close

Вход

Забыли?

вход по аккаунту

?

835

код для вставкиСкачать
T his d isse r ta tio n has been
m ic r o film e d e x a ctly as receiv ed
62-27
COLEMAN, M arion T raw ick, 1 926SEROLOGICAL STUDIES OF TETRAHYMENA
PYRIFORMIS WITH THE AID OF FLUORESCENT
ANTIBODIES.
E m ory U n iv e rsity , P h .D ., 1960
B io lo g y - G en etics
University Microfilms, Inc., Ann Arbor, Michigan
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
SEROLOGICAL STUDIES OP TETRAHYMENA PYHIFORMIS
WITH THE AID OP FLUORESCENT ANTIBODIES
By
Marion Trawick Coleman
B. S., University of Georgia, 1948
M. A., Emory University, 1958
A Dissertation submitted to the Faculty of the Graduate
School of Emory University in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
1960
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
SEROLOGICAL STUDIES OF TETRAHYMENA PYRIFORMIS
WITH THE AID OF FLUORESCENT ANTIBODIES
Approved for the Department
Adviser
/
^
Date
^ ^ ^ / 1 ''
7
Accepted:
Dean of the Graduate School
Date:
\^j)Ljib
______ _
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
In presenting this dissertation as a partial fulfillment
of the requirements for an advanced degree from Emory Uni­
versity, I agree that the Library of the University shall
make it available for inspection and circulation in ac­
cordance with its regulations governing materials of this
type.
I agree that permission to copy from, or to publish,
this dissertation may be granted by the professor under
whose direction it was written, or, in his absence, by the
Dean of the Graduate School when such copying or publica­
tion is solely for scholarly purposes and does not involve
potential financial gain.
It is understood that any copy­
ing from, or publication of, this dissertation which
involves potential financial gain will not be allowed
without written permission.
(<x
/A-
V
/
■/si*?.
<t1
<•
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
NOTICE TO BORROWERS
Unpublished theses deposited in the Emory University
Library must be used only In accordance with the stipula­
tions prescribed by the author in the preceding statement.
The author of this dissertation Is:
Marion Trawick Coleman (Mrs. George W. Coleman)
794 Scott Circle
Decatur, Georgia
The director of this dissertation is:
Dr. Charles Ray, Jr.
Department of Biology
Emory University
Atlanta 22, Georgia
Users of this dissertation not regularly enrolled as
students at Emory University are required to attest ac­
ceptance of the preceding stipulations by signing below.
Libraries borrowing this dissertation for the use of their
patrons are required to see that each user records here
the information requested.
Name of user
Address
Date
Type of use
(Examination only
or copying)
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
DEDICATION
This dissertation is affectionately dedicated, in
partial fulfillment of lasting obligations, to the family
and friends who have so generously'bestowed upon me the
precious gift of time.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
ACKNOWLEDGMENTS
The writer wishes to express sincere appreciation to
the following individuals:
Dr. Charles Ray, Jr., for
direction of the research and for assistance in the prepa­
ration of the plates; Dr. Morris Goldman, Communicable
Disease Center, United States Public Health Service, for
counsel on technical problems and for his thoughtful
criticism of the dissertation; Margaret May Wells for
encouragement and timely assistance in various phases of
the work.
The research was accomplished during tenure as a
Cooperative Graduate Fellow of the National Science Founda­
tion.
The Investigation was supported in part by a
research grant from the National Science Foundation to
Dr. Charles Ray, Jr., a research grant (PHS-E795) and a
training grant (2E 37) from the National Institutes of
Health to Dr. Chauncey G. Goodchild.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
TABLE OF CONTENTS
Page
I.
I N T R O D U C T I O N .................................
1
II.
MATERIALS AND METHODS
. . . . . . ..........
.............
A.
Experimental Organisms
B. Preparation of Antigens for Injection
C. Preparation of A n t i s e r u m ..........
D.
Serum F r a c t i o n a t i o n .................
E.
Labeling Globulin Fractions ........
F. Sorption and Extraction Procedures
.
G. Staining with Fluorescent Antibody
.
H. Controls for Staining Specificity . .
I.
Experimental Design .................
J.
Scoring and S t a t i s t i c s ............
K.
Fluorescence Equipment
............
L.
Photomicrography
...................
11
12
12
13
14
15
17
19
22
23
25
26
27
III.
OBSERVATIONS AND R E S U L T S ....................
A.
Fixing and Staining Methods ........
B. Specificity of Staining Reactions . .
C. Effects of Duration of Conjugation
.
D.
Comparison of S t r a i n s ..............
E.
P h o t o m i c r o g r a p h y ...................
28
28
31
36
40
43
D I S C U S S I O N ..........
ATechnical Factors ...................
B*
Biological Implications ............
46
46
58
CONCLUSIONS
.............................
66
S U M M A R Y ....................................
68
LITERATURE C I T E D ............................
72
P L A T E S ......................................
80
IV.
V.
VI.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
L IS T
OF TABLES
Page
TABLE
TABLE
TABLE
I
II
III
TABLE
IV
TABLE
V
TABLE
VI
TABLE
VII
TABLE VIII
TYPICAL STAINING REACTIONS WITH
SORBED FLUORESCENT GLOBULINS ........
32
TESTS FOR INHIBITION OF FLUORESCEIN
STAINING BY UNLABELED GLOBULINS
...
35
REACTIONS OF S-I X S-II CONJUGANTS •
........... 37
TO STAINING MIXTURE A
REACTIONS OF S-I X S-II CONJUGANTS
TO STAINING MIXTURE B ........... 39
MATING SYSTEM OF MATING TYPES I VII, VARIETY 1 (NEW TESTER STRAINS)
.
41
FLUORESCENCE OF NEW TESTER CONJU­
GANTS IN RESPONSE TO STAINING
MIXTURE A
........................ 42
MATING REACTIONS OF S-I, S-II,
NT-I, NT-II, AND NT-III STRAINS
...
FLUORESCENCE OF S-I, S-II, NT-I,
NT-II, AND NT-III CONJUGANTS IN
RESPONSE TO STAINING MIXTURE A . . . .
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
44
45
INTRODUCTION
Morphological mutants and other recognizable heredi­
tary markers In Tetrahymena pyriformls are limited in
number, making it difficult to study the genetics of the
species.
The detection of heritable strain-specific dif­
ferences which are not limited to special cases presents a
problem.
This Investigation was undertaken in an attempt
to provide a marker of general applicability for use In
studies of the various problems associated with conjuga­
tion, nuclear differentiation, and the life cycle of
Tetrahymena pyriformls.
The precise nature of serological methods and the
exquisite specificity of antigen-antibody reactions enhance
their usefulness as genetic markers.
The use of serologi­
cal techniques in microbial genetics is expanding (Cushing
and Campbell, 1957).
The wealth of naturally occurring
variation in antigenic systems and the apparently direct
relation of genotype to antigenic constitution are features
which attract geneticists to study antigenic variations.
Most serological studies of Tetrahymena and other
ciliates, which will be described in subsequent paragraphs,
have been based on the ability of specific antiserums to
1
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
*•
immobilize living ciliates (Rossle, 1905).
The Coons
fluorescent antibody technique (cf. reviews by Coons, 1956
and 1958), which will be discussed in detail, seemed to
offer promise of combining immunological specificity with
high sensitivity for detecting antigenic differences in
strains of Tetrahymena.
The use of labeled antiserums
prepared against strains of Tetrahymena was expected to
provide a visual marker not limited to those strains
characterized by distinctive cytological conditions such
as absence of a micronucleus, haploidy, and tetraploidy.
A consequence of the dearth of suitable genetic
markers in Tetrahymena is reflected in the disparity be­
tween the bulk of nutritional and metabolic literature on
the genus and the few publications concerned with pure
genetics.
The voluminous literature on the genus Tetra­
hymena is reviewed by Corliss (1954, 1957), and Elliott
(1959a) has reported on recent investigations with the type
species, T. pyriformls.
The discovery of conjugation (Elliott and Nanney,
1952) and of mating types (Elliott and Gruchy, 1952) stimu­
lated genetic and cytogenetic research on Tetrahymena.
Knowledge of the breeding system, which is fundamental to
any genetic analysis, was provided by Gruchy (1955) from
studies of clones collected from various parts of the
United States and Canada.
Over 40 mating types are known
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
in the 9 non-interbreeding varieties of this species.
Two major contributions to a knowledge of inheritance
in Tetrahymena are the work of Nanney and his associates
(Nanney et al., 1953, 1955; Nanney, 1955, 1959a) on mating
types and that of Ray (1956) on meiosis and nuclear be­
havior during conjugation.
Knowledge of mating type deter­
mination is especially valuable because, without this
foundation, it is difficult to interpret analyses of other
traits.
The first Mendelian factors demonstrated in cili­
ates were genes controlling mating type potentialities in
Paramecium aurella (Sonneborn, 1939).
Analysis of the mating type system in variety 1 of
T. pyriformls revealed a pattern of mating type determina­
tion and inheritance similar to that of the group A system
in Paramecium aurella (cf. Beale, 1954).
Nanney, Caughey,
and Tefankjian (1955) found that a series of alleles at a
single chromosome locus, 'mt, of T. pyriformls determines
the array of mating type potentialities and their fre­
quencies of expression in a population.
Variety 1 consists of 7 known mating types, each of
which will conjugate, under appropriate conditions, with
every mating type other than its own.
This clear-cut
picture is complicated, however, by occasional selfing,
mating within a clone.
Selfers may appear in immature
clones following conjugation and later stabilize into pure
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
mating types (Allen and Nanney, 1958).
Selfers are oc­
casionally found in natural collections (Elliott, 1959b)
and sometimes appear in laboratory populations.
Wells
(1958) reported the occurrence of intra-clonal mating
(selfing) in three laboratory strains of mating types I,
II, and III, variety 6, which had previously been non­
self lng strains.
The second major investigation of importance to Tetra­
hymena genetics is the cytogenetic study of Ray (1956).
His careful analysis of chromosomal behavior during meiosis
and nuclear reorganization provided a foundation for in­
telligent interpretation of future genetic studies.
Serotype potentialities in ciliates have been shown to
be under genetic control (Sonneborn, 1948; Sonneborn and
LeSuer, 1943; Finger, 1957).
Nanney (1959b, 1959c) ex­
plained serotype determination in Tetrahymena pyriformls as
the result of differentiations of macronuclear subnuclei
and their subsequent assortment.
The immunology of the
ciliates has been most thoroughly explored in Paramecium by
Sonneborn and his associates (cf. Beale, 1957).
Harrison
and Fowler (1946) used immobilization reactions as a sero­
logical tool to compare the antigenic characters of preconjugant, conjugating, and exconjugant individuals of
Paramecium bursaria.
In many cases, the exconjugants dis­
played antigenic characters strikingly different from the
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
preconjugant cultures from which they were derived.
The
authors found a sharp alteration in antigenic character
which was detectable only after conjugation between two
individuals had proceeded for approximately two-thirds of
the normal conjugation time.
Among the early investigators of antigens in Protozoa,
Robertson (1939a, 1939b) described the reactions of living
Glaucoma (= Tetrahymena) to antiserums from immunized rab­
bits.
Treatment with homologous antibodies immobilized the
ciliates and also caused them to clump together.
Harrison
and Fowler (1945) confirmed observations of immobilization
response to antiserum in another strain of Tetrahymena.
Antigenic relationships among strains of Tetrahymena
have been demonstrated by Kidder, Stuart, McGann, and
Dewey (1945) and Tanzer (1941).
Kidder and associates
examined 6 strains of T. gelell (= T. pyriformls) and two
of T. vorax by cross-adsorption techniques.
Antigenic
differences between the two species were marked but intra­
specific strain differences were mostly quantitative.
Tanzer (loc. clt.) compared complement fixation and immobi­
lization reactions of three ciliates designated as
Colpldlum campylum, C . striatum, and Glaucoma pyriformls,
which are now considered to be three strains of T. pyrlformls (cf. Loefer et al., 1958).
Two of the strains ap­
peared to be more closely related serologically to each
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
6
other than to the third strain.
Loefer e_t al. (lo c . c i t .) reported separation of 31
strains of T. pyriformls into 14 serological "groups" on
the basis of immobilization reactions.
Continued investi­
gations (Margolin, Loefer, and Owen, 1959) demonstrated
differences in serotype potentialities in the same strains
when cultivated at various temperatures.
(1959)
Elliott and Byrd
reported that, in some instances, mating types in a
single variety were distinguished from each other by im­
mobilizing antiserums.
Certain investigators have employed criteria other
than immobilization as serological tools to study protozoan
antigens.
These include antibody absorption (van Wagten-
donk and van Tijn, 1953; van Wagtendonk et^ a l ., 1956),
precipitation and complement fixation (Bernheimer and
Harrison, 1940), and gel diffusion (Finger, 1956; Preer and
Preer, 1959).
Fluorescent antibody methods have been applied to
protozoological research in a number of instances.
Goldman
has used the method for differentiating species of
Entamoeba (Goldman, 1953, 1954, 1959, 1960) and for diag­
nosis of infection with Toxoplasma gondii (Goldman, 1957a,
1957b).
The following parasitic Protozoa have been stained
with fluorescent antibody (cf. Cherry et al., I960):
Entamoeba histolytica. Entamoeba coll. Toxoplasma gondii.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
7
Trichomonas vaginalis, Trypanosoma cruzl, Plasmodium
berghel, and Anaplasma marglnale.
Beale and Kacser (1957) undertook to study the anti­
gens of Paramecium aurella by means of the fluorescent
antibody method.
They demonstrated the specificity of the
reaction of paramecia to fluorescent antibody in pairs of
conjugating animals.
When complementary mating types of
dissimilar serotypes were induced to conjugate, the members
of a pair exhibited a differential uptake of fluoresceinlabeled antiserum after conjugation had proceeded for two
hours or more.
A green fluorescence appeared around the
surface of the homologous member but the heterologous
member showed no surface fluorescence.
Fluorescent labeling of antibodies is a method of
marking antibodies so that reactions with antigen can be
observed microscopically.
It depends on the fact that
antibody molecules can be tagged by chemical linkage with
dye molecules without damage to the original immunological
properties of the antibody (Marrack, 1934).
The
fluorescent antibody technique as developed by Coons and
associates (Coons et al., 1942; Coons and Kaplan, 1950)
involved conjugating antiserum globulins with fluorescein
isocyanate which imparts a yellow-green fluorescence.
Silversteln and his associates (Silverstein, 1957;
Silverstein et al., 1957) reported the use of orange-
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
fluorescLng rhodamine B isocyanate to label antibody.
By
using rhodamine labeled antibody in conjunction with a
different antibody labeled with fluorescein isocyanate,
they were able to stain different organisms in the same
smear with contrasting fluorescent colors.
Chadwick,
McEntegart, and Nairn (1958a, 1958b) used lissamine rhoda­
mine B 200 conjugates as plasma tracers and as specific
immunological stains.
Double tracer experiments were car­
ried out using conjugates of both rhodamine and fluores­
cein.
Smith, Marshall, and Eveland (1959) described a
counterstalning method that gave a contrasting nonspecific
orange fluorescence when rhodamine-conjugated normal serum
was used in conjunction with immunospecific fluoresceinlabeled antibody systems.
The isocyanates of fluorescein and rhodamine are dif­
ficult to prepare and are unstable.
The synthesis of
labeling reagents was greatly simplified by the development
(Riggs e_t a_l., 1958) of isothiocyanate compounds for use in
coupling fluorescent dyes with immune globulins.
Stable,
solid isothiocyanates of fluorescein and rhodamine B are
presently available from commercial sources.
Marshall,
Eveland, and Smith (1958) confirmed the superiority of
fluorescein isothiocyanate (Riggs) for fluorescent antibody
work.
The isothiocyanate compound is said to be superior
in stability, ease of conjugation, and degree of
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
fluorescence.
From the knowledge of Immobilization systems In Tetra­
hymena and the reports In the literature of successful use
of fluorescent antibodies with other Protozoa, it appeared
that suitable modifications in fluorescent antibody tech­
niques could be developed for use with Tetrahymena.
Vital
staining with the fluorochrome, acridine orange, had
proved remarkably successful (Coleman, 1958).
Tetrahymena
was known to be free of interfering autofluorescence in
the wavelengths employed.
Consequently, the fluorescent
antibody program was undertaken for the purpose of testing
the potentialities of the method with application to anti­
genic systems in T. pyriformls.
Since much of the work on genetic analysis has been
done on variety 1, mating types I and II, representative
strains of these types were selected.
It was proposed to
distinguish the two strains by staining with two specific
antiserums labeled with fluorescent dyes of contrasting
color, fluorescein and rhodamine.
The importance of sexual
methods of reproduction for genetic recombination implies
the critical nature of events during the process of conju­
gation.
With these considerations in mind, an investiga­
tion was planned to test by immunofluorescence the anti­
genic characters of conjugating populations at different
intervals of time following initiation of conjugation
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
within the culture.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
MATERIALS AND METHODS
This section will describe the entire program of re­
search, giving details of the various techniques in sequen­
tial order.
In cases where several alternative methods
were tested, the procedure which was finally selected as
standard operating procedure will be described first.
Subsequently, the alternative trials will be set forth.
Methods of handling the experimental organism, Tetrahymena
pyriformls, are given first.
Preparation of sufficient
numbers of the Protozoa for use as antigens for injection
is described.
The immunological program was launched by
the preparation of antiserums in rabbits; these antiserums
were fractionated and the globulin fractions were conju­
gated with fluorescent dyes.
The dye-globulin conjugates
were subjected to various sorption and extraction proce­
dures to reduce nonspecific fluorescence.
Descriptions of
controls for staining specificity are included.
The
methods of performing actual tests for antigen-antibody
reactions and the scoring and statistical analysis of these
tests are described.
Information on fluorescence equipment
and data on photomicrography conclude this section.
11
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
12
Experimental Organisms
Strains of Tetrahymena pyriformls, variety 1, mating
types I and II, obtained from Dr. A. M. Elliott were main­
tained in axenic culture in a peptone-tryptone medium
(Elliott and Hayes, 1953).
These strains will be referred
to as S-I (serotype I) and S-II throughout the paper.
Stock cultures were kept in log phase of growth by trans­
ferring 1 ml every 3 to 5 days into 5 ml of sterile medium.
Cultures were grown at room temperature (23-25° C).
Con­
jugation was induced by placing together complementary
mating types in wells of 10-hole depression slides.
to mixing for conjugation,
Prior
the ciliates were washed 4 to 5
times in glass distilled water.
Additional strains, representing Inbred lines of each
of the 7 mating types In variety 1, were furnished by Dr.
Charles Ray for comparison of reactions to antiserums pre­
pared against S-I and S-II organisms.
These strains are
referred to as new testers (NT-I, NT-II,
....
NT-VII).
Preparation of Antigens for Injection
Mass cultures of T.~ pyrif ormls were grown in 2,000 ml
erlenmeyer flasks containing approximately 330 ml of
sterile medium inoculated with one tube (5 ml) of 2- to 3day-old stock cultures.
At the end of three days the
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
15
i organisms were harvested by centrifugation In 50 ml cen! trifuge tubes at 1200 rpm for 10 minutes.
The sedimented
organisms were washed with distilled water and recentrl! fuged three times to remove all traces of medium.
Packed
cells were resuspended in 0.15 M saline to give a ratio of
: packed cells to final volume of 1:10.
The suspension was
alternately frozen and thawed three times.
The resulting
brei was dispersed in 5 ml aliquots and stored in the deep
freeze.
Preparation of Antiserum
Doses of 1 ml of thawed saline brei of whole ciliates
were administered intravenously to young adult rabbits
weighing 6 to 10 pounds.
Injections were given three
times a week for a total of nine injections.
Pour rabbits
were used for each of two strains of Tetrahymena, S-I and
S-II.
Test bleedings of rabbits were made once during the
course of inoculations and final bleedings were taken 10
days following the final injection.
Blood was drawn from
the heart and allowed to clot at room temperature in 50 ml
centrifuge tubes.
The tubes were rimmed with an applicator)
stick to release the clot.
After overnight refrigeration,
the clot was discarded and the serum was cleared by centri-■
fugation in an International refrigerated centrifuge.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
I
14
Samples of serum were Incubated at 56° C for 30 minutes to
|inactivate complement.
Serum sterilized by Seitz filtra-
!tion was stored in vials In the deep freeze without
j
preservatives.
Serum Fractionation
The globulin fraction from whole serum was obtained by
precipitation with saturated ammonium sulfate (Kabat and
Mayer, 1948).
To a measured amount of whole serum in an
Ice bath, an equal volume of saturated ammonium sulfate
(542 g/liter) was added slowly from a pipette while the
serum was rotated constantly.
stand overnight at 5°C.
The mixture was allowed to
Following centrifugation at 5° C,
the supernatant was discarded.
The precipitated globulin
was dissolved in a measured quantity of distilled water.
The volume of distilled water was a little less than the
original volume of whole serum.
In an ice bath the globu­
lin was reprecipitated by adding a volume of saturated
ammonium sulfate equal to the volume of distilled water re­
quired to dissolve the first precipitate.
Three precipita­
tions were carried out to remove any hemoglobin originally
present in the serum.
Globulin solutions were dlalyzed In
cellophane tubing against 0.85)6 saline to remove all traces
of ammonium sulfate.
Dialysis was continued at 5° C until
the dialysate was free from sulfate as determined by
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
I
j
15
testing with barium chloride solution.
Labeling Globulin Fractions
The total protein content of globulin solutions to be
labeled was determined by the biuret method (Hiller, 1953).
The total protein concentration was adjusted to 1% by dilu­
tion with 0.9% saline or by evaporation through a dialysis
membrane.
An amount of carbonate-bicarbonate buffer
(0.5 M, pH 9.0) equal to 1/10 the volume of globulin solu­
tion was added.
Two different fluorescent dyes, fluorescein isothiocyanate (Baltimore Biological Laboratory, lot #12331) and
rhodamine B isothiocyanate (Nutritional Biochemicals Cor­
poration, #8532), were used to label samples of globulin.
The dye powder (0.05 mg per mg of protein) was added to
globulin chilled in an ice bath.
overnight in the cold.
The solution was shaken
The dye-globulin conjugate was
dialyzed against 0.85# saline for a day or two and then
against buffered saline (0.01 M phosphate, pH 7.5) until an
overnight dialysate of 100 ml showed no fluorescence.
Con­
jugates were stored in 5 ml portions at -20° C in the deep
freeze.
A working solution was kept at 5° C in the refrig­
erator.
Another conjugation procedure (Riggs £t al., 1958;
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Marshall e_t al., 1958) was also employed for rhodamine B
isothiocyanate as follows;
10 ml of 0.85$ saline, 3 ml
carbonate-bicarbonate buffer (0.5 M, pH 9.0), and 2 ml
reagent grade acetone were combined in an erlenraeyer flask.
The mixture was cooled in an acetone-dry ice bath until
crystals of ice formed.
To the cooled mixture was added,
with stirring, 10 ml of globulin solution (1$ protein con­
centration) .
This mixture was again cooled until ice
crystals formed.
To this cooled mixture was added, with
stirring, 1.5 ml acetone containing the required amount of
rhodamine B isothiocyanate.
After the addition of the
solution of dye in acetone,
the mixture was stirred or
shaken at 5° C overnight.
The dye-protein conjugate was
then dialyzed against phosphate buffered saline.
Globulins of normal (pre-immune) rabbit serum and of
antiserums to S-I and S-II were labeled with fluorescein
isothiocyanate.
Other aliquots of these globulins were
labeled with rhodamine B isothiocyanate.
These 6 conju­
gates are designated as anti-l-Fl, anti-I-Rh, anti-H-Fl,
anti-II-Rh, NRS-Fl, NRS-Rh.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
17
j
Sorption and Extraction Procedures
I
;Standard Procedure
j - - - - - - - - - - - - - - - - - |
To remove antibodies common to both strains, leaving
Iantibodies specific for the homologous strain, antiglobu­
lins (labeled or unlabeled) were sorbed with disrupted
cells of the heterologous strain as follows:
Mass cultures
of T. pyrlformls of the appropriate strain were harvested
and washed twice in distilled water and a third time in
buffered distilled water (pH 7.2 - 7.5).
Packed cells were
alternately frozen and thawed to disrupt them.
Sorptions
were accomplished by mixing globulin or globulin-dye conju­
gate with an equal volume of the thawed ciliate brei.
After the mixture stood for one hour at 5° C, the globulin
was recovered by reclaiming the supernatant following high­
speed centrifugation.
Two one-hour sorptions were carried
out.
Additional methods reported in the literature for
decreasing nonspecific fluorescence were tested.
(1)
Labeled globulins were sorbed with monkey liver
powder (Coons and Kaplan, 1950) at concentrations of 100 mg
of powder per ml of globulin solution.
The sample of
monkey liver powder was furnished by Dr. Morris Goldman.
The dry liver powder was washed with buffered saline before
sorption to mitigate change in pH.
were carried out at 5° C.
Two one-hour sorptions
The globulin was recovered each
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
18
time by removing the supernatant following high-speed
centrifugation.
(2) Globulins were incubated overnight at 5° C in the
presence of living Tetrahymena of heterologous strains.
The ciliates were removed by repeated centrifugation.
(3) Following conjugation of globulins with fluores­
cent dyes, the labeled protein solution was reprecipitated
with saturated ammonium sulfate until all detectable
fluorescence was associated with the precipitate and none
with the supernatant.
The final precipitate was redis­
solved with distilled water and the solution was dialyzed
against normal saline to remove all traces of ammonium
sulfate.
(4) Powdered activated charcoal, Norite (0.05 g per ml
of conjugate), was added to labeled globulins to remove dye
not chemically attached to protein.
stirred for one hour at 5? C.
The mixture was
The charcoal was removed by
centrifuging (Chadwick, McEntegart, and Nairn, 1958b).
(5) Following the method of Dineen and Ada (1957) for
removing free fluorescein derivatives, dye-globulin conju­
gates were extracted with one volume of ethyl acetate.
The
emulsion formed was broken by low-speed centrifugation for
5 minutes.
The supernatant acetate was discarded.
The
dissolved acetate in the remaining aqueous phase was re­
moved by use of a water vacuum pump.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
19
(6) Globulins were subjected to various combinations
ii of the treatments described above.
j
i
i
Staining with Fluorescent Antibody
; Standard Procedure
Ciliates to be stained were centrifuged by hand in 15
ml tubes.
pipette.
All excess wash water was removed with a microThe packed cells were killed and fixed for 5
minutes or longer in absolute methyl alcohol.
Methanol
was removed by centrifuging and washing with buffered dis­
tilled water (pH 7.2).
The ciliates were again concen­
trated and resuspended in the centrifuge tube with a
mixture of labeled globulins.
Staining mixture A, which was thought to contain two
specific Immunologic reagents, consisted of equal parts of
1:20 dilutions of fluorescein-labeled S-I antiglobulin and
rhodamine-labeled S-II antiglobulin.
When it became appar­
ent that none of the rhodamine conjugates possessed im­
munologic specificity, staining mixture B was prepared with
equal parts of diluted S-II antiglobulin labeled with
!
fluorescein and normal (pre-immune) globulin labeled with
1
■
rhodamine.
Staining mixture C combined normal globulin
labeled with fluorescein and normal globulin labeled with
rhodamine.
Staining mixture D was a triple stain of S-I
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
j
20
|and S-II antiglobulins labeled with fluorescein and normal
|globulin with rhodamine.
The staining reaction usually re-
jquired from 15 to 30 minutes at room temperature.
The re-
|
action was stopped by washing with buffered distilled water
to remove unreacted globulin conjugates.
A sample drop of
stained ciliates was placed between an ordinary glass slide
and cover slip for microscopic examination.
A number of other methods of staining were also
tested.
(1)
Living ciliates washed free of media were incu­
bated with fluorescent antiserums.
(2)
A modification of the Chatton-Lwoff technique of
gelatin embedding (cf. Corliss, 1953) was employed.
of animals was placed on a warmed slide.
A drop
A smaller drop of
10# gelatin (35° C) was added with a warm pipette, mixed,
and spread over the surface of the slide.
The slide was
cooled to 5° C for two minutes, then rinsed in cold buf­
fered distilled water.
The slide was covered with labeled
antiserum and incubated for 30 minutes.
washed off in cold distilled water.
Antiserum was
A cover slip was
mounted in buffered glycerin.
(3)
Drops of living Tetrahymena in distilled water or
suspensions of fixed ciliates in absolute methanol were
spread over glass slides and allowed to dry at room tem­
perature.
The "smear” was flooded with fluorescein-labeled
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
21
| globulin and incubated in a moist chamber for 30 minutes
I
at room temperature.
The unbound globulin conjugate was
washed off with buffered distilled water.
A cower slip
was mounted over the "smear” with buffered glycerol.
I
(4)
The following fixatives, in addition to absolute
methanol, were tested:
Nissenbaum’s fluid (Nissenbaum,
1953), tertiary butyl alcohol, osmium tetroxide fumes,
dilute aqueous solution of osmium tetroxide, and acetone.
Nissenbaum's fluid or tertiary butyl alcohol served to at­
tach the ciliates to cover slips for staining.
Ciliates
were suspended over osmium tetroxide fumes in hanging
drops.
Acetone and aqueous osmium tetroxide were applied
to packed cells in centrifuge tubes.
(5)
Methanol-fixed Tetrahymena were prestained with
aqueous acridine orange (1:100,000) (Coleman, 1958) as a
counterstain and then treated with fluorescein-labeled
antibody.
(6)
An indirect staining technique (cf. Cherry at
al., 1960) involved treating fixed ciliates with unlabeled
specific anti.serum followed by fluorescein-labeled anti­
globulin prepared in sheep against rabbit globulin.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
22
!
i '
Controls for Staining Specificity
The following procedures were carried out to establish
Immunologic specificity of the staining reaction:
I
j
(1)
A useful control to demonstrate staining speci-
|ficity is failure of conjugates prepared from heterologous
antiserums or normal serum to stain the specimen.
ciliates were fixed and stained with:
Rh, NRS-F1, or NRS-Rh.
S-I
anti-II-Fl, anti-II-
S-II ciliates were stained with*
anti-I-Fl, anti-I-Rh, NRS-F1, or NRS-Rh.
S-I and S-II
specimens were each stained with staining mixture A (antlI-Fl and anti-H-Rh) and staining mixture B (anti-II-Fl and
NRS-Rh).
(2)
S-I x S-II conjugants were stained with each of
the 6 dye-globulin conjugates and the two staining mix­
tures, A and B.
(3)
Inhibition (Coons and Kaplan, 1950; cf. Cherry
et al., 1960) refers to the ability of specific unlabeled
antibody to inhibit fluorescent staining with specific
labeled antibody.
Normal serum or globulin is not capable
of Inhibiting specific fluorescence.
Methanol-fixed S-I
specimens were incubated overnight at 5° C with unlabeled
anti-I globulin, while other specimens were incubated with
i
;
unlabeled normal globulin.
.
The unlabeled globulins were
washed off and the pretreated ciliates were stained with
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
’
i
|
23
anti-I-Fl.
S-II specimens were treated with unlabeled and
labeled antl-TI globulins or with unlabeled normal globulin
and anti-II-Fl•
Rhodamine conjugates were also tested
after specimens had been exposed to unlabeled globulins.
i
(4)
S-I x S-II cbnjugants were incubated with appro-
i
!prlate unlabeled globulins before staining with staining
mixtures A, B, or D.
(5) Labeled globulins were diluted with unlabeled
normal serum and unlabeled specific antiglobulins, respec­
tively.
The unlabeled antiserum in mixture with homologous
fluorescent antibody should inhibit the specific staining,
whereas normal or heterologous serum should not (Goldman,
1957a).
(6)
Labeled globulins were diluted with supernates
from Tetrahymena brei (soluble antigen), which should re­
duce the staining power of the conjugate by removal of
specific antibody (Goldman, 1953).
Experimental Design
Protozoa from 2-day-old S-I and S-II cultures were injduced to conjugate as described above.
j
Conjugating pairs
j
were fixed with absolute methanol at intervals of 1, 2, 3,
j
:
I
5, 6, 8, 10, 12, 16, and 18 hours following onset of conju­
gation.
The fixed conjugants were stained with the mixture
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
24
of labeled antibodies (A) according to the standard proce­
dure outlined above.
Samples of each stage were examined
microscopically and counts were made of 1,000 pairs in
each stage.
S-I x S-II conjugants at 1, 4, 10, 12, and 18 hour
stages were stained with staining mixture B.
1,000 were made of each of these stages.
Counts of
S-I and S-II
controls were maintained separately in distilled water for
the same length of time as the conjugating animals.
Some
vegetative animals were stained at the time animals were
mixed for conjugation; other vegetative controls were
stained after 16 hours In distilled water (corresponding
to the 12-hour stage of conjugation) and after 22 hours
(equal to 13-hour conjugants).
S-I x S-II conjugants were stained with mixtures C and
D by the same method.
Typical reactions were recorded, but
no counts were m ade.
Nuclear behavior in living S-I x S-II conjugants was
observed by fluorochroming with aqueous acridine orange
(1:100,000) (Coleman, 1958).
Stages from 9 to 12 hours and
later were studied.
The 7 new tester strains representing the 7 mating
types In variety 1 were first mixed in all possible crosses
to test for conjugation.
Conjugants from each of these
crosses, as well as vegetative clones of each mating type,
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
25
were stained with mixture A.
Typical reactions were re­
corded, but no counts were made.
Comparisons were made of serotype expression and mat­
ing reactions by conjugating S-I, S-II, and new tester
strains in all possible combinations and staining the con­
jugants with mixture A.
Scoring and Statistics
Each conjugating pair was scored according to the
color of fluorescence emitted by the co-conJugants.
Pairs
consisting of one green fluorescing member and one orange
fluorescing member were scored as F-R (fluoresceln-rhodamine); both orange co-conjugants as R-R; and both green
co-conjugants as F-F.
For statistical considerations, R-R and F-F counts
were combined to obtain the total number of atypical stain­
ing reactions in a sample of 1,000 pairs.
Comparison of
the number of atypical staining reactions at various inter­
vals of time following the beginning of conjugation was
accomplished by the use of the Brandt-Snedecor formula for
chi square (Batson, 1956).
By use of this formula it is
possible to partition the total chi squares into individual
chi squares of mathematically independent comparisons.
|
I
i
i
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
26
i
Fluorescence Equipment
i
Stained preparations were examined with a conventional
! Zeiss microscope with bright-field condenser.
The Osram
I
| HBO 200 maximum pressure mercury vapor arc lamp served as
I the light source.
The HBO 200 is rich in ultraviolet and
blue-violet radiations.
To narrow the spectral range,
primary filters were used which selectively transmitted
only the shorter exciting radiations and absorbed the
longer wavelengths.
A glass heat-absorption filter (Schott
KG 1) was placed between the light source and the primary
filter to prevent cracking the latter.
Secondary filters
were located in the eyepieces as barriers to absorb the
exciting wavelengths and to transmit the fluorescent light
of longer wavelengths emitted by the stained specimens.
The following combinations of exciter-barrier filters
were effective:
(1) Two Schott BG 12 (3 mm each) UV pass
filters with Tjy- and blue-excluding ocular filters.
(2)
On© Schott BG 12 (3 mm) and 1 Schott TJG 2 (2.5 mm) with UVexcluding ocular filters.
Additional ocular filters were
required to obtain effective contrast in black-and-white
photography.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
27
!
Photomicrography
I
An inclined binocular photo tube (Zeiss) facilitated
j attachment of a Leitx Micro-Ibso with 1/3 X cone to the
| microscope.
Color transparencies were obtained from
super-Anscochrome and high-speed Ektachrome film with ex­
posures of 1 and 2 minutes.
A TTV-excluding filter was
located in the ocular of the Micro-Ibso.
Black-and-white
photomicrographs were taken on Kodak Tri-X Pan film.
In
addition to the UV-excluding filter, a Corning #9780
(2 mm) filter and Kodak wratten gelatin filter #B-58 were
placed in the ocular.
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
O B SE R V A T IO N S AND R E SU L T S
In this section will be reported the results of the
investigation of techniques for testing antigenic reac­
tions of Tetrahymena pyriformis to fluorescent antibody,
for controlling nonspecific fluorescence, and for estab­
lishing suitable controls for the fluorescent antibody
tests.
The appearance of fluorescent specimens in response
to various treatments will be described.
Data will be pro­
vided on the reactions of conjugating pairs of Tetrahymena
to labeled antibodies as related to the length of time fol­
lowing Initiation of conjugation within the culture.
The
S-I and S-II strains are compared with the 7 new tester
strains for their mating reactions and expression of sero­
type .
Fixing and Staining Methods
Living ciliates in distilled water exuded a translu­
cent gelatinous material.
No living ciliates showed surface
fluorescence on exposure to any labeled globulins.
Instead,
fluorescence was limited to vacuoles within the cytoplasm.
In early conjugating cultures single animals displayed
fluorescence within vacuoles but conjugating pairs did not.
28
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
29
The technique of gelatin embedding gave inconsistent
results.
Some ciliates exhibited marginal and ciliary
fluorescence to a satisfactory degree but, in the same
preparation, most were faint or non-fluorescent.
Dried smears of Tetrahymena treated with fluorescein
conjugates displayed green fluorescence; those exposed to
rhodamine conjugates had an orange fluorescence.
Without
fixation, a considerable amount of distortion resulted
from drying; attempts to study cytological detail were
fruitless.
No specificity of staining was demonstrated;
both S-I and S-II ciliates stained with either homologous
or heterologous globulin conjugates.
There was no differ­
ential uptake of mixed fluorescein- and rhodamine-labeled
globulins.
Instead, dried conjugating ciliates stained
different shades of orange and green blends.
Absolute methanol was the only completely satisfactory
fixative tested.
Satisfactory intensity of fluorescence
was never obtained following fixation with osmium tetroxide
fumes or with dilute aqueous solutions of osmium tetroxide.
Ciliates stained after Nissenbaum’s or tertiary butyl
alcohol fixation showed faint marginal and ciliary fluores­
cence.
Acetone fixation was not conducive to satisfactory
fluorescent staining; intensity was low and separate orange
and green fluorescence was not distinct.
Methanol-fixed ciliates suspended in solutions of
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
30
labeled antibody displayed uniform fluorescence of satis­
factory Intensity.
S-I or S-II specimens treated with
homologous, heterologous, or normal rhodamine conjugates
!exhibited overall orange fluorescence which appeared to be
concentrated along the klnetles and the bases of the cilia.
Any fluorescein conjugate produced green fluorescence with
an accumulation of fluorescent material around the pel­
licle, along the cilia, and at the tips of the cilia of
both S-I and S-II strains.
Staining mixtures A and B, consisting of mixtures of
rhodamine- and fluorescein-labeled globulins, produced a
striking appearance in moat S-I x S-II conjugants.
The
typical pairs had one orange member and one green member.
Orange fluorescence appeared to be confined to the body of
the di l a t e s ;
the pellicle and cilia also fluoresced in the
green member.
Careful inspection revealed that the green
fluorescence appeared to be superimposed on a basic orange
background.
No difficulty was experienced, however, in
distinguishing orange and green animals in well-stained
samples.
In a very few cases, both members of a pair ap­
peared orange, or both were green.
triple conjugants were encountered.
A few instances of
The three co-conju-
gants were either two green and one orange, or two orange
and one green.
color were seen.
No cases of three co-conjugants of the same
Animals undergoing fission were stained a
i
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
31
single color, either orange or green, but these were easily
distinguished from single colored conjugating animals by
their characteristic shape and manner of attachment to each
other.
|
Fluorochroming with acridine orange as a counterstain
I
i to fluorescein produced brilliant orange fluorescence,
which obscured the fluorescence of labeled antiserums.
The Indirect staining method using unlabeled specific
antiserum to S-I or S-II followed by, or in conjunction
with, fluorescein-labeled anti-rabbit globulin gave non­
specific green fluorescence in all tests.
Specificity of Staining Reactions
Table I records the typical staining reactions of S-I
and S-II vegetative animals and S-I x S-II conjugants to
each of the 6 dye-globulin conjugates and to staining mix­
tures A, B, C, and D.
Antiglobulins had been sorbed with
breis of heterologous ciliates.
Although the two fluores-
celn-antlglobulln conjugates stained co-conjugants differi
entially, the difference in intensity was not sufficient to!
I
jdistinguish between S-I and S-II vegetative animals in a
j
:
I
mixed population.
'
Fluorescein-normal globulin also stainedj
;S-I, S-II, and S-I x S-II organisms.
Each of the rhodamine'
Iconjugates stained both strains of ciliates and conjugating
|pairs with the same Intensity.
By counterstainlng with a
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
TABLE I
TYPICAL STAINING REACTIONS
WITH SORBED FLUORESCENT GLOBULINS
S-I
S-II
S-I x S-II
anti-I-Fl
F
F
F-f
anti-I-Rh
R
R
R-R
anti-II-Fl
F
F
F-f
anti-II-Rh
R
R
R-R
NRS-FI
F
F
F-F
NRS-Rh
R
R
R-R
Mixture A
F
R
F-R
Mixture B
R
F
F-R
Mixture C
R
R
R-R
Mixture D
F
F
F-F
F = green fluorescence of fluorescein stain
n
n diminished intensity
f =
«
«
n rhodamine stain
R = orange
NRS = normal (pre-immune) rabbit serum
Mixture A = anti-I-Fl + anti-II-Rh
"
B = anti-II-Fl + NRS-Rh
n
C = NRS-FI + NRS-Rh
"
D = anti-I-Fl + anti-II-Fl + NRS-Rh
* Reactions in which specificity is demonstrated are
underlined.
32
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
33
rhodamine conjugate in mixtures A, B, and D, specific
staining with fluorescein antiglobulins was obtained for
both conjugants and vegetative animals (see Plate I, Pig.
1; Plate II, Fig. 2; Plate III, Pigs. 3, 4, 5).
Specifici­
ty of rhodamine staining was never demonstrated.
Staining with globulins from complement-inactivated
serums did not appear to be more specific than the stain­
ing reactions when serums had not been inactivated.
Attempts were made to improve the staining specificity
of labeled globulins by removing dye not chemically bound
to protein.
Extensive dialysis, repeated precipitation
with ammonium sulfate, and ethyl acetate extraction failed
to eliminate nonspecific staining.
Sorption with activated
charcoal was much quicker than the other methods and, when
followed by sorption with appropriate Tetrahymena breis,
appeared to enhance the specificity of globulins in the
counterstaining technique.
Sorption of globulin-dye conjugates or sorption of u n ­
labeled globulins with liver powder before labeling did not
appreciably diminish the nonspecific fluorescence of S-I
and S-II strains.
Sorption with heterologous Tetrahymena
breis or overnight incubation of fluorescein-labeled globu­
lins with living ciliates of heterologous strains led to
differential staining intensity in co-conjugants.
In order to demonstrate the specificity of staining
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
34
reactions, tests were made to see if pretreating ciliates
with unlabeled homologous globulins could prevent staining
by labeled globulins.
The specific fluorescence of fluo­
rescein in the presence of a rhodamine-globulin counter­
stain was inhibited by pretreating the ciliates with
homologous unlabeled globulin (Plate II, Pig. 2).
The
inhibition could not be demonstrated for fluorescein
stains without a rhodamine counterstain.
Rhodamine fluo­
rescence could not be inhibited by any pretreatment.
Table II presents the results of inhibition reactions to
fluorescein conjugates.
Although aqueous rhodamine used as a simple fluorochrome produced the same orange fluorescence as rhodaminelabeled antibody, substitution of aqueous rhodamine for
the protein conjugate in staining mixtures A and B did not
give a consistent staining pattern.
The clear-cut demon­
stration of inhibition by unlabeled antibody was not
achieved when aqueous rhodamine was substituted as a
counterstain.
Dilution of labeled globulins with unlabeled specific
antiglobulina failed to inhibit staining to a greater
degree than did equal dilution with normal globulin.
Dilution of labeled globulins with soluble antigen
from Tetrahymena brei did not reduce the staining power of
the conjugate appreciably.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
TABLE II
TESTS FOR INHIBITION OF FLUORESCEIN STAINING
BY UNLABELED GLOBULINS
I Specimen
Unlabeled
Globulin
anti-I
anti-I
antl-I
NRS
S-I
S-I
S-I
S-I
S-II
S-II
S-II
S-II
anti-II
anti-II
anti-II
. NRS
anti-I
anti-II
anti-I +
anti-II
NRS
S-I x S-II
S-I x S-II
S-I x S-II
S-I x S-II
Mixture A
■
B
■
D
NRS
=
=
=
=
Labeled
Globulin
Inhibitlon
Fluorescein
fluorescence
antl-I-Fl
Mixture A
Mixture D
A or D
0
+
+
0
+
0
0
+
anti-II-Fl
Mixture B
Mixture D
A or D
0
+
+
0
+
0
0
+
Mixture A
Mixture B
+
+•
0
0
Mixture D
A, B, or D
+
0
0
+
anti-I-Fl + anti-II-Rh
anti-II-Fl + NRS-Rh
anti-I-Fl + anti-II-Fl +NRS-Rh
normal (pre-lmmune) rabbit serum
I
i
35
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
36
I .............................
j
Effects of Duration of Conjugation
Whereas the great majority of S-I x S-II conjugants
reacted to the dual staining mixture (A) by showing one
i green and one orange member, when large numbers of pairs
1
|were observed, occasional exceptions were found.
In these
iatypical cases both members of a pair (co-conjugants)
jfluoresced the same color.
Some pairs stained orange but
showed no green fluorescence; other pairs displayed green
pellicles and accumulation of fluorescein along the cilia
of both members.
At intervals from 1 to 18 hours follow­
ing the initiation of conjugation within the culture,
counts were made of 1,000 pairs.
Data from these counts
(Table III) showed an overall increase in atypical staining
pairs which was more pronounced In the later stages.
These
data are represented graphically in Plate IV, Pig. 7.
Analysis of the data presented in Table III by the
Brandt-Snedeoor formula for chi square (Batson, 1956)
Indicated that there is a true increase in the frequency
of pairs with both members fluorescing the same color when
the period from 1 to 18 hours is considered as a whole.
I
Comparison of the period from 1 to 10 hours following con- ,
j
j
Jugation with the 12 to 18-hour period revealed a differj
;
'
ence which is statistically significant at the 0.1 per cent!
i
level. There is no significant variation within either of
;the periods, 1 to 10 or 12 to 18, separately.
There la an
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
TABLE III
REACTIONS OP S-I X S-II CONJUGANTS TO STAINING MIXTURE A
Atypical
Reactions
Hours
After
Conjugatlon
P-R
R-R
F-F
Total
1
2
3
5
6
8
10
12
16
18
996
991
997
984
992
992
986
1,901
946
939
3
8
3
13
4
6
6
97
31
57
1
1
0
3
4
2
8
2
23
_4
1,000
1,000
1,000
1,000
1,000
1,000
1,000
2,000
1,000
1.000
4
9
3
16
8
8
14
99
54
61
10,724
228
48
11,000
276
Totals
Pluorescence Reactions
R-R + F-P
Mixture A = Anti-I-Pl + anti -II-Rh
one green co-conjugant, one orange
P-R
both co-conjugants orange
R-R
both co-conjugants green
P-P
37
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
38
\
' '
!actual Increase In the count for the 12-hour stage as comi
pared with the count for the 10-hour stage, which Is also
j significant at the 0.1 per cent level.
!
The data quoted above refer to reactions of S-I x S-II
i
conjugants to fluorescein-labeled anti-I globulin with a
i
rhodamine counterstain (staining mixture A). The specific
fluorescein label was reversed in staining mixture B
(anti-II-Fl + NRS-Rh).
Samples of S-I x S-II conjugants
at 1, 4, 10, 12, and 18-hour stages were also scored ac­
cording to their staining reactions to mixture B (Table
IV).
Again, a significant increase in frequency of identi­
cal staining co-conjugants was demonstrated and the criti­
cal period was between the 10-hour and 12-hour stages
(Plate V, Fig. 8).
The two types of atypical staining pairs, R-R and F-F,
did not Increase at the same rate during the course of con­
jugation.
More R-R than F-F pairs were observed with both
of the staining mixtures, A and B.
The possible signifi­
cance of this difference will be discussed later.
Samples of the S-I x S-II conjugants were stained in
the living condition with acridine orange.
Acridine orange I
stained nuclei bright green and cytoplasmic inclusions
orange.
The macronuclear anlagen stage in conjugants was
easily recognized by the brilliant fluorescence of the old
disintegrating macronucleus in the posterior end of one or
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
i
TABLE IV
REACTIONS OP S-I X S-II CONJUGANTS TO STAINING MIXTURE B
Hours
After
Conju­
gation
Atypical
Reactions
Fluorescence Reactions
F-R
R-R
F-F
Total
R-R + F-F
1
997
2
1
1,000
3
4
998
1
1
1,000
2
10
992
8
0
1,000
8
12
975
16
9
1,000
25
18
910
87
_3
1,000
90
Totals
4,872
114
14
5,000
128
Mixture B
F-R
R-R
F-F
=
=
=
=
Anti-II-Fl + NRS-Rh
one green co-conjugant, one orange
both co-conjugants orange
both co-conjugants green
39
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
40
both co-conjugants.
None of the living conjugants were
!found in the anlagen stage at 9 hours following conjugai
tion.
Only one pair was found in anlagen stage at 9-1/2
!hours following conjugation, but by 10 hours several
anlagen pairs could be seen.
The pairs possessing anlagen
:were quite numerous by 12 hours and continued to increase
in abundance through the 18-hour period of observation.
S-I and S-II controls were maintained separately in
distilled water for lengths of time equal to the total
period from mixing for conjugating cultures.
No selfing
was observed in either S-I or S-II cultures.
S-I controls
stained green with staining mixture A and orange with B.
There was no change in staining reactions of 1,000 vegeta­
tive animals observed during and after 22 hours of starva­
tion.
S-II controls stained orange with A and green with B
both before and after extended starvation.
Comparison of Strains
The mating system in the 7 new tester strains is il­
lustrated in Table V.
Each strain conjugated with every
other strain but there was no selfing within a strain.
Table VI gives the staining reactions of the new tester
strains to staining mixture A.
as did S-I.
Only NT-III stained green
Conjugants of NT-III with any of the other new
tester strains had the same appearance (green and orange
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE V
MATING SYSTEM OP MATING TYPES I - VII, VARIETY 1
(NEW TESTER STRAINS)
I
II
III
IV
V
VI
I
0
II
+
0
III
+
+
0
IV
+
+
+
0
V
+
+
+
+
0
VI
+
+
+
+
+
0
VII
+
+
+
+
+
+
+ = conjugation observed
0 = no conjugation observed
41
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
VII
0
TABLE VI
FLUORESCENCE OF NEW TESTER CONJUGANTS
IN RESPONSE TO STAINING MIXTURE A
I
I
II
II
III
IV
V
VI
VII
(R)
R
(R)
III
F-R
F-R
(F)
IV
R
R
F-R
(R)
V
R
R
F-R
R
(R)
VI
R
R
F-R
R
R
(R)
VII
R
R
F-R
R
R
R
(R)
R 3 both co-conjugante orange
(R) = no conjugation, vegetative animala orange
(F) 3 no conjugation, vegetative animals green
F-R 31 one green eo-conjugant, one orange
Mixture A = anti-I-Fl + anti-II-Rh
i
42
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
43
!members) as S-I x S-II conjugants.
All other conjugants
of NT strains were orange.
Mating reactions between S-I, S-II, NT-I, NT-II, and
i
iNT-III strains are recorded In Table VII.
S-I and NT-I
i
jare both mating type I; S-II and NT-II are both mating
j
I type II. Staining reactions of the conjugants among S-I,
S-II, NT-I, NT-II, and NT-III strains (following exposure
to mixture A) are found in Table VIII.
Photomicrography
Color transparencies were poor representations of the
true orange and green contrasting fluorescence observed
microscopically.
Color prints from these transparencies
were completely unsatisfactory because the two colors did
not reproduce well.
In order to demonstrate the contrast­
ing fluorescence of green and orange co-conjugants on
black-and-white film, filters which blocked the transmis­
sion of orange wavelengths and passed green fluorescence
were located in the ocular of the Micro-Ibso.
Thus a
contrast in intensity was recorded (Plate III, Pigs. 3, 4,
i5, 6).
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE VII
CO
i
H
MATING- REACTIONS
OP S-I, S-II, NT-I, NT-II, AND NT-III STRAINS
NT-I
S-II
NT-II
S-I
0
S-II
+
0
NT-I
0
+
0
NT-II
+
0
+
0
NT-III
+
+
+
+
NT-III
+ = conjugation observed
0 * conjugation not observed
44
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0
TABLE VIII
FLUORESCENCE OF S-I, S-II, NT-I, NT-II, AND NT-III
CONJUGANTS IN RESPONSE TO STAINING MIXTURE A
S-I
S-I
(F)
S-II
F-R
NT-I
S-II
NT-II
(R)
(F)(R)
R-R
(R)
NT-II
F-R
(R)
R-R
(R)
NT-III
F-F
F-R
F-R
NT-I
NT-III
(F)(R)
(F)
F-R = one green co-conJugant, one orange
R-R = both co-conjugants orange
F-F = both co-conjugants green
(R) = no conjugation, vegetative animals orange
(F) = no conjugation, vegetative animals green
Mixture A * anti-I-Fl + anti-11-Rh
45
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION
Technical Factors
Direct staining of suspensions of fixed Protozoa with
fluorescent antibody was the method employed to detect
antigenic differences in two strains of Tetrahymena pyrlformls, variety 1.
These were strains of mating types I
and II In which selfing had not previously been detected.
The question of whether to stain living or fixed
ciliates was resolved by the finding that living ciliates
did not take up fluorescent conjugates except by ingesting
material which fluoresced within food vacuoles.
Only vege­
tative animals in early conjugating cultures had fluores­
cent material thus localized; the conjugants, which did not
have food vacuoles, were entirely lacking In fluorescence.
The same animals, vegetative or conjugating, stained with
typical orange or green fluorescence following fixation with
absolute methanol.
Goldman (1953) found that amebae were
refractory to labeled antibody in the living condition but
stained readily following fixation.
;
Beale and Kacser
i
(1957) reported that living Paramecium treated with dilute
fluorescein-labeled whole antiserum acquired fluorescence
In a thin layer around the entire surface and in globules
46
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
at the tips of the cilia in addition to fluorescence of
ingested material within food vacuoles.
Failure of living
Tetrahymena to react with fluorescent antiglobulins is be­
lieved to be due to protection by a gelatinous exudate
formed when the ciliates were suspended in distilled water.
The protection afforded is thought to be purely physical
and unrelated to any specific neutralization of antibodies.
Loefer et ad. (1958) interpreted a similar exudation by
Tetrahymena as a generalized defense mechanism which pro­
tected test organisms against agglutination by antiserum.
Individual Tetrahymena were not tested for changes in
antigenic fluorescence before and during conjugation, since
fixation was necessary for fluorescent antibody staining.
Antiserums were prepared in rabbits against the two
strains, S-I and S-II, and samples of whole serum were
heat-treated to inactivate complement.
This was done be ­
cause of the report by Sinclair (1958) on the role of com­
plement in certain immune reactions of Tetrahymena pyrlformls.
The staining reactions of complement-inactivated
serum conjugates did not appear any different, however,
from those which had not been inactivated and the procedure
was abandoned for further work.
Only the globulin fraction rather than whole serum was ,
labeled because less labeling agent is required and because j
it was thought that nonspecific staining might result from
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
j
48
labeling other serum proteins not associated with anti­
body.
The selection of the fluorescent labeling reagents
i
i
was based on reports in the literature of contrasting
;fluorescent dyes used as double tracers for histological
i
i localization of antigenic substances (Silverstein, 1957;
Chadwick ejb al., 1958a).
The advantage of contrasting
fluorescent colors can be obtained with fluorescein and
rhodamine because their fluorescent emissions are far
enough apart to provide good visual contrast.
The light
emitted by fluorescein is yellow-green with a peak at
540 mu.
The emission spectra of rhodamine compounds lie
above 550 mu, producing red fluorescence.
The isothio-
cyanates of the two fluorochromes were used because of
their stability and ease of conjugation with protein.
Some antiglobulins prepared against S-I and S-II
strains of Tetrahymena were labeled with fluorescein and
others with rhodamine with the expectation of achieving
two immunologically specific fluorochromes of contrasting
color.
It appeared from the literature that the proposal
was entirely reasonable.
Silverstein (1957) used a
rhodamine-labeled anti-pneumococcus serum and a fluoresi
cein-labeled anti-S typhosa serum to stain a smear contain-i
ing both organisms.
The two species were easily recognized
not only by morphology but also by the distinctive
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
49
J fluorescent color of each.
In addition, Chadwick ejb al.
I (1958a) reported that serum conjugates with llssamlne
rhodamine and fluorescein could be used together, either
as simple plasma labels or as immunological stains.
The original intention of using two immunologieally
i
| specific labels of contrasting color to stain different
strains of Tetrahymena was thwarted by the discovery that
! rhodamine-labeled normal globulin behaved exactly the same
as antiglobulins against either serotype.
None of the
rhodamine conjugates possessed any immunological staining
specificity.
At first it was thought that improper conju­
gation of dye and protein was responsible.
The process
was repeated and a different labeling procedure was tried:
the dye powder was first dissolved in a small amount of
reagent acetone before it was added to the protein solu­
tion.
The nonspecific staining of rhodamine conjugates
was not affected.
Nonspecific fluorescence, i. e., fluorescence other
than that associated with the immunological reaction which
is being studied, is one of the major problems troubling
workers with the fluorescent antibody method.
The problem
of nonspecific fluorescence involves two types of phenom­
ena.
One is the Innate fluorescence of many biological
materials.
The second, and the more generally troublesome
cause of unwanted fluorescence, is that due to nonspecific
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
binding of dye-protein conjugates.
Since autofluorescence was not stimulated in Tetra-
j hymena by the wavelengths employed, this type of interferI ence was not a problem.
Nonspecific staining reactions
I
were encountered, however, when globulins labeled with
i
either fluorescein or rhodamine were used to stain Tetra­
hymena .
For example, normal (pre-immune) globulin conju­
gates, as well as the antiglobulins to S-I and S-II,
stained the ciliates.
Another nonspecific reaction was
the uptake of labeled antiglobulins by heterologous as
well as homologous strains of Tetrahymena.
The difficulties with nonspecific staining were
finally resolved by using a nonspecific rhodamine conjugate
as a counterstain to limit the green fluorescence of
fluorescein antiglobulins to those reactions which were
immunospecific.
This solution was reached after careful
consideration of the various theories in the literature
regarding nonspecific fluorescence and after attempts to
repeat the methods in the literature that supposedly remove
nonspecific reactions.
One factor which has been considered as a possible
source of nonspecific fluorescence is the presence of free i
i
fluorescein derivatives in conjugates.
Dialysis removes
some fluorescein which dissociates from protein, but side
reactions can produce fluorescein derivatives not readily
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
51
removed by dialysis.
Dineen and Ada (1957) recommended
rapid extraction with ethyl acetate to remove free dye
quickly and without a drop in antibody titer.
Chadwick
et a l . (1958a) found sorption with activated charcoal u se­
ful in reducing nonspecific staining with lissamine
j
rhodamine conjugates.
Attempts were made to purify the conjugates prepared
to stain Tetrahymena by extensive dialysis and also by re­
peated precipitation with ammonium sulfate.
Sorption with
activated charcoal gave results equal to or better than
acetate extraction and required fewer manipulations with
the conjugates.
Thorough dialysis followed by sorption
with charcoal was adopted as standard treatment for conju­
gates to insure the removal of unbound dye derivatives.
Even when purified conjugates were used, nonspecific
staining continued to be a problem.
Other workers have
also found that labeled antiserum is taken up by tissues
or objects which do not contain any of the specific antigen
under investigation.
Coons and Kaplan (1950) found empiri­
cally that such reactions could be removed by sorption withi
tissue powder, usually liver powder; sorption with liver
powder is thought to remove a common tissue antigen crossreacting with antibody in the conjugate.
In the present
investigation sorbing conjugates with monkey liver powder
did not have an appreciable effect on their staining
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
j
52
specificity.
Sorption of globulins or serums with heterologous,
serologically related antigens Is a common means of improv­
ing specificity in serologic systems other than fluorescent
antibody.
The problem of specificity is likely to be more
acute In fluorescent antibody work because it is a very
sensitive technique.
Cross-staining reactions may Inter­
fere in situations In which cross-immobilization does not
occur.
In order to remove antibodies common to both S-I
and S-II serotypes, globulins were sorbed either before or
after conjugation with breis of the heterologous ciliates,
leaving antibodies specific for the homologous strain.
When fluorescein conjugates sorbed in this manner were
used to stain conjugating pairs of ciliates, a difference
in intensity of fluorescence of the members could be de­
tected, but the difference was too slight to be of practi­
cal use in distinguishing strains of vegetative organisms
in a mixed culture.
Even this degree of specificity could
not be achieved with rhodamine conjugates; both co-conjugants continued to fluoresce with the same Intensity.
Smith and his associates (Smith et al., 1959) at­
tempted to turn the apparent liability of nonspecific
fluorescence into an asset by using a normal serum conju­
gated with lissamine rhodamine as a counterstain In fixed
tissue preparations.
The rhodamine-labeled normal serum
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
53
was mixed with fluorescein-labeled antiserum before stain­
ing the tissues.
The authors reported that the appearance
of the specific staining with fluorescein-conjugated anti­
serum was enhanced by a contrasting orange background
where there was no immunological reaction.
The counter-
staining method was said to eliminate nonspecific staining
with the fluorescein dye.
The nonimmunological reaction
of the rhodamine-labeled normal serum with the tissues was
interpreted as a protein-protein physicochemical interac­
tion .
A fluorescent serum protein was also used as a sero­
logically nonspecific stain by King, Hughes, and Louis
(1959).
These workers distinguished normal from neoplastic
cells with various serum proteins labeled with either
fluorescein or rhodamine.
They demonstrated that the non­
specific staining phenomenon was completely independent of
any explanation based on antigen-antibody reactions.
The
reaction was interpreted as a physicochemical interaction
between proteins which depended upon differences in iso­
electric points.
When adequate differentiation of S-I and S-II strains
of T. pyrlformls failed to materialize with either fluores­
cein or rhodamine antiglobulins used separately, the two
stains were combined (staining mixture A).
S-I vegetative
ciliates exhibited a green fluorescence in response to
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
54
stain A that was not present in the orange-colored S-II
vegetative organisms stained in the same way.
Conjugants
stained with mixture A presented a striking appearance with
one green and one orange member of a pair.
That the goal of two specific contrasting stains had
not been realized, however, was clearly demonstrated by the
results of tests for staining specificity.
The fluorescein
staining reactions were iimnunologically specific, but those
of the rhodamine-labeled antiglobulin were not.
Subse­
quently, normal (pre-immune) globulin labeled with rhoda­
mine was used as a counterstain in staining mixtures B and
D.
Evidence for the immunological specificity of fluores­
cein staining in the combination of conjugates was
demonstrated in two ways:
(1)
Using the same globulins
as in Stain A with the fluorescent labels reversed (stain­
ing mixture B) produced green fluorescence in only one cocon Jugant, presumably of the serotype corresponding to the
fluorescein antiglobulin in each case.
With staining mix­
ture C, fluorescein-labeled normal globulin did not stain
any ciliates in the presence of a rhodamine-globulin conju- ;
gate.
The triple stain, mixture D, consisting of rhoda­
mine-labeled normal globulin and fluorescein-labeled antiglobulins against both serotypes, caused a green pellicle
and ciliary fluorescence in both co-conjugants.
All single
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
;
55
vegetative animals in the culture were stained with fluo­
rescein also.
(2)
The specific green fluorescence in conjugants and
vegetative specimens was inhibited by prior exposure to u n ­
labeled homologous antiglobulin, whereas pretreatment with
unlabeled normal globulin did not inhibit fluorescein
staining (Plate II, Pig. 2).
Pretreatment with both u n ­
labeled antiglobulins prevented any green fluorescence
with the triple stain (D).
Although the rhodamine conjugate served to eliminate
the nonspecific staining with fluorescein and provided a
contrasting orange fluorescence in heterologous organisms,
no specificity could be demonstrated for the rhodamine
stain Itself.
Pretreatment with unlabeled globulins did
not inhibit rhodamine fluorescence.
The remarkable ability
of rhodamine-labeled normal globulin to impart specificity
to the fluorescein staining reaction has precedent in the
work of Smith e_t al. (1959), who used lissamine rhodamine
to counterstain fixed tissue preparations.
The complete
lack of specificity of rhodamine-labeled antiglobulins is
contrary to the findings of Silverstein (1957) and Chadwick
ct al. (1958a), who were able to obtain immunospecific
staining with both fluorescein and rhodamine conjugates.
The explanation for the complete absence of staining speci­
ficity in rhodamine conjugates is not readily apparent.
R eproduced with permission of the copyright owner. Further reproduction prohibited w ithout permission.
Taking into consideration the findings of other work­
ers and the results of inhibition tests with Tetrahymena
serotypes, the staining procedure which was employed as
standard for the Investigation of antigenic reactions dur­
ing conjugation is Interpreted as follows:
The rhodamine
globulin component of the staining mixture functioned as a
counters tain to suppress the nonspecific fluorescence of
the fluorescein conjugate In heterologous organisms and to
enhance its specific immunofluorescence In homologous in­
dividuals by color contrast.
The differential reactions of ciliates to the dual
staining mixtures evidently Involves both an immunological
antigen-antibody reaction and a protein-protein physico­
chemical interaction.
That the rhodamine staining reaction
is indeed a protein-protein physicochemical interaction
rather than a simple aqueous fluorochroming was confirmed
by tests with an aqueous solution of rhodamine.
Aqueous
rhodamine did impart to Tetrahymena a brilliant orange
fluorescence, but aqueous rhodamine lacked the capacity to
confer specificity upon the fluorescein-staining reactions
as a counterstain.
Mayersbach (1959) investigated the problem of nonspe­
cific staining reactions In frozen tissues and came to the
conclusion that they occur through an interaction based on
electrostatic attractions; that the relatively basic tissue
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
57
proteins have a binding capacity for the relatively acid
serum.
Hughes (1958) had postulated earlier that nonspe­
cific staining was due to physicochemical Interactions in
which basic proteins in tissues attract labeled serum.
Mayersbach suggested that any treatment which lowered the
isoelectric point of tissue proteins would decrease the
nonspecific uptake of conjugates by tissue sections.
If the explanation of the nonspecific reaction of
rhodamine conjugates with Tetrahymena has a similar basis,
manipulation of isoelectric points by methods such as
freeze drying or using different chemical fixatives could,
perhaps, abolish nonspecific uptake by heterologous cili­
ates.
With the present state of knowledge, such investiga­
tions would be largely of an empirical nature.
The system of counterstaining T. pyriformls with
fluorescein- and rhodamine-labeled globulins may be con­
sidered a competition in which an equilibrium has been
reached between two staining mechanisms:
an Immunological
reaction and a protein-protein physicochemical interaction.
Since these results were mostly obtained by empirical means,
it is possible that the equilibrium could be affected by
additional factors influencing isoelectric points, or anti­
body or antigen excess.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
58
Biological Implications
Statistical analysis has clearly established that
there was a significant quantitative difference in the
staining reactions of a population of conjugating Tetra­
hymena which was related to time, but there remains the
problem of interpreting the biological meaning of this
phenomenon.
(1)
Two possible explanations are given here.
The serotype of each strain remained stable through­
out conjugation, and co-conjugants which stained the same
color are the result of intra-clonal mating (selfing).
(2)
Only individuals of complementary mating type conju­
gated but one member of an atypical pair underwent anti­
genic change during the course of conjugation.
Although selfing had never been observed in the two
laboratory strains, S-I and S-II, and could not be detected
in washed controls maintained separately in distilled water
for the same length of time as the mixed mating types, the
possibility remains that selfing could have been induced in
the conjugating culture.
When Ray (1958) examined conju­
gants between tetraploid and diploid strains of variety 6,
he found that the pairs formed early in conjugation were
true conjugants between individuals of complementary mating
type, while pairs formed about 5 hours later were mostly
between individuals of the tetraploid clone.
The
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
59
tetraploid clone was one which arose spontaneously in the
laboratory during asexual reproduction.
The ability to
self evidently was acquired at the same time the spontane­
ous ploidy occurred.
Although it had not been observed in
this clone before, following ploidy, selfing was noted
when the animals were suspended in distilled water.
S-I and S-II are mature laboratory strains far removed
from conjugation; they are unlikely to demonstrate matingtype instability as a consequence of immaturity.
When
selfing is believed to be age-induced, it is demonstrable
by suspending the ciliates in distilled water and observing
pairing within the clone.
Selfing cannot be so demonstra­
ted in either S-I or S-II cultures.
Plate IV, Pig. 7, and Plate V, Pig. 8, show that the
two types of atypical staining pairs, R-R and F-P, did not
Increase at the same rate during the course of conjugation.
Far more pairs stained with the nonspecific rhodamine than
with the immunospeciflc fluorescein label.
This disparity
is not inconsistent with the hypothesis of selfing.
It is
quite possible that one strain would possess a greater de­
gree of selfing than the other.
If this were the case, a
reciprocal relationship of R-R and P-P frequencies should
have been demonstrated when the specific fluorescein label
was reversed for the two antiserums.
However, staining
with the B mixture revealed that the R-R pairs still far
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
60
outnumbered the F-F reactions (Plate V, Fig. 8).
Although
some selfing may have occurred, it cannot account for the
excess of R-R pairs with both staining mixtures.
The second hypothesis to explain identical fluores­
cence of co-conjugants is that of antigenic change during
the course of conjugation.
This hypothesis assumes that
all pairing was the result of mating S-I with S-II Indi­
viduals.
The atypical staining pattern, then, was the
consequence of a change in serotype by one of the co-conjugants during conjugation.
That mating type and serotype
are not identical is shown by comparison of the 7 new
tester strains with S-I and S-II strains.
Although S-I
and NT-I belong to the same mating type (Table VII, p. 44),
they do not possess the same antigens (Table VIII, p. 45).
NT-III Is related serologically to S-I, but the two strains
are of different mating types.
The observed change In
immunofluorescence could well have been a change In sero­
type which took place in true conjugants.
Both Paramecium (cf. Beale, 1954) and Tetrahymena
(Margolin £t a l . , 1959) are known to possess alternating
systems of antigens and many environmental factors are
known to influence which system is expressed.
The finding
that most atypical pairs stained only with the nonspecific
rhodamine label In either staining mixture could be inter­
preted as a change from S-I serotype to some other
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
61
alternative antigen of unknown identity.
It is not known
whether the loss of an antigenic component is reversible.
In the F-F cases, a known antigen was gained by one member.
Some factors which influence serotype transformation
are temperature changes, starvation, exposure to antiserums, ultraviolet, or X-radiation (Margolin £t a l .,
1959).
There is some evidence (loc. clt.) that young
axenic cultures in log growth phase differ antigenically
from older cultures.
Since antiserums in the present in­
vestigation were prepared against cultures in log growth
phase and individuals in the same phase were used for de­
tecting antigenic response, it is probable that this factor
did not enter into the atypical staining reactions.
With
the exception of starvation, no other environmental altera­
tions known to influence serotype expression were intro­
duced.
Since starvation is the agent employed to induce
conjugation, this treatment in itself could be sufficient
cause for antigenic changes.
When S-I and S-II strains
were washed and mixed for conjugation, controls of each
strain were kept separately in distilled water.
These con­
trols were stained with mixture B immediately upon washing
and again after 16 and 22 hours starvation.
Since conjuga­
tion in the mixed dishes did not begin until 4 hours after
mixing, 16-hour control organisms, correspond in length of
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
62
starvation to 12-hour conjugants.
Vegetative controls
maintained for 22 hours correspond to 18-hour conjugants.
Staining reactions of the starved S-I and S-II ciliates
were identical to those of the freshly washed organisms.
Therefore, it appears that starvation did not induce the
atypical staining reactions.
A more plausible explanation for the time-dependent
change in antigenicity seems to lie in some Intrinsic
physiological mechanism associated with the process of
conjugation.
In the serologic study of conjugation in
Paramecium bursarla, Harrison and Fowler (1946) observed
a sharp change in sensitivity of individuals to immobiliz­
ing antiserums during conjugation.
The change in antigenic
character was detectable only after the pairs had been In
conjugation for a period equal to two-thirds of the normal
conjugation period.
Wichterman (1953) noted that these
changes in antigenicity were correlated with the time of
pronuclear transfer during conjugation.
Harrison and
Fowler favor transfer of cytoplasm between conjugants as
the mechanism of antigenic transfer.
The significant change in immunofluorescence of T.
pyrlformls conjugants occurred between 10 and 12 hours fol­
lowing the beginning of conjugation in the culture, and the
number of atypical reactions continued to increase through­
out the period studied (18 hours).
Ray (1956) observed
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
63
that nuclear reorganization at conjugation in variety 1
takes 9 to 10 hours from pairing to formation of the new
macronucleus.
In the present study, living S-I x S-II
conjugants were stained with acridine orange and observed
by fluorescence microscopy to determine the stages of
nuclear events.
Acridine orange stains nuclei bright green
and cytoplasmic inclusions orange.
The macronuclear
anlagen stage in conjugants is easily recognized by the
brilliant fluorescence of the old disintegrating macro­
nucleus in the posterior end of one or both co-conjugants.
Only one pair was found in anlagen stage at 9 hours follow­
ing conjugation, but by 10 hours several anlagen pairs
could be seen.
The anlagen pairs were quite numerous by
12 hours and continued to increase thereafter.
Thus the
change in antigenicity was correlated with the time of de­
velopment of macronuclear anlagen.
This is not to imply
that there is a causal relationship between the developing
macronucleus and the antigenic change.
The principle of fluorescent antibody methodology is
deceptively simple.
Theoretically, fluorescent antibodies
should be applicable to any system involving an antigenantibody reaction.
Experience with the complex antigen
system of Tetrahymena indicates some of the complications
which can arise.
Rigorous controls to provide proof that
observed fluorescence is due to specific antigen-antlbody
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
reactions are requisite.
In addition to direct staining and inhibition, one
other variation in immunofluorescent methods was attempted,
the indirect staining method (cf. Cherry at al., 1960).
The indirect staining method would be advantageous for
Tetrahymena research because it requires only one labeled
reagent, anti-rabbit globulin, which can be obtained com­
mercially.
Reactions of numerous unlabeled antiserums
could be detected with a single fluorescein conjugate.
In
the indirect test the binding of unlabeled antibody to
antigen is visualized by a secondary reaction between the
unlabeled antibody (primary reagent) and a fluorescent
antibody (secondary reagent) prepared against the primary
reagent.
A preliminary trial of indirect staining with u n ­
labeled S-I antiglobulin followed by fluorescein antirabbit globulin did not prove successful because no stain­
ing specificity could be demonstrated.
Since the indirect
method involves additional manipulations and two separate
serological reactions, there are additional opportunities
for nonspecific reactions.
In view of the difficulties
which were encountered with nonspecific reactions by direct
staining, it appears unlikely that further explorations
with the indirect method would be profitable.
The development of suitable techniques for application
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
65
of fluorescent antibodies to individual ciliates should
open new avenues of approach to the immunology of Tetra­
hymena .
Useful information on the nature of antigenic
material in these ciliates might be obtained by using
chemically fractionated components of Tetrahymena rather
than whole ciliates to stimulate antibody production.
It would be interesting to compare the results of Im­
munofluorescence with Immobilization in parallel experi­
ments.
The present study could be extended by preparing
additional specific antiserums to determine if other
strains possess similar patterns of antigenic change dur­
ing conjugation.
It should be possible to study the in­
fluence of factors such as temperature shock or enzyme
treatment on serotype transformation by introducing them
at the critical period from 10 to 12 hours following onset
of conjugation.
Because of the labile nature of antigens In the genus
Tetrahymena, and because of the degree of nonspecific
staining which was encountered, the fluorescent antibody
method cannot be recommended unconditionally as a genetic
marker of,general applicability to problems other than
those of an immunological nature.
Any work with fluores­
cent antibody will require careful attention to specificity
controls and caution in the interpretation of immunochemi­
cal reactions.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CONCLUSIONS
Under the experimental conditions of this investiga­
tion, the following conclusions appear to he justified:
(1)
Fluorescent antibody methods can be used to
study the complex antigen system of populations of Tetra­
hymena pyrlformis.
Strain specific differences in these
ciliates can be detected by fluorescent antibody tests.
(2)
Living Tetrahymena did not take up labeled anti­
body except by Ingestion.
Absolute methanol was a satis­
factory fixative which was conducive to adequate staining.
(3)
It was found that all rhodamine staining reac­
tions were serologically nonspecific.
The cause was not
determined.
(4)
Adequate specificity of immunofluorescence was
not obtained with either fluorescein or rhodamine conju­
gates used separately.
(5)
Useful staining mixtures were achieved by combin­
ing a specific antiglobulin labeled with fluorescein isothiocyanate and a normal globulin labeled with rhodamine B
isothiocyanate.
The rhodamine counterstain limited the
green fluorescence of fluorescein to homologous strains
and provided an effective color contrast in heterologous
organisms.
66
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
67
(6)
Comparison of mating reactions and serotype
fluorescence in different strains of variety 1 demonstrated
that strains of the same mating type were not necessarily
of the same serotype, and that different mating types could
be of the same serotype.
(7)
During conjugation of the two strains studied,
many of the co-conjugants appeared to undergo a change in
serotype expression.
The change became detectable in
significant numbers of conjugants between 10 and 12 hours
following onset of conjugation.
(8)
Controls indicated that the serotype change was
not induced by starvation.
(9)
The time of serotype change within a conjugating
population of Tetrahymena was coincident with the occur­
rence of macrcnuclear reorganization in the conjugants.
(10)
Rigorous controls of staining specificity must
be maintained in order to interpret correctly the validity
of fluorescent antibody reactions.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
SUMMARY
The fluorescent antibody technique was used to detect
antigenic similarities and differences In strains of Tetra­
hymena pyrlformls, variety 1.
Antiserums were prepared in
rabbits against two strains, S-I and S-II, representing
mating types I and II of variety 1.
These and other
strains belonging to the 7 mating types of this variety
were stained with serum globulins conjugated with either
fluorescein isothiocyanate or rhodamine B isothlocyanate.
The ciliates were fixed with absolute methanol before
staining because living Tetrahymena did not take up fluo­
rescent globulins except by ingestion Into food vacuoles.
Dye-globulin conjugates were sorbed with activated
charcoal and with breis of heterologous ciliates before use
as cytochemical stains.
In order to obtain satisfactory
specificity of staining, it was necessary to use a rhodamine-globulin conjugate as a nonspecific counterstain for
fluorescein-antiglobulin conjugates.
All rhodamine conju­
gates gave immunologically nonspecific staining reactions.
The specificity of fluorescein staining was demonstrated by
reversing the fluorescent labels of globulins in polyvalent
staining mixtures and by inhibition with unlabeled anti­
bodies.
The rhodamine counterstain prevented nonspecific
68
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
69
green fluorescence and enhanced the appearance of fluores­
cein-stained ciliates with contrasting orange fluorescence
in heterologous organisms.
Evidence from reactions of
strains other than S-l and S-II showed that serotype and
mating type were not necessarily correlated.
Typical conjugating pairs of S-I x S-II strains
stained with the mixtures of fluorescein- and rhodaminelabeled globulins had one green fluorescing member and one
orange member.
A few pairs stained atypically with both
members green, or both orange.
The number of atypical
pairs increased during the time of conjugation with a sig­
nificant rise between 10 and 12 hours after the start of
conjugation in a population.
Macronuclear anlagen stages
were noted in conjugating populations at the time of sero­
type change.
Selfing was considered to be an unlikely
explanation for the atypical staining reactions.
Evidence
indicated that starvation was not the inducing agent of
serotype changes during conjugation^
The change in stain­
ing reactions of conJugants was attributed to a change in
antigenicity brought about by physiological mechanisms ac­
companying the process of conjugation.
An attempt to apply indirect staining methods with
fluorescent antibody to Tetrahymena was not pursued because
of nonspecific reactions.
It was concluded that stringent controls must be
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
70
maintained in order to establish valid interpretations
based on fluorescent antibody reactions of Tetrahymena.
An attempt was made to evaluate the methods for future ap­
plication to research with Tetrahymena.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
L IT E R A T U R E
C IT E D
71
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
L IT E R A T U R E
C IT E D
Allen, S. L., and D. L. Nanney 1958 An analysis of
nuclear differentiation In the selfers of Tetrahymena. Amer. Naturalist, 92: 139-160.
Batson, H. C. 1956 An introduction to statistics in the
medical sciences.
Burgess Publishing Co.,
Minneapolis.
Beale, G. H. 1954 The genetics of Paramecium aurelia.
Cambridge University Press" Cambridge.
1957 The antigen system of Paramecium aurelia.
Int. Rev. Cytol., 6: 1-23.
__________ , and H. Kacser 1957 Studies on the antigens of
Paramecium aurelia with the aid of fluorescent
antibodies.
J. Gen. Microbiol., 17; 68-74.
Bernheimer, A. W . , and J. A. Harrison 1940
antibody reactions in Paramecium:
group.
J. Immun., 39: 73-83.
Antigenthe aurelia
Chadwick, C. S., M. G. McEntegart, and R. C. Nairn 1958a
Fluorescent protein tracers: a trial of new
fluorochromes and the development of an alterna­
tive to fluorescein.
Immunology, 1: 315-327.
__________
1958b Fluorescent protein tracers. A simple
alternative to fluorescein.
Lancet, 1: 412-414.
Cherry, W. B., M. Goldman, T. R. Carski, and M. D. Moody
1960 Fluorescent antibody techniques in the
diagnosis of communicable diseases.
Public
Health Service Publication No. 729, United States
Government Printing Office, Washington, D. C.
Coleman, M. T. 1958 Fluorescence microscopy in cytological studies of Tetrahymena pyriformis (Ehrenberg)
Fur gas on. M. A. thesis, Emory University
Library.
72
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
73
Coons, A. H. 1956 Histochemistry with labeled antibody.
Int. Rev. Cytol., 5: 1-23.
__________
1958 Fluorescent antibody methods.
In "Gen­
eral Cytochemical Methods", ed. by J. F.
Danielli. Academic Press, Inc., New York, pp.
399-422.
__________ , H. J. Creech, R. N. Jones, and E. Berliner
1942 The demonstration of pneumococcal antigen
In tissues by the use of fluorescent antibody.
J. Immun., 45: 159-170.
Coons, A. H., and M. H. Kaplan 1950 Localization of anti­
gen In tissue cells.
II. Improvements in a
method for the detection of antigen by means of
fluorescent antibody.
J. Exp. Med., 91; 1-13.
Corliss, J. 0. 1953 Silver Impregnation of ciliated
protozoa by the Chatton-Lwoff technic.
Stain
Techn., 28: 97-105.
__________
1954 The literature on Tetrahymena: Its
history, growth, and recent trends.
J. Protozool., 1: 156-169.
________
1957 The literature on Tetrahymena;
through 1956.
Ibid., 4 (Suppl.); T5.
1954
Cushing, J. E., and D. H. Campbell 1957 Principles of
Immunology. McGraw-Hill Book Company, Inc.,
New York.
Dineen, J. K., and G. L. Ada 1957 Rapid extraction with
ethyl acetate of free fluorescein derivatives
from fluorescein Isocyanate-globulin conjugates.
Nature, 180: 1284.
Elliott, A. M. 1959a Biology of Tetrahymena.
Microbiol., 13: 79-96.
__________
Ann. Rev.
1959b A quarter century exploring Tetrahymena. |
J. Protozool., 6: 1-7.
I
__________ , and J. R. Byrd 1959 Serotypes in eight varie­
ties of Tetrahymena pyriformis.
Ibid., 6
(Suppl. ) 1 5 " (abs'trf)."
--__________ , and D. F. Gruchy 1952 The occurrence of mat­
ing types in Tetrahymena. Biol. Bull., 103: 301.j
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
74
__________ , and R. E. Hayes 1953 Mating types In Tetra­
hymena . Ibid., 105: 269-284.
.
______ , and D. L. Nanney 1952 Conjugation In Tetra­
hymena . Science, 116: 33-34.
Finger, I.
1956 Immobilizing and precipitating antigens
of Paramecium. Biol. Bull., Ill: 358-363.
__________
1957 The inheritance of the immobilization
antigens of Paramecium aurelia, variety 2.
J.
Genetics, 551 361-374.
Goldman, M. 1953 Cytochemical differentiation of
Endamoeba histolytica and Endamoeba coll by
means of fluorescent antibody.
Amer. J. Hyg.,
58: 319-328.
__________
1954 Use of fluorescein-tagged antibody to.
identify cultures of Endamoeba histolytica and
Endamoeba coll. Ibid., 59: 318-325.
__________
1957a Staining Toxoplasma gondii with fluores­
cein-labelled antibody.
I . The- reaction in
smears of peritoneal exudate.
J. Exp. Med.,
105; 549-556.
__________
1957b Staining Toxoplasma gondii with fluores­
cein-labelled antibody.
I I . A new serologic test
for antibodies to Toxoplasma based upon inhibi­
tion of specific staining. Ibid., 105:
557-573.
__________
1959 Microfluorimetric evidence of antigenic
difference between Entamoeba histolytica and
Entamoeba hartmanni"
Proc. S o c . Exp. Biol. Med.,
102: tss- tst:---------
__________
1960 Antigenic analysis of Entamoeba histo­
lytica by means of fluorescent antibody.
1. In­
strumentation for microfluorimetry of stained
amebae. Exp. Parasit., 9: 25-36.
Gruchy, D. F. 1955 The breeding system and distribution
of Tetrahymena pyrlformis. J. Protozool., 2:
178—185.
Harrison, J. A., and E. H. Fowler 1945 An antigenantibody reaction with Tetrahymena which results
in dystomy. Science, 1(521 65-66.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
75
__________
1946 A serologic study of conjugation In
Paramecium bursaria.
J. Exp. Zool., 101:
425444.
Hiller, A.
1953 Practical clinical chemistry.
C. Thomas, Publisher. Springfield.
Charles
Hughes, P. E.
1958 The significance of staining reactions
of preneoplastic rat liver with fluoresceinglobulin complexes.
Cancer Res., 18:
426-432.
Kabat, E. A., and M. M. Mayer 1948 Experimental immunochemistry.
Charles C. Thomas, Publisher.
Springfield.
Kidder, G. W . , C. A. Stuart, V. G. McGann, and V. C. Dewey
1945 Antigenic relationships in the genus,
Tetrahymena. Physiol. Zool., 18: 415-425.
King, E. S. J., P. E. Hughes, and C. J. Louis 1959 D if­
ferential fluorescence staining of normal and
neoplastic tissues: Use of various serum pro­
teins.
Cancer, 12: 741-752.
Loefer, J. B., R. D. Owen, and E. Christensen 1958 Sero­
logical types among thirty-one strains of the
ciliated protozoan Tetrahymena pyrlformls. J.
Protozool., 5: 209-21*7.
Margolin, P., J. B. Loefer, and R. D. Owen 1959 Immobi­
lizing antigens of Tetrahymena pyriformis.
J.
Protozool., 6: 207-215.
Marrack, J. 1934 Nature of antibodies.
292-293.
Nature, 133:
Marshall, J. D., W. C. Eveland, and C. W. Smith 1958
Superiority of fluorescein isothiocyanate (Riggs)
for fluorescent antibody technic with a modifica­
tion of its application.
Proc. Soc. Exp. Biol.
Med., 98; 898-900.
Mayersbach, H. 1959 Unspecific interactions between serum
and tissue sections in the fluorescent-antibody
technic for tracing antigens in tissues.
J.
Histochem. Cytochem., 7: 427.
Nanney, D. L. 1956 Caryonidal inheritance and nuclear
differentiation. Amer. Naturalist, 90: 291-307.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
76
1959a Genetic factors affecting mating type
frequencies in variety 1 of Tetrahymena pyriformls. Genetics, 44: 1173-1184.
1959b Serotype determination in Tetrahymena
"pyriformis, variety 1. R e c . Genetics S oc. Arner.
28: 69 (abstr.).
1959c Nuclear differentiation in serotype de­
termination in Tetrahymena. National Academy of
Sciences, abstract of paper presented at Bloom­
ington, Indiana, Nov. 16-18, 1959.
_, and P. A. Caughey 1953 Mating type determina­
t i o n in Tetrahymena pyriformis.
Proc. Natl.
Acad. Sci.', 39:
1057-1063.
_, P. A. Caughey, and A. Tefankjian 1955 The
'genetic control of mating type potentialities
in Tetrahymena pyriformis.
Genetics, 40: 668680.
Nissenbaum, G. 1953 A combined method for the rapid fixa­
tion and adhesion of ciliates and flagellates.
Science, 118: 31-32.
Preer, J. R., Jr., and L. B. Preer 1959 Gel diffusion
studies on the antigens of isolated cellular com­
ponents of Paramecium.
J. Protozool., 6: 88-
100 .
Ray, C., Jr. 1956 Meiosis and nuclear behavior in Tetrahymena pyriformis. Ibid., 3: 88-96.
__________
1958 Tetraploidy in Tetrahymena pyriformis.
X International Congress of Genetics, Proc., 2:
229-230 (abstr.).
Riggs, J. L., R. J. Selwald, J. Burckhalter, C. M. Downs,
and T. G. Metcalf 1958 Isothiocyanate compounds
as fluorescent labeling agents for immune serum.
Amer. J. Path., 34: 1081-1097.
Robertson, M. 1939a A study of the reactions in vitro of
certain d i l a t e s belonging to the GlaucomaColpldlum group to antibodies in the sera of rab­
bits immunized therewith.
J. Path. Bact., 48:
305-322.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
77
__________
1939b An analysis of some of the antigenic
properties of certain ciliates belonging to the
Qlaucoma-Colpldlum group as shown in their
response to immune serum.
Ibid., 48: 323-338.
Rossle, R.
1905 Spezifische Sera gegen Infusorien.
Hyg. Bakt., 54: 1-31.
Arch.
Silverstein, A. M.
1957 Contrasting fluorescent labels
for two antibodies.
J. Histochem. Cytochem., 5;
94-95.
__________ , W. C. Eveland, and J. D. Marshall, Jr. 1957
Rapid identification of organisms with fluores­
cent antibodies of contrasting colors.
Bacterio­
logical Proceedings, Soc. Amer. Bacteriologists:
147 (abstr.).
Sinclair, I. J. B. 1958 The role of complement in the
immune reactions of Paramecium aurelia and
Tetrahymena pyriformis. Immunology, 1: 291-299.
Smith, C. W., J. D. Marshall, Jr., and W. C. Eveland 1959
Use of contrasting fluorescent dye as counter­
stain In fixed tissue preparations.
Proc. Soc.
Exp. Biol. Med., 102: 179-181.
Sonneborn, T. M. 1939 Paramecium aurelia; mating types
and groups lethal Interactions; determination
and inheritance. Amer. Naturalist, 73: 390-413.
;
__________
1948 The determination of hereditary antigenic
differences in genically identical Paramecium
cells.
Proc. Nat. Acad. Sci., 34: 413-418.
__________ , and A. LeSuer 1948 Antigenic characters in
P. aurelia (variety 4): determination, inheri­
tance and Induced mutations.
Amer. Naturalist,
82: 69-78.
Tanzer, C.
1941 Serological studies with free-living
protista.
J. Immun., 42: 291-312.
van Wagtendonk, W. J., and B. van Tijn 1953 Cross reac­
tion of serotypes 51A, 51B, and 51D of Paramecium I
aurelia, variety 4. Exp. Cell Res., 5: 1-9.
;
I
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
78
__________ , R. Litman, A. Reisner, and M. L. Young 1956
The surface antigens of Paramecium aurelia.
J.
Gen. Microbiol., 15: 61T-619":
Wells, C.
1958 Intra-clonal mating in strains of variety
6, Tetrahymena pyriformis.
ASB Bull., 5: 17
(abstr.).
Wichterman, R. 1953 The Biology of Paramecium.
Blakiston Company, Inc., New York.
The
I
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATES
79
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
PLATE
Figure 1.
I
Direct staining with fluorescent anti­
body.
Fixed S-I x S-II conjugants
were suspended in staining mixture B,
fluorescein-labeled antiglobulin pre­
pared against strain S-II combined with
a counterstain, rhodamine-labeled
normal globulin.
Co-conjugants dis­
played contrasting green (fluorescein)
and orange (rhodamine) fluorescence.
Heavy black stippling represents green
fluorescence and light gray stippling
represents orange fluorescence.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE I
DIRECT STAINING WITH
FLUORESCENT ANTIBODY
S-I X S-I
STAINING
M IXTU R E B
FLUORESCENT CO-CONJUGANTS
gig- 1
81
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE
II
Ftg- 2
Test:
Control:
Inhibition of specific fluorescence.
S-I x S-II conjugants were incubated with
a mixture of unlabeled (no stippling)
antiglobulins prepared against each strain.
Subsequently, the ciliates were exposed to
staining mixture A, fluorescein-labeled
anti-I globulin and rhodamine-labeled antiII globulin.
(Rhodamine normal globulin
would have given the same reaction.)
No
green (fluorescein) fluorescence resulted.
Both members had orange (rhodamine) fluores­
cence. Specific fluorescein staining was
inhibited by unlabeled antibody but non­
specific rhodamine fluorescence was not
suppressed.
Unlabeled normal globulins did not inhibit
either fluorescein or rhodamine staining.
Stained co-conjugants continued to
fluoresce in contrasting green and orange
colors. Heavy black stippling represents
green fluorescence and light gray stippling
represents orange fluorescence.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE
NH IBITION
TEST
OF
II
SPECIFIC
FLU O R E S C E N C E
CONTROL
S-I x S -I
S-I x S - I
+
+
UNLABELED
UNLABELED
UNLABELED
NORMAL
.
GLOBULIN
+
*
*
STAINING MIXTURE A
STAINING MIXTURE A
g-lg- 2
83
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE
III
Figure 3
Contrasting fluorescence in vegeta­
tive Tetrahymena. The upper animal
took up nonspecific rhodamine conju­
gate; the lower animal took up specific
fluorescein conjugate.
Figure 4
Contrasting fluorescence in conjugating
Tetrahymena. The upper member took up
nonspecific rhodamine conjugate; the
lower member took up specific fluores­
cein conjugate.
Figure 5
Atypical F-F conjugants.
Both members
took up specific fluorescein conjugates.
Figure 6
Atypical R-R conjugants. Both members
took up nonspecific rhodamine conjugate.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE
I 'll
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission
PLATE
Figure 7.
IV
Atypical staining reactions of S-I x
S-II conjugants to staining mixture A,
fluore-scein anti-I globulin plus
rhodamine anti-II globulin.
(Rhoda­
mine normal globulin could have been
substituted.)
The number of atypical
pairs per 1,000 showed a significant
increase between 10 and 12 hours follow­
ing initiation of conjugation within the
culture.
R-R pairs were more frequent
than F-F pairs.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
•Of
ATYPICAL STAINING REACTIONS OF S -I x S-3E
CONJUGANTS TO STAININtj MIXTURE A
NUMBER OF PAIRS
•0•
•0
40
to
HOURS
Slg* 7
87
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE V
Figure 8.
Atypical staining reactions of S-I x
S-II conjugants to staining mixture 3,
fluorescein anti-II plus rhodamine
normal globulin.
The number of atypi­
cal pairs per 1,000 showed a significant
Increase between 10 and 12 hours follow­
ing initiation of conjugation within the
culture.
R-R pairs were more frequent
than F-F pairs.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PLATE V
••
ATYPICAL STAMNG REACTIONS OF S - I x S - I
CONJUGANTS TO STAINMG MIXTURE B
?• ,
••
to
10
I I
HOURS
Fig. 8
89
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Документ
Категория
Без категории
Просмотров
5
Размер файла
3 221 Кб
Теги
835
1/--страниц
Пожаловаться на содержимое документа