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This is to certifg that the

thesis entitled
Differentiation Between Typhoid V1 and W
Antigens by 2, 3, S Triphenyltetrazolium
Chloride Incorporated in a Medium.

presented hg

Grace Allen

 

has been accepted towards fulfillment
of the requirements for

 

M. S. degree in Bacteriolog
W\.Wc
Major professor
May 16, 1951

Date

 

0-169

 

 

DIFFERENTIATION EETWEEN TYPHOID VI AND w ANTIGENS
BY 2,5,5-TRIPHENYL TETRAZOLIEM CHLORIDE
o

INCORP RATED IN A MEDIEM

By

Grace Maghakian Allen

“at,

A 71*‘1.—\ '1-

A TLILD .LS

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

for the degree of
MASTER OF SCIENCE

Department of Bacteriology and Public Health

1951

 

@Gf/g/
ng/f

ACKNOWLEDGEMENT
INTRODUCTION.

HISTORY . . . .
TETRAZOLIULI . .
MATERIALS . . .
METHODS . . . .
DISCUSSION. . .
smmmw. ...
BIBLIOGRAPHY. .

TABLE

OF CONTENTS

Page

ll
14
17
24
28
30

ACKNOWLEDGEMENT

The author wishes to express sincere appreciation
to Dr. H. J. Stafseth, Professor and Head of the Department
of Bacteriology and Public Health for his corrections of
this manuscript and to Dr. W. N. Mack, Associate Professor
of Bacteriology and Public Health, for his advice and
counsel. '

The author also wishes to express sincere gratitude to
Dr. I. F. Huddleson, Professor of Bacteriology and Public
Health for his patient instruction of procedures used in
this study and to Dr. W. N. Ferguson, Associate Director
of the Salmonella Typing Laboratory, Michigan Department
of Health, for his helpful suggestions and for supplying
the required cultures.

The author also wishes to thank Mr. Roland C. Fulde,
Graduate Assistant in the Department of Bacteriology and
Public Health, Michigan State College, for the preparation

of photographs used herein.

 

 

 

III.

INTRODUCTION

The reduction in morbidity due to typhoid fever in
various parts of the world has been one of the most
striking phenomena in modern public health experience.
Despite our modern methods of sanitation and public health
programs, typhoid fever continues its manifestation as
isolated epidemics in rural communities and institutions.
In view of this fact, any information concerning the
etiologic agent, Salmonella typhosa, is received as a
possible basis for better control of the disease.

The discovery of V1 antigen and its association with
the virulence of S. t hosa, (Felix and Pitt, 1934) in-
stituted a new field of speculation and research on that
organism. The practical results of these investigations
are generally evident in our procedures for laboratory
diagnoses, specific therapy and prophylaxis. Of these,
the introduction of routine bacteriophage typing (Craigie
and Yen, 1958) has served as an invaluable aid to the
epidemiologist. This method permits the identification of
V1 antigen in a culture. It also permits the identification
of the specific type responsible for producing the disease
in a case, carrier or epidemic.

Basically, the technique for bacteriophage typing
requires introducing the effects of one living agent upon
another. The simplest alternative for detecting Vi antigen,

would be direct observation of characteristic colonial

differences between strains of typhoid bacilli containing
Vi and W antigens. Several authors have reported these
differences. However, their distinction is necessarily
reserved for those who are highly experienced and
therefore capable of accurate judgement.

It is desirable to place at our disposal a relatively
tractable method for recognizing the presence of Vi or W
antigens in a culture. The utilization of 2,3,5-tripheny1
tetrazolium chloride has recently been adopted (Levine
1949, Huddleson 1950) for differentiating bacterial
variation by colonial observation. In this study, this

salt was applied to S. typhosa with the same purpose.

 

l

HISTORY

One of the earliest reports describing the visible
effects of variation in typhoid organisms was contributed
by Savage (1901). He observed clumping of S. typhosa in
old broth cultures simulating agglutination, in contrast
to the bacilli which.remained dispersed and in active
movement in young broth cultures. This was reported due
to frequent subcultivation rather than prolonged incu-
bation. He attributed the pseudo-clumping as having
some bearing on the susceptibility of the race of S. typhosa.

Essentially the same phenomenon was later detected
by Arkwright (1922), Andrewes (1922), White (1929) and
Grinnell (1930). Their observations ultimately raised
the question regarding the comparative efficacy of
vaccines prepared from rough and smooth colonies. Grinnell
suggested that some strains utilized for typhoid vaccines
may have originated from the rough, less potent variety
and others from the smooth, virulent types.

Such evidence was conducive to more thorough investi-
gation by Felix and Pitt (1934a), who reported that rough-
ness in colonies indicated low virulence, while smoothness
was an indication of high virulence. However, the mere
presence of smooth "0" or somatic antigen did not neces-
sarily indicate high virulence. They determined virulent
strains by agglutination tests. Those strains of S.

typhosa which demonstrated inability to agglutinate with

smooth "0" antibodies, were termed O-inagglutinable, and

proved to be highly virulent. O-agglutinable strains were
not as virulent. Since the strains were not distinguish-
able by smooth or rough origin, it was indicated that some
unknown factor was essential for the resistance to 0 anti-
gen antibody. In that same year, Felix and Pitt (1934)
were able to isolate a new antibody independent of the O
and H antibodies. The symbol "Vi" was used to designate
the new antibody. The correspOnding antigen was termed
Virulence Antigen, or "Vi Antigen".

Further experiments by Felix and Pitt (1935), Felix,
Krikorian and Reitler (1955), and Felix et a1 (1955),
provided for better understanding of the Vi antigen.
Independence of the Vi antibody was established by absorption
tests. This was confirmed by Dyachenko (1936). 'The V1
antibody was more protective than 0 antibody, and the
combination of Vi and 0 antibody neither increased nor
diminished that protective capacity. An explanation for
this protective ability was offered by Bhatnagar (1935)
who reported that inagglutinable strains were highly
resistant to phagocytosis, while agglutinable strains were
highly susceptible.

The controversy developed as to whether Vi antigen was
actually representative of virulence. Felix (1936) maintained
that it was, despite the fact that Vi antigen was present
in avirulent rough variants of virulent smooth strains.

AKEuffmann and Orskov (1936) related virulence to toxic

conditions in mice. Large doses of living Vi forms were
reported to cause acute intoxication followed by death

in two days. With smaller, non-intoxicating doses it was
not possible to demonstrate any difference between Vi and
non-Vi forms of the same strain, with respect to severity
of infection. They concluded that Vi antigen plays no part
in virulence of typhoid bacilli for mice. Almon (1943)
attributes the toxic effect of Vi antigen in large doses
to superior multiplication of the Vi organism. The
situation of Vi substance near the surface of the antigen
has also been suggested as a possible key to the genesis
of virulence.

It was noted that some Vi properties could be altered
simply by routine transfer of a culture. Changes were
determined by agglutination and absorption tests. The
transformation from Vi to non-Vi was gradual and involved
an intermediate condition in which the original resistance]
to O agglutination was lost but agglutination in Vi anti-
serum was retained. Kauffmann (1935) introduced a termin-
ology pertaining to these changes. Those organisms con-
taining Vi antigen and demonstrating 0 inagglutinability
were termed "V" forms (viel). Intermediate conditions
were termed *VQW" forms (transition). Finally, when all
Vi characteristiCS were lost the organisms were called
"W" forms (wenig). Kauffmann believed that V-W transition
was reversible but this condition is known to be rare.

Felix and Pitt (1934a) demonstrated the presence of

Vi antigen by agglutinin absorption tests, as previously
mentioned. Serological selection of this type entails
considerable time and labour. Kauffmann's (Craigie and Bran-
don, 1936) slide agglutination method has proved less
complex and more accurate.

It was not until Craigie and Brandon (1936) discovered
a Vi bacteriophage, that a more rapid and reliable method
for detecting Vi antigen was introduced. It was noted
by these workers that the Vi form of S. typhosa was
sensitive to a bacteriophage isolated from the stool of
infected persons. Sensitivity was manifested by lytic
areas on the surface of inoculated plates. W forms re-
mained invulnerable to bacteriophage activity. Mixed
cultures of VSW forms were detected by their presentation
of typical "moth-eaten" areas where inhibition of Vi and
non-inhibition of W forms were simultaneously present.

By examining the various strains of bacteriophage
active against S. tvohosa, Craigie and Yen (1938) were
able to isolate a group of four types of Vi bacteriophages
(Table I). It was observed in particular that 'Phage II
propagation on a strain of S. _yphggg resulted in the de-
velopment of a highly selective, lytic activity for the
substrate strain. The advantages of this response were
utilized toward determining the identity of Vi strains in
unknown cultures.

It was assumed by Craigie and Yen (1938), that varie-

ties of S. typhosa occurred which differed only in their

TABLE I

Properties of the Four Vi Bacteriophages

 

 

Vi Relative Thermal Neutralization Lytic activity
bacterio- particle death by antiphage for Vi forms of
phage size point serum ‘S. typhi
I II III IV
Type I Large 67-7000 - - - - Lyses all Vi
'phage forms
Type II Medium 69-7200 - - - - Develops a high
'phage selective lytic
activity for the
type of S. typhi
on which it is
propagated
Type III Small 61—6400 - - - - Lyses majority
'phage of Vi forms
Type IV Medium 59-6200 - - - - Lyses majority
'phage of Vi forms with

exceptions of

Types F and D1

 

From the above information,

deduced several facts:

Craigie and Yen (1938)

l. The four bacteriophages are specific for the

Vi form of S. typhosa.'

2. Serologically, the four types of bacteriophage

are distinct.

3. A variation occurs in the lytic action on Vi

form strains, particularly in the case of

Type II bacteriophage.

Vi antigen components. Consequently, they postulated that
‘Phage II exists as a series of mutants differing from each
other in selective affinity for different Vi strains of S.
typhosa. The loss of unrelated mutant particles with
concurrent development of a specific mutant on the substrate
strain, probably initiated the increase in specificity of
bacteriophage.

By continuous selective propagation of these mutants
on a substrate strain, it was possible to obtain bacterio-
phage preparations which manifested a highly developed
affinity for the homologous Vi strain of S. tvphosa.
Applying this method of selective propagation, Craigie and
Yen (1938) produced the following eleven bacteriophages:

A, B1, B2, C, D1, D Fl’ G, H and L. At present, the

29 El,
complete list of known types and subtypes includes A, B1’

32, B3, C, D1, D2, D4HP56, D5, D6, El, E2, F1, F2, c, H,

J, K, L1, L2, M, N, O and T. The reactions of these
bacteriophages against their corresponding Vi type strains
are tabulated in Table II.

The specificity of Vi type strains may be questioned
since Type A displays sensitivity to all Vi bacteriophage
preparations. However, this is of rare occurrence. Craigie
and Felix (1947) recently indicated that this may be due
to dissociation from the original Vi type or types present
in a carrier.

The application of bacteriophage typing in diagnostic

laboratories has established its value for accurate deter-

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mination of the presence of Vi antigen as well as the
specific type present.

The differentiation between Vi and W forms by visual
means has been more recently described by Landy (1950).
With the optical arrangement properly adjusted, Vi forms
appear very dense and reddish—coppery in color, while the
W forms are translucent and appear grayish green. The
degree of denSity of the colony and the ceppery appearance
is associated with the quantity of Vi antigen present. It
has been previously stated that this procedure demands
the efforts of those who are highly skilled and therefore
competent in detecting the subtle points for differentia-

tion.

ll

TETHAZOLIUM

In recent years a new compound, 2,5,5-triphenyl tetra-
zolium chloride, has gained the interest of bacteriologists

as a prospective indicator of colonial differences in

'bacterial variants. For the purpose of simplicity the

:3alt will hereafter be referred to as tetrazolium.

The advantages of tetrazolium were originally dis-

c¢3vered in Germany by a botanist (Lakon, 1942). He applied

tlae salt as a means of determining the viability of seeds.
Idss practical value was recognized in this country and
soon adopted for purposes of a similar nature.

iattson et a1 (1947) revealed that living tissues

VViJll reduce the colorless tetrazolium to form an insol-

‘UJDILe, triphenyl formazan which is red. They reported

‘tlazat the reduction is produced by the following reaction:

 

-‘N l+cffloo + a. H‘ H'N—Nac'“l‘||-=N*H+ “-
.9.
L \
/ /

12

The reaction was described as a result of enzyme
activity present in living tissue. It was indicated more
specifically, that this activity was probably a dehydrogenase
system requiring coenzymes I and II as the electron
acceptor. Thus, variations occurring in the enzyme activity
of an organism, will reduce the salt accordingly.

Lederberg (1948) found that tetrazolium was instrumental
in determining the presence of fermenters and non-fermenting
variants of Escherichia co;i. Non-fermenting variants
stained red, whereas the fermenters produced no change.

Levine and Garber (1950) speculated on the effects

of tetrazolium on dissociants of Pasturella pestis. They

 

found that smooth and rough colonies could be readily
identified when cultivated on tryptose agar containing
tetrazolium. Smooth colonies were approximately 2 mm. in
diameter, round, and exhibited sharply defined carmine
red centers. Rough colonies were irregular in outline
and presented by a diffused pink color.

That same year, Huddleson (1950) published a more
conclusive report of results obtained in subjecting
various bacterial species to tetrazolium. He observed
differences in color and opacity of the borders surround-
ing the colonies as well as varying degrees of color in-
tensity within the borders. He particularly described the
effects produced on the species of Erucella. The S phase

colonies of Brucella suis stained dark red in the central

 

portion, while the borders were light opaque yellow. In

15

contrast, Brucella abortus stained lighter red in the central
portions with light, almost transparent, bluish.green
borders. Finally, Brucella melitensis stained the lightest
red in the central portions with light opaque pink borders.
Huddleson's work was not restricted to the variations
occurring in the Brucella species, but included various

phases occurring within the species of Salmonella, Shigella,

 

Escherichia, Alcaligenes, Streptococcus and some aerobic
spore-forming bacilli.

In view of these results, tetrazolium was employed
as a possible indicator in the differentiation between

thm Vi and W forms of §. tgphosa.

14

MATERIALS

The four Vi-type cultures of E. typhosa: A, D4HP56,

E1, and F1, and their homologous Vi bacteriophage strains

in concentrated form, originated from the Connaught Medical

Research Laboratories, University of Toronto, Toronto,

Canada. They were subsequently received by this laboratory

through the Michigan State Department of Health, Lansing,

Michigan. Type H901 was received upon request from Dr. W.

Ferguson of the Michigan State Department of Health.

All the Vi-type cultures were maintained on Dorset

Ifigg medium.(3 parts whole egg, 1 part saline, slanted

arnd inspissated). Stock strains were transferred to new

slants at five month intervals following passage on nutrient

aggar and tests for specificity. 'Eacto' Tryptose Agar

slnants were used in making daily transfers.

A preliminary test on each concentrated Vi-‘phage
tyjpe was determined to obtain its Critical Test Concen-
‘treition (C.T.C.) which is the greatest dilution of bacterio-
Phage that will produce inhibition on the homologous

Strain of §_. tzohosa. This resulted in diluting each

bacteriophage to 1:100.
The following media were prepared for bacteriophage

tyDing, as recommended by Craigie and Yen (1938):

15

Agar medium

'Bacto' nutrient broth (dehydrated) 20.00 gms.
Sodium chloride 7.50 gms.
'Bacto' agar 20.00 gms.
Distilled water to 1000.00 ml.

'Phage dilution broth
'Bacto‘ nutrient broth (dehydrated) 8.00 gms.

Distilled water to 1000.00 ml.

'Phage culture broth

'Bacto' nutrient broth (dehydrated) 15.00 gms.
Sodium chloride 7.00 gms.
Distilled water to 1000.00 ml.

The tetrazolium, in powder form, was obtained from the
khantclair Research Corporation, Montclair, New Jersey. This
nmaterial was stored in a dark place as accompanying instruction
Slpecified. A 1 percent aqueous solution was prepared and
Strerilized at 1150 C for 15 minutes. This was added to
'Iiacto' tryptose agar in the following manner: 1 ml. of
3- IDercent aqueous solution of tetrazolium.was transferred
t”) 100 m1. of sterile, melted tryptose agar. The agar was
coOled before adding the tetrazolium solution. This com-

bination was mixed well to insure uniform distribution.
T1163 mixture was then poured in Petri dishes approximately
0.5 cm. in depth, and incubated at 57° C for about twenty
h(Furs to remove excess moisture from their surfaces.

Tube 1 McFarland's nephalometer was used as a turbidi-

16

metric standard for estimating the dilution of a culture
suspension. The density of Tube 1 McFarland's nephalometer
represents a bacterial suspension approximating three
hundred million organisms (Todd and Sandford, 1948).
Dilution blanks containing 99 ml. of sterile physio-
logical saline solution was used to reduce the concentration

of organisms.

17

METHODS

All cultures were typed to determine the presence,
or absence of Vi antigen, concurrently with inoculation
on tetrazolium-agar plates. Typing procedures were
duplicated as recommended by Craigie and Yen (1938) in
every detail.

A small amount of the growth on tryptose agar slants
was inoculated into 2 ml. of nutrient broth and allowed to
incubate at 57° C for 2-4 hours. At the end of this period,
nutrient agar plates were marked for areas which were in-
oculated in the following manner. A loopholder with a
standard platinum loop (2.75 mm. in internal diameter),
was supported between the thumb and first two fingers
like a pen. A full loopful of the broth culture was ob-
tained by inserting the loop horizontally without touching
the sides of the tube and quickly removing it while in the
horizontal position. With the forearm and side of the hand
resting on the bench, the drop was transferred to the
marked area on the agar plate by merely touching the surface
of the agar. The loop was then horizontally moved about
an imaginary circle by manipulating the fingers over an
area approximately 15 mm. in diameter. Thus, the drop is
allowed to spread in a natural way. The 100p was actually
used as a guide and never allowed to come in contact with

the surface of the agar. This caution is to be observed

18

to prevent pseudo-lytic areas.
When the fluid had become absorbed, by the agar, (5-10

minutes), the bacteriophage was applied to the center of

the inoculated area. In this case, the loopholder was sup-
ported between the thumb and fingers in a horizontal
position. The 100pful of bacteriophage was lowered until
it almost touched the surface of the agar, at which time,
it was rotated into a vertical position allowing the
bacteriOphage to flow onto the center of the inoculated
area. Once again, actual contact of the loop and agar
was avoided.

When the plates were thoroughly dried, they were
incubated for 2 hours at 57° C. They were then removed
to the refrigerator overnight followed by reincubation for
4-6 hours. The reactions were observed for lytic areas

against a dark background.

The method of interrupted incubation is preferable

in view of the following advantages. Bacteriophage

. reactions may be observed at their optimum activity before
they are obscured by overgrowth by some strains. The in-
terval of storage in the cold room permits diffusion of
the bacteriophage into the surrounding normal culture.

As previously stated, cultures were typed concurrently
with inoculation of tetrazolium-agar plates. The procedure
for inoculation was as follows: a small amount of growth

was removed from a tryptose agar slant and inoculated into

19

a tube containing 10 m1. of sterile, physiological saline
solution. The organisms were dispersed in the solution

with the aid of the loop and if necessary, by agitating

the tube. This was compared with the Tube 1 nephalometer.

If the suspension was too light or too heavy, it was
corrected by a heavier inoculation of organisms or dilution
with sterile saline as required. With the aid of a graduated
pipette, 0.1 ml. of the culture suspension was transferred to
a dilution blank containing 99 ml. of sterile, physiological
saline solution. After mixing well, 0.1 ml. was removed

and transferred into a second dilution blank, which was

also mixed well. Einally, 0.5 ml. was removed from the
second blank and transferred onto the tetrazolium-agar

plate. This was rotated to insure uniform expansion of

the suspension. The plate was allowed to dry at room
temperature on a table or board with a level surface.

When the suspension had been absorbed, the plates were
inverted and incubated at 57° C for 48 hours. The resultant
red colonies were examined for various color patterns as
recommended by Huddleson (1950). A plate was placed "under
a low power (x12) dissecting microscope which was illum-
inated by reflected, oblique light from the new Spencer
micr0300pe lamp, Model #755. The light was focused at an
angle of 40° on a small, concave mirror placed in front of
the microsc0pe. The rays were reflected at an oblique angle
from the mirror so as to strike the under surface of the

Petri plate resting on the glass stage."

20

To stimulate the transformation from the Vi form to
the W form, several series of cultures corresponding to
the stock strains were transferred daily, to fresh tryptose

agar slants.

21

TABLE III

Color Patterns of Type A Strain

 

 

 

Time Vi 'phage Colonies Color patterns
typing Borders Central portions
4 weeks CL S N, LYO IDCR
8 weeks CL S N, LYO IDCR
12 weeks CL S N, LYO UR
16 weeks CL S N, LYO UR

 

CL - Confluent lysis LYO - Light Yellow Opaque

S - Smooth IDCR - Interior Deep Crimson Red
N - Narrow UR - Uniform.Red
TABLE IV

Color Patterns of Type D4HP56

 

 

 

Time Vi 'phage Colonies Color patterns
typing Borders Central portions
4 weeks CL S N, LYO IDCR
8 weeks - S N, Bl 0 UR
12 weeks - R0 W, BT R
16 weeks - R0 W, BT R

 

CL - Confluent lysis LYO - Light Yellow Opaque

- - No lysis IDCR- Interior Deep Crimson Red
S - Smooth UR - Uniform Red

N -~Narrow BT - Blue, transparent

W - Wide R - Radial

El 0 - BIuish Opaque RO - Rough

22

TABLE V

Color Patterns of Type E1 Strain

 

 

 

 

Time Vi 'phage Colonies Color_patterns
typing Borders Central portions
4 weeks CL S N, LYO IDCR
8 weeks f S N, LYO UR and R
12 weeks - S N, Bl 0 UR and R
16 weeks - S W, BT UR and R
CL - Confluent lysis LYO Light Yellow Opaque

- - No lysis IDCR Interior Deep Crimson Red
é - Partial lysis UR - Uniform Red
N _ Narrow B1 0 - Bluish Opaque
W _ Wide BT - Blue Transparent
s - Smooth R - Radial
TABLE VI

Color Patterns of Type F1 Strain

 

 

 

Time Vi 'phage Colonies Coigr patterns
typing Borders Central portions
4 weeks CL S N, LYO IDCR
8 weeks 5 s N, LYO UR and R
12 weeks - Ro W, ET UR and R
16 weeks 0 0 0 ' 0

 

CL - Confluent lysis N - Narrow

- - No lysis w - Wide
5 - Partial lysis LYO — Light Yellow Opaque
0 - No growth IDCR - Inter Deep Crimson Red
S - Smooth 1 UR - Uniform Red
R0 - Rough BT - Blue Transparent
R - Radial

23
TABLE VII

Color Patterns of Type Fl Strainw

 

 

 

Time Vi 'phage Colonies Color_patte3ns
typing Borders _Central portions

4 weeks CL S N, LYO IDCR
8 weeks CL S and Ro N, LYO IDCR and R
12 weeks CL S and Ro N, LYC IDCR and R

 

CL - Confluent lysis LYO - Light Yellow Opaque

S - Smooth IDCR - Interior Deep Crimson Red
Ro - Rough R --Radial

N - Narrow

* This strain was requested from the Michigan State
Department of Health when its. preceding corres-
ponding strain had become non-viable.

TABLE VIII

Color Patterns of Type H 901 Strain

 

 

 

 

Time Vi 'phage Colonies Color patterns
typing Borders Central portions
Upon
receipt - S and R0 W, BT UR
- - No lysis W - Wide
S - Smooth BT - Blue Transparent

Ro - Rough UR - Uniform Red

 

Smooth and Rough Vi Antigen Colonies

 

W Colonies

 

V4W Colonies

 

Single Vi Antigen Colony Among W Colonies

(Taken with two different time exposures)

 

w Colonies with Radial Pattern

24

DISCUSSION

It may be seen from the preceding tables that various
color patterns are demonstrated during increased periods
[of subcultivation. At the beginning of the study, when
types A, D4HP56, E1, and F1 exhibited confluent lysis,
comparatively identical color patterns were expressed.
Collectively, the significant features were presented by
light yellow Opaque borders accompanied by an innermost
deep, crimson red center. The edges of these centers
were sharply defined and were surrounded by an area of
lighter colored red. This indicates the presence of V1
antigen. V

As the study progressed and Vi antigen gradually was
destroyed, the well defined central areas became success-
ively less defined until they displayed a uniform light
red color throughout to the borders. At the same time, the
borders became less opaque assuming translucency. The light
yellow of the opaque borders finally became bluish when
completely translucent. The bluish, translucent patterns
indicate strains which are devoid of Vi antigen,the W form.

0f the individual strains, Type A particularly per-
sisted in retaining the Vi characteristics. At no time did
the borders lose their opacity, although central areas be-

came light red throughout to the borders after 8 weeks of

continuous subcultivation. The gradual recession of the
deep crimson centers were observed as the initial mani-

festation of Vi antigen deterioration.

25

Types El and F1 produced a similar pattern of results
during the course of the study. Both strains completely
lost their Vi characteristics after a period of 12 weeks.
This was represented by a progressively wider border, be-
coming more translucent with age after their centers lost
the significant deep, crimson red. The varying degrees
of these patterns are indicative of the transitional,

V;W forms.

Type D4HP56 became devoid of the V1 antigen after
only 4 weeks of subcultivation. This too, was demonstrated
by wide, bluish, transparent borders with light red centers.
The colonies of this strain assumed roughness comparatively
early until their irregularity could be easily detected
without the aid of a microscOpe. The early loss of Vi
antigen of Type D4HP56 may be due to its variability in
type. It has been suggested (Henderson and Ferguson,

1949) that Type D4HP55 cultures, upon storage, tend to
change their reactions with Type II ‘Phage to Type D1.

The possibility that Type D4HP56 carries a latent bacterio-
phage which produces comparable reactions when tested on
Vi type stock strains and W form cultures of S. Eypgl, may
account for the comparatively early demonstration of the

W form.

The non-significance of rough and smooth colonies in
relation to Vi antigen is clearly presented. Both types of
colonies showed Vi, W and VQW forms without distinguishing.

Type H 901 was received at the close of the study.

26

This is a fixed, classic W form which has been used as a
standard strain in research laboratories for many years.
The color patterns of this strain were therefore compared
with those W forms which were induced by subcultivation.
Essentially the same patterns were presented. The only
shades of difference detected were found in slightly

wider borders with more translucency among colonies of
Type H 901.

I It is noted that a number of plates produced a radial
pattern in the central portions. There was no apparent
reason for which this could be attributed. Eoth'Vi and

W forms demonstrated this pattern. It was suspected that
these patterns were characteristic of the transitional,
V—W forms, since they were first noticed in cultures which
were partially lysed when typed with homologous bacterio-
phage strains. However, variations of the basic pattern
occurred in pure Vi and W forms. Further, when plates
were re-examined after storage in the refrigerator for
periods of 2, 5, and 4 months, it was noted that the radial
patterns became more pronounced. They also commenced to
make their appearance in colonies which did not demonstrate
them originally.

It is possible that a reversion to the colorless
characteristic of tetrazolium was in process. The reaction
producing color is due to an oxidation-reduction system
involving enzymatic activity. Perhaps these organisms

which were gradually losing the activity of their enzyme

27

systems, or, as in the other cases, which might be weak
in the activity of their enzyme systems resulted in the
salt resuming its. oxidized form. If a growth estimate
could be calculated to determine the decrease in viable
organisms, or the degree of activity present, some

correlation might be presented to explain these effects.

It is also possible that the deep, crimson red centers
present in colonies of the Vi form were the result of
superior activity of their enzyme systems. The V1 forms
have been suggested to be more active in growth and repro-
duction, a factor contributing to their virulence. A
colony of organisms multiplying more rapidly would elicit
more activity of their enzyme systems.

The object in inoculating the plates with a suspension
was mainly to obtain a limited number of well isolated
colonies. Also, if plates were streaked rather than
poured with suspensions, a slightly but evident growing

out from the sides of the colonies occurred simulating
the pattern made by the streak lines. This would tend to

obscure the true pattern of the colonies.

The plates required 48 hours incubation for complete
and accurate color development. 'Upon observation of
colonies at earlier periods of incubation, it was noticed
that all forms presented colonies which were indistinguish-

able from one another.

28

SUMMARY

A study is presented to demonstrate the use of 2,5,5-
triphenyl tetrazolium chloride as an indicator, which
when incorporated in a medium, will manifest a visible
distinction between the colonies of typhoid Vi and W
antigens.

The results show that those strains possessing
completeness of the Vi antigen, conform to a significant
pattern. This pattern may be described as demonstrating
a sharply defined, deep, crimson red center, surrounded
by an area of lighter red. A border encircling the colony
is narrow, and in all cases opaque, with slight color

variations ranging from light yellow to light rose.

The W forms, completely devoid of the Vi antigen,
present patterns which are readily distinguished from
those of the Vi forms. The sharply defined, deep, crimson
red center is completely absent. Rather, the central
portion is a uniform light red. A border encircling the
colony is about two to three times wider than that of the
Vi forms, and in all cases translucent, with slight color
variations ranging from light blue to light green.

The V-W forms are demonstrated by a combination of
patterns associated with those of the individual forms in
their pure state. That is, a section of the colony may be
typically representative of.a W form, while the remainder

of the colony is clearly characteristic of a Vi form.

There were a considerable number of colonies which
presented a radial pattern. An account of the possible
circumstances conducive to such a pattern is presented
in the discussion of this paper.

Types A, D4HP56, El’ and F were used in this study.

1
They were all received in the Vi form. By continuous
subcultivation, it was possible to induce degradation to
the W form. Type A exclusively persisted in its Vi form.
Bacteriophage typing procedure was applied to deter-

mine the presence or absence of Vi antigen. A method for

the preparation of tetrazolium salt in agar is described.

29

l.

5.

7.

10.

11.

12.

15.

14.

30

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