was termed bacteriostasis. Churchman (2) further noted that when
bacteria were exposed to gentianviolet or related dyes, there were
four phases of bacterial inhibition, namely; cessation of motility,
inhibition of reproduction, suSpension of animation, and inhibition
of sporulation. Anilin dyes show these four types of inhibition without
killing the bacteria although there is no doubt that some dyes actually
kill some bacteria.

Several theories have been suggested to explain the selective
bacteriostatic action of dyes. It is agreed that both chemical and
physical factors are involved in the action of dyes but the exact mech-
anisms of these factors are not known. Changes in physical environ-
ment greatly inﬂuence the inhibitory action of dyes as shown by
Churchman (3), in 1926, in his studies on the influence of age in relation
to bacterial resistance to dye. He proved that older cultures are much
less resistant to gentian violet than young ones. McCalla and Clark,
in 1941, (4) demonstrated that at pH values higher than the iso-electric
point, basic dyes are absorbed while at lower values acid dyes are
absorbed. As the bacterial systems were made more acid, the hydro-
gen ion adsorption increased and the ability of the bacterial cell to
take up crystal violet decreased. Ingraham (5) demonstrated by a

series of experiments that the action of gentian violet was due to a

certain degree on the oxidation-reduction potential. The effectiveness
of the dye depended on the initial oxidation-reduction potential. The
dye is toxic only during the lag phase when the cells are adjusting the
oxidation-reduction potential. She also showed that agents such as
serum, bile, or dead cells which favor a reduction of potential are
known to counteract the action of dyes. Fisher (6) pointed out in 1944
that the bacteriostatic action of triphenylmethane dyes may be con-
nected with the respiratory metabolism of the micro -organism.

The paper is primarily devoted to the use of the various cul-
tural media containing dyes for the enteric colon group of organisms.
As early as 1902 Conradi and Drigalski (7) suggested the use of mala-
chite green for inhibitory purposes. In 1913, Browning, Gilmore, and
Mackie (8) confirmed Loeffler’s work with brilliant green in which it
was found that Escherichia coli was inhibited to a greater extent than

 

Eberthella typhosa. Endo (9), in 1903, employed basic fuchsin, decolor-

 

ized with sodium sulfite, for the isolation of various colon organisms.
In 1926, Wilson and Blair (10) evolved a medium for the isolation of

E. typhosa containing glucose, bismuth and sodium sulfite. Various
modifications of this medium have been suggested in order to obtain
better results. In 1936, Jones (11) suggested the use of brilliant green
eosin lactose agar at concentrations that would inhibit E. g_o_l_i but would
allow the growth of E. typhosa. Eosin methylene blue agar for differ-
entiating the colon from the typhoid-dysentary group of bacteria was
preposed in 1916 by Holt-Harris and Teaque (12). Levine (13), in 1918,
suggested a simplified medium using this same principle. S and S, a
relatively new medium, has been developed by Difco laboratories in
Detroit, Michigan.

The constant search for new types of media is the result of
the many shortcomings of the present formulations. Some of these
media are based on the principle that sufficient concentration of
certain dyes be used to reduce the growth of the non—pathogenic gram
negative organisms found in fecal material. However in the proper
concentration the dye has a slight inhibitory effect on the pathogens
so that when few organisms are present as in carrier cases growth
fails thus giving false negatives. Other media allow the growth of

such organisms as the E. coli and Aerobacter aerogenes as well as the

 

pathogens but use lactose and acid indicators to differentiate the
organisms. False negative results occur under these conditions also.
The use of enrichment media before the differential and selective
medium has enabled the workers to detect the pathogens when they
occur in small numbers, but such a procedure has many disadvantages,
such as a long period of time before results are obtained, and increased
cost due to labor and materials.

During this period of experimentation with media for enteric

pathogens, bacteriologists were also experimenting with media for

 

growth of E. 9211 and _A. aerogenes to determine the sanitary condition
of water. The first medium to be used for the quantitative test for

E. 9311 in water was glucose broth suggested by Theobald Smith in
1893 (14). In 1912 lactose bile broth became standard medium for the
presumptive test. The use of lactose broth for the presumptive test
was recommended in 1917 by the Standard Methods Committee for

the examination of water and sewage. Hall and Ellelson (15), in 1919,
suggested the addition of gentian violet to lactose broth for water
examination. Brilliant green lactose medium was suggested, in 1920,

by Muer and Harris (16). Jordan (17) as well as others have tested a

variety of media containing brilliant green. Horwood and Heifertz (18),
Ruchhoft (19), Mallmann and Darby (20), Stark and England (21), and
Ruchhoft and Norton (22) and others have found that presumptive media
containing brilliant green and crystal violet are too toxic to the
colon—aerogenes group thus giving a number of false negative results.
Black and Klinger in 1936, (23) tested several media by the Butterfield
(24) dilution method, the results of which were based on gas production.
They found the media, ranked in order of decreasing sensitivity were,
standard lactose broth, buffered lactose broth, fuchsin broth, methylene
blue brom cresol purple broth, brilliant green bile broth, crystal violet
broth and formate ricinoleate broth. It was found that brilliant green
lactose bile broth equaled standard lactose broth in sensitivity with

A; aerogenes and intermediate strains but not with E. coli.

 

 

Brilliant green lactose bile broth, Endo's medium, eosin
methylene blue agar have been used for the partial confirmatory test.
McCrady (25) has shown that many errors occur with use of E.M.B.
and Endo's media. Mallmann and Darby (26) showed that some organ—
isms placed in brilliant green lactose bile broth or streaked on
E.M.B. agar caused the organisms to lose their ability to ferment
lactose or inhibited these organisms from growing.

It is evident that the dyes now in use are somewhat toxic to
gram negative bacteria. Although most of the enteric organisms
do grow in the presence of these dyes, it is questionable upon initial
isolation from fecal material and sewage contaminated water supplies
whether all of the viable'cells of these organisms grow.

Several years ago in this laboratory Darby (27) in a study
of dyes found that ethyl purple showed less toxicity to some Gram
negative organisms than did such dyes as brilliant green and crystal

violet. These studies were never completed. The limited data
gathered at that time indicated that further studies might be fruitful.
Accordingly the study was initiated to determine more completely
the value of this dye as a bacteriostatic agent for the gram positive
bacteria. The literature is void of any research concerning the use
of ethyl purple in medium work. It has been used, however, as a
stain for spirochetes, islets of Langerhans, and nervous tissue. The

structural formula of ethyl purple 6B is:

.Ca,”
6" ” ON‘CLH:
am i, a O 35‘ c. H
C3,”; O~:C:H:

PROCEDURE:

 

The work consisted of showing the effect of ethyl purple on
a representative group of gram negative and gram positive organisms.
Growth curve studies were carried out on the gram negative organisms.
Ethyl purple was compared to brilliant green, crystal violet, and lauryl
tryptose broth. The organism on which the growth curve studies were
made were 24 hour agar cultures of E. _<_:_9__1_i_, E. typhosa and Salmonella

 

typhimurium.

 

In the first experiment in which lauryl tryptose broth and
lactose broth were compared it was shown that the lag phase was not
as long with lauryl tryptose broth. Since LTB was also shown to be
superior to lactose broth in testing water samples by Darby and
Mallmann (28) it was used as the control and base medium. In the
experiments 100 ml. samples of the LTB were prepared to which was

added just prior to inoculation varied concentrations of the dye. These

samples were then seeded with E. coli, §_. typhimurium and E. typhosa

 

so as to give approximately 0-30 organisms per ml. Duplicate plate
counts were made on the samples at the time of seeding and at 2, 4, 6,
and 24 hours. These plates were counted after 24 hours incubation.
Two trials were made in each case. The results are shown in The
Appendix. Each graph represents one trial.

The second phase of the experimental work was to determine

the effect of ethyl purple on a group of gram positive organisms.

 

Bacteriostatic studies were done on Micrococcus caseolyticus, Staph-

ylococcus and Bacillus subtilis. The base medium was tryptose broth

 

to which was added brilliant green, crystal violet or ethyl purple to
obtain concentrations of 1—1 million, 1~2.5 million, 1-5 million, 1-8
million, 1—10 million and 1-25 or 50 million. One -tenth ml. and

1/10 ml. of 1-10, 1-100, 1-1,ooo, 1-10.000 and 1—1oo,ooo dilution of a
suSpension of organisms from a 24 hour agar slant were planted into
5 tubes of each of the various concentrations of the dye. Plate counts
were made on the .1 ml. amount to determine the approximate number
of organisms inoculated in the tubes. The tubes were incubated and
read at 24 and 48 hours for turbidity.

RESULTS :

I. Growth curve studies of S_. typhimurium

 

Tables II, III, and IV show the growth curves of §_. typhimurium

 

in media containing brilliant green, crystal violet and ethyl purple
respectively. These data show that with brilliant green and crystal
violet there is marked inhibition at a dilution of 1-1 million where as
the break appeared at 1 -333 thousand concentration of ethyl purple.

The reduction in growth rate when ethyl purple was at a concentration

of 1-125 thousand was similar to that of brilliant green at a level of 1-1

million. Graph I shows how closely the growth rate of .Si' typhimurium

 

in the medium containing a 1-1 million concentration of ethyl purple
compared to growth rate of the organism in the control medium. Graphs

I and II demonstrate that the growth rate of S_. typhimurium was much

 

slower in media containing brilliant green or crystal violet. It can be
concluded from the data that ethyl purple is not as toxic to this organism

as brilliant green or crystal violet.

II. Growth curve studies of E. whosa

The results of the growth curve experiment on E. typhosa can be
found in table V, VI, and VII. Brilliant green at a concentration of 1 -1
million was considerably more toxic than ethyl purple was at a conc en—
tration of 1-333 thousand. Ethyl purple proved to be less toxic at a
concentration of 1-333 thousand than did crystal violet at a concentration
of 1 -500 thousand. The crystal violet at this concentration was so
toxic, in fact, that in 24 hours the counts were not anywhere near as
great as their control. Graphs III and IV show the difference in these
curves. Therefore ethyl purple was obviously less toxic to E. ghosa
than was brilliant green or crystal violet.

III. Growth curve studies of E. 39E
The data on the effect of brilliant green, crystal violet, and
ethyl purple on E. g_o_1_i are presented in tables VIII, IX, and X. Brilliant
green was more inhibitive to E. 32E at a concentration of 1-1 million
than was ethyl purple at a concentration of 1 -125 thousand. At a concen-
tration of 1 ~333 thousand ethyl purple was less toxic to the organism
than was crystal violet at a level of 1-500 thousand. Graphs V and VI show that
brilliant green or crystal violet caused slower growth of the organism
than did ethyl purple.

IV. Bacteriostatic studies on Bacillus subtilis

 

Tables XI, XII, and XIII show the bacteriostatic effect of brilliant
green, crystal violet and ethyl purple on E. subtilis. A study of these
tables show that the organism failed to grow with any of the dyes at
a concentration of 1-1 million. In a concentration of 1—2.5 million
there was no growth with crystal violet but one positive tube with brilliant
green and 2 positive tubes with ethyl purple. At a concentration of 1-5
million a high percentage of the tubes were positive with all dyes. Thus
these data indicate that the bacteriostatic effect of these dyes were
not significantly different. It might be noted that the initial inoculation

in the different media was 45,000 organisms.

V. Bacteriostatic studies on Micrococcus caseolyticus

 

The bacteriostatic effect of brilliant green, crystal violet, and
ethyl purple on E. caseolyticus may be found in tables XIV , XV and XVI.

 

Crystal violet had the greatest bacteriostatic effect on this organism
since there was no growth even in the highest dilution of 1 ~50 million.
Ethyl purple was one hundred percent bacteriostatic up to and including
a concentration of 1-5 million. The brilliant green, however was not

as effective since one tube showed growth in as high a concentration

of brilliant green as 1 -1 million. At 1-5 million of brilliant green there
was a high percentage of positive tubes. It is interesting to note that
although there was a heavier inoculant of organisms than with E. subtilis
ethyl purple and crystal violet were more bacteriostatic against E.

caseolytic than with E. subtilis. Ethyl purple, therefore, showed excellent

 

bacteriostatic prOperties against this organism.

VI. Bacteriostatic studies on Staphylococcus aureus

 

The results of the studies made on this Gram positive organism
may be found in tables XVII, XVIII and XIX. In a 1-1 million concentra-
tion of the dyes there was no growth in any of the tubes. There was
scattered growth in the 1-2.5 million concentration. Ethyl purple was
slightly less toxic to the _S_. aureus than was brilliant green and crystal
violet. However, ethyl purple demonstrated good bacteriostatic prOper-

ties against this organism.

DISCUSSION:

 

The usual methods of measuring dye toxicity is by streaking
agar plates and noting the total growth. By comparing the amount of
growth the degree of toxicity can be determined. This procedure is
adequate when the difference in toxicity of various substances are great.
In these studies it was believed that the difference in toxicity would be
too small to measure by this method. Since the difference although
small might be significant the method of using an initial minimum
inoculant and following growth curves over the early growth phase was
used. This method gives a more accurate determination of the toxicity.
Furthermore in the isolation of bacteria where growth must start from
minimal numbers, and frequently from single cells, the effect of dye
would be expected on the first generation of cells. If the dye were too
toxic these few cells may not grow, therefore there would be no isola-
tion obtained, This technique is fully justified by the results obtained.
For example mo st of the growth curves show that there was much var-
iation in the rate of growth up to and including the 6 hour seeding.
However by the 24 hour period the organisms had adjusted themselves

to the dye so that most of the population levels were similar. Thus

10

if the agar streak method had been used, the growth at 24 hours would
have indicated no significant difference between the dyes.

The technique of using growth curves for measuring toxicity
of dyes offers a new method for evaluating dyes and other inhibitory
agents. Darby and Mallmann (29) used this technique successfully in
the deveIOpment of tryptose lactose broth for the detection of colon
organisms.

Ethyl purple with the three test organisms, namely; E. whosa,

E. typhimurium, and E. coli, was found to offer less toxicity than did

 

brilliant green and crystal violet as shown by the reduction in growth
rates. On the other hand the bacteriostatic titers against three Gram

positive organisms, namely; E. subtilis, 2:1. caseolyticus, and _S_. aureus,

 

were not significantly different for the crystal violet, brilliant green or
ethyl purple broths.

It would appear that ethyl purple is an effective bacteriostat
against gram positive organisms and is comparable to brilliant green
and crystal violet. Since the dye could be used as a bacteriostatic
agent against gram positive organisms and at the same time could be
used in a dilution that would be less inhibitive to gram negative organ-
isms than brilliant green or crystal violet, it could be used successfully
in media work. The fact that ethyl purple with the test organism, _S_.
typhimurium at a 1 -1 million dilution allowed a growth rate during the

 

first six hours equal to that of the lauryl tryptose broth control further
indicates its usefulness. This is true especially since ethyl purple was
one hundred per cent bacteriostatic at this concentration.

It has been demonstrated that the substitution of methyl groups
in a hexa methyl tri amino tri phenol methane dye by ethyl groups causes
a decreased toxicity toward Gram negative organisms but little or no

change in toxicity toward Gram positive organisms.

11

It is also interesting to note that in brilliant green which contains
four ethyl groups that a greater toxicity toward gram negative organisms
is exhibited. No explanation for these findings are offered at this time.

SUMMARY:

 

It was shown that ethyl purple is less toxic to Gram negative
organisms than is crystal violet or brilliant green. This difference was
demonstrated by determining growth curves on E. typhimurium, E. _typhosa,

 

and E. coli. The experiments further indicate that at concentrations that
have little or no toxicity to these Gram negative organisms, ethyl purple
is bacteriostatic to such Gram positive organisms as E. subtilis, E.

caseolyticus and E. aureus. It is suggested that further work should

 

be done with media containing ethyl purple for water analysis and enteric

isolations .

12

REFERENCES CITED:

 

l. Churchman, John W. The Selective Action of Gentian Violet on

01

10.

11.

12.

13.

14.

Closely Related Bacterial Strains. Jour. Exptl. Med. 16:
221-247. 1912.

. Churchman, John W. Inhibitory PrOperties of Anilin Dyes. Stain

Technol. 1(1): 27-32. 1926.

Churchman, John W. Relation of Age of Bacteria to Bacteriostatic
Properties of Anilin Dyes. Stain Technol. 1(3): 103-104. 1926.

McCalla, T. M. and Francis E. Clark. Dye Adsorption by Bacteria
at Varying Hydrogen Ion Concentrations. Stain Technol. 16(3):
95-100. 1941.

. Ingraham, Mary A. The Bacteriostatic Action of Gentian Violet

and its Dependence on the Oxidation Reduction Potential. J our.
Bact. 26: 573-598. 1933.

Fisher, E., O. Hoffman, E. Prado and R. Bone. On the Mechanism
of Bacteriostasis with Triphenylmethane Dyes. Jour. Bact. 48:
439-466. 1944.

Conradi, H. and V. Drigalski. Ueber eim Verfahren zum Nachweir
der Typhusbacillen. Zeitschrift fur Hygiene. 39: 283 -300. 1902.

Browning, Gilmour and Mackie. The Isolation of Typhoid Bacilli
from Feces by Means of Brilliant Green in Fluid Medium. Jour.
Hyg. 13: 335-342. 1913.

Endo, S. Ueber ein Verfahren zum Nachweis der Typhus bacillen.
Centralbl. f. Bakteriol. Originale. 35: 109-110. 1904.

Wilson, W. J. and M. Blair. Sodium Sulphite Affording an Enrich—
ment and Selective Medium for the Typhoid—Paratyphoid Groups
of Bacteria. Jour. Path. and Bact. 29: 310. 1926.

Jones, E. R. The Use of Brilliant Green Eosin Agar and Sodium
Tetrathionate Broth for the Isolation of Organisms of the Typhoid
Group. Jour. Path. and Bact. 42(2): 455—467. 1936.

Holt —Harris, J. E. and O. Teague. A New Culture Medium for the
Isolation of Bacillus Typhus from Stools. Jour. Infect. Dis.
18: 596-600. 1916.

Levine, Max. Differentiation of B. coli and E. aerogenes on a
Simplified Eosin Methylene BIﬁe Agar J our. Iaect. Dis. 23:
43-47. 1918.

Smith, Theobald. Ann. Rept. State Bd. of Health of N. Y. 13:
712. 1893.

15.

16.

l7.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

13

Hall and Ellefson. Further studies on Gentian Violet as a Means of
Eliminating Spurious Presumptive Tests for B. coli in Water.
Jour. Amer. Water Works Assoc. 6: 67. 19T9.

Muer, T. C. and R. L. Harris. Value of Brilliant Green in Eliminat—
ing Errors Due to the Anerobes in the Presumptive Test for
E. coli. Amer. Jour. Publ. Health. l-: 874. 1920.

Jordan, Harry E. Brilliant Green Bile as the Detection of the Colon-
Aerogenes Group. Jour. Amer. Water Works Assoc. 18: 337. 1927.

Horwood, M. P. and A. Heifertz. A Comparative Study of Certain
Presumptive Test Media. Jour. Bact. 27: 57-58. 1934.

Ruchhoft, C. C. Comparative Studies of Standard Methods and
the Brilliant Green Bile Medium on Lake Michigan Water at
Chicago. Jour. Amer. Water Works Assoc. 16: 778. 1926.

Mallmann, W. L. and C. W. Darby. The Use of Sodium Lauryl
Sulfate Lactose Tryptose Broth as a Primary Medium for Detec-
tion of the Coliform Group. Amer. Jour. Publ. Health. 31:
127-134.

Stark, C. N. and C. W. England. A Note on the Use of Crystal
Violet in Presumptive Tests for Water Pollution. J our. Bact.
23: 36. 1932.

Ruchhoft, C. C. and John E. Norton. Study of Selective Media for
Coli—aerogenes Isolation. J our. Amer. Water Works Assoc.
27: 1134. 1935. .. '

Black, Luther A. and Mary E., Klinger. A Comparison of Media
for the Detection of Escherichia -Aerobactor. Jour. Bact. 31(2):
171-179. 1936.. ' ‘

Butterfield, C. T. Experimental Studies of Natural Purification
in Polluted Waters VII. The Selection of a Dilution Water for
Bacteriological Examinations. Publ. Health Repts. 48: 681-
691. 1933.

McGrady, M. H. A Practical Study of Procedures for the Detec-
tion of the Presence of Coliform Organism in Water. Amer.
Jour. Publ. Health. 27: 1143-1258. 1937.

Mallmann, W. L. and C. W. Darby. Uses of Lauryl Sulphate
Lactose Broth for the Detection of Coliform Organisms.
Amer. Jour. Publ. Health. 31: 127-134.

Darby, C. W. Studies on Primary and Selective Media for Coli-
form Organisms. Thesis for Master of Science Degree.
Michigan State College. 1943.

Darby, C. W. and W. L. Mallmann. Studies on Media for Coli—
form organisms. Jour. Amer. Water Works Assoc. 31(4):
689-706. 1939.

14

29. Mallmann, W. L. and C. W. Darby. Uses of Lauryl Sulphate Lactose
Broth for the Detection of Coliform Organisms. Amer. J our.
Publ. Health. 31: 127-134.

Table I - A Comparison of Growth Rates for Lactose Broth and

Lauryl Tryptose Broth using Escherichia coli as a Test Organism.

 

Hours Comparison of Lactose Broth to Lauryl Tryptose Broth.

 

 

Incub. Eactose Eauryl Tryptose

Broth Broth

.01 1* .009

o .019 .045
2 . 65 . 95
4 7.3 49.00

6 300,000 600,000

24

 

* Bacteria per ml. in thousands.

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Table XI - Bacteriostatic Action of Various Concentrations

of Brilliant Green on Bacillus Subtilis

 

 

Dilutions of organisms*

 

 

 

Dye Hours Undil. 1-10 1-100 1-1t 1—10t 1-100t
Dilutions Incub. Number of positive tubes

1 24 o** o o o o o
m 48 o o o o o o
24 o o o o o o
2'51“ 48 2 o o o o o
24 5 o o o o 0
5m 48 5 1 2 1 o o
24 5 5 5 4 o o
3'3“” 48 5 5 5 5 4 2
10m 24 5 5 4 4 1 o
48 5 5 5 5 4 0
25m 24 5 5 5 5 4 1
48 5 5 5 5 4 1
24 5 3 5 - 5 4 1
TLB 48 5 3 5 5 5 1

 

i «f W5

 

* Initial inoculation of 45,000 organisms.
** Number of positive tubes out of five inoculated.

Table XII - Bacteriostatic Action of Various Concentrations

of Crystal Violet on Bacillus subtilis

 

 

Dilutions of or ganisms*

 

Dye Hours Undil. li—lO 1-100 1—1t l-lOt l-lOOt

 

 

Dilutions Incub. Number of positive tubes
24 o** o o o o 0
1m 48 o o o o o o
24 o - o o o o o
2°51” 48 o o o o o 0
5m 24 5 5 5 5 1 o
48 5 5 5 5 4 2
24 5 o o o o o
8°31“ 48 5 4 5 5 2 1
24 5 5 5 o o 0
50m 24 5 5 5 5 4 o
48 5 5 5 5 5 1
24 5 3 5 5 4 1
TLB 48 5 3 5 5 4 1

‘ tor—15mm“. ”1.11 '18:! 4.1-425?
.. q ‘

 

* Initial inoculation of 45,000 organisms.
** Number of positive tubes out of five inoculated.

Table XIII - Bacteriostatic Action of Various Concentrations

of Ethyl Purple on Bacillus subtilis

 

 

Dilutions of organisms*

 

Dye Hours Undil.

 

 

1-10 1-100 l-lt 1-10t 1-100t
Dilutions Incub. Number of positive tubes
24 o** o o o o 0
1m 48 0 o o o o o
24 o 1 1 o o o
2'51“ 48 o 1 1 o o o
24 4 3 2 1 o 0-
5m 48 5 5 3 1 o o
24 1 2 5 4 4 2
8°31“ 48 1 2 5 4 4 2
24 1 2 2 5 5 2
10m 48 1 2 2 5 5 2
50m 24 5 5 5 5 4 3
48 5 5 5 5 5 4
24 5 3 5 5 4 1
TLB 48 5 3 5 5 4 1

 

* Initial inoculation of 45,000 organisms.
** Number of positive tubes out of five inoculated.

 

Table XIV - Bacteriostatic Action of Various Concentrations

of Brilliant Green on Micrococcus caseolyticus

 

 

-‘ .

Dilutions of organisms*

 

 

 

Dye Hours Undil. 1-10 1-100 l-lt 1-10t 1—100t
Dilutions Incub. Number of positive tubes

24 1M 0 o o o 0
1m 48 1 o o o o o
24 4 o o o o . o
2°51“ 48 5 o o o o 0
5m 24 5 5 5 o o o
48 5 5 5 4 2 o
8 3m 24 5 5 5 5 5 1
° 48 5 5 5 5 5 5
10m 24 5 5- 5 5 5 3
43 5 5 5 5 5 4
24 5 5 5 5 4 0
25m 48 5 5 5 5 5 5
24 5 5 5 5 5 4
TLB 48 5 5 5 5 5 5

 

* Initial inoculant 175,000 organisms.
** Number of positive tubes out of five inoculated.

 

Table XV - Bacteriostatic Action of Various Concentrations

of Crystal Violet on Micrococcus caseolyticus

 

 

Dilutions of organisms*

 

 

 

Dye Hours Undil. 1-10 1-1oo l-lt 1-10t 1-100t
Dilutions Incub. Number of positive tubes
1m 24 o** o o o o o
48 o o o o o o
24 o o o 0 o o
2'5““ 48 o o o o o o
24 o o o o o 0
5m 48 o o o o o o
24 o o o o o o
8°31“ 48 o o o o o o
24 o o o o o 0
10m 48 o o o o o 0
50m 24 o o o o o o
48 o o o o o o
24 5 5 5 5 5 5
“‘3 48 5 5 5 5 5 5

 

* Initial inoculant 175,000 organisms
** Number of positive tubes out of five inoculated.

 

Table XVI - Bacteriostatic Action of Various Concentrations

of Ethyl Purple on Micrococcus caseolyticus

 

 

Dilutions of organisms*

 

 

 

 

Dye Hours. Undil. 1-10 1-100 l-lt l-lOt 1400: it? .
Dilutions Incub. Number of positive tubes g
24 0** 0 0 o 0 0
1m 48 0 0 0 0 0 0 j;
24 0 0 o 0 0 0 [i
2'51“ 48 0 0 0 0 0 0 .
24 0 0 0 0 0 0
5m 48 0 2 0 0 0 0
24 5 4 0 0 0 o
8'31“ 48 5 5 5 3 1 2
24 1 0 1 0 0 0
10m 48 1 5 2 1 o 0
50m 24 5 5 5 5 5 5
48 5t 5 5 5 5 5
24 5 5 5 5 5 5
TLB 48 5 5 5 5 5 5

 

* Initial Inoculant 175,000 organisms.
** Number of positive tubes out of five inoculated.

 

Table XVII - Bacteriostatic Action of Various Concentrations

of Brilliant Green on Staphylococcus aureus

 

 

Dilutions of organisms*

 

Dye Hours Undii. 1-10 1-100 1-1t 1—10t 1-100t

 

 

. Dilutions Incub. Number of positive tubes '
24 0** 0 0 0 0 0
1m 48 0 0 0 o 0 0
24 0 0 0 0 0 0
2°51“ 48 1 0 0 0 0 o
24 5 0 0 0 0 0
5m 48 5 1 0 o 0 0
8 3m 24 5 0 0 0 0 0
° 48 5 5 1 0 0 0
10 24 5 5 5 5 2 0
m 48 5 5 5 5 5 5
25m 24 5 5 0 0 0 0
48 5 5 5 0 0 0
24 5 5 5 5 5 5
TLB 48 5 5 5 5 5 5

 

* Initial inoculant was 2,300,000 organisms.
** Number of positive tubes out of five inoculated.

Table XVIII - Bacteriostatic Action of Various Concentrations

of Crystal Violet on Staphylococcus aureus

 

 

Dilutions of organisms*

 

Dye Hours Undil. 1-10 1-100 l-1t 1-10t 1-100t

 

 

Dilutions Incub. Number of positive tubes

1m 3: 3* 3 3 3 3 3
um 33 3 3 3 3 3 3
sm 3: 3 3 3 3 3 3
am 33 3 3 3 3 3 3
mm 3: 3 3 3 3 3 3
5... 33 3 3 3 3 3 3
333312 i: 2 2 3 8 8 3
5m

TLB if; g 2 E, 2 ‘2 g

 

* Initial inoculant is 2,300,000 organisms.
** Number of positive tubes out of five inoculated.

Table XIX - Bacteriostatic Action of Various Concentrations

of Ethyl Purple on Staphylococcus aureus

 

 

Dilutions of organisms*

 

Dye Hours Undil. 1-10 1-100 l-lt 1-10t l-lOOt

 

Dilutions Incub. Number of positive tubes

1m 33 3’” 3 3 3 3 3
23m 33 3 3 3 3 3 3
5m 33 3 3 3 3 3 3
8-3m 33 3 3 3 3 3 3
10m 33 3 3 3 3 3 3
50m 24 5 5 5 5 5 O

48 5 5 5 5 5 1
TLB 33 3 3 3 3 3 3

 

* Initial inoculum - 12,000,000 organisms per one -tenth milliliter.
** Number of positive tubes out of five inoculated.

GRAPH I - E. typhimurium growth curves with 1—1 million concentration

 

of ethyl purple and brilliant green.

---LTB control curve for B. G.
—Brilliant green curve l—lM
---LTB control curve for E. P.

”Ethyl purple curve l-lM

Population (in logarithms)

 

0 2 4 6

Hours incubation

Papulation (in logarithms)

GRAPH II - E. typhimurium growth curve with l-lM cone. of ethyl

 

purple and crystal violet

—--LTB control for C. V.
——Crystal violet curve 1-500T

~~LTB control curve for E. P.

--Ethyl purple curve l-333T

 

 

0 ' 2 4 8

Hours incubation

Papulation (in logarithms)

GRAPH III - E. typhosa growth curves with 1—333 thousand concentration

of ethyl purple and l -500 thousand concentration of crystal violet.

---LTB control curve
--- Crystal violet curve 1 -500T
--- LTB control curve for E. P.

“Ethyl purple curve 1-333T

—“2- -‘ can--.”

M’

0 2 j 6

Hours incubation

Population (in logarithms)

GRAPH IV - E. typhosa growth curves with 1-333 thousand concentration
of ethyl purple and 1-l.6 million concentration of brilliant green.

-‘-LTB control curve for B. G.
---Brilliant green curve 1-1.6M
~----LTB control curve for E. P.

_Ethyl purple curve 1 -333T

Hours incubation

GRAPH V - E. coli growth curves with 1-333T concentration of ethyl
purple and l ~500T concentration of crystal violet.

--- LTB control curve for C. V.
”Crystal violet curve 1-500T
---LTB control curve for E. P.

wsEthyl purple curve 1-333T

Population (in logarithms)

 

 

 

0 2 4 6

Hours incubation

Population (in logarithms)

GRAPH VI- E. coli growth curves with 1-333 thousand concentration

of ethyl purple and 1-1 million concentration of brilliant green.

““ LTB control curve for B. G.
“Brilliant green curve l-lM
~' WLTB control curve for E. P.

“Ethyl purple curve 1-1M

 

 

 

0 2 4 6

Hours incubation

ROOM use ONLY)"-
Mﬁcqtg

MR3153

 

 

 

 

.0 .0! 3.33.1311

MICHIGAN STATE UNIVERSITY I

l llll HI 3333355
031162 3322

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