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IMMUNOFLUORESCENCE AS A METHOD FOR THE RAPID
IDENTIFICATION OF STREPTOCOCCUS FAFJALIS
IN WATER
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
Doctor of Philosophy
By
Robert L. Abshire, B.S., M.S.
Denton, Texas
August, 1970
TABLE OF CONTENTS
Page
LIST OF TABLES.
LIST OF FIGURES..--...--.---....--- ..- ....-................. i
Chapter
I. INTRODUCTION............................... .1
II. REVIEW OF LITERATURE--.-.-.........................6
III. MATERIALS AND METHODS.....--------............32
IV. RESULTS......---..-..------..........................79
V. DISCUSSION...--.-----------.---...................125
VI. SUMMARY.......---..--..--.-.--...-.....-................131
BIBLIOGRAPHY-...-.-.-.-.-.-.-.-.-.-...-.-.-.-.-.-.-.-.-.-.-.-..................134
LIST OF TABLES
Table Page
I. Stains of Bacteria Employed in the Study.,.,........34
II. Axide Detrose Broth Medium---------.--.............65
III. M-Enterococcus Agar..................................66
IV. Agglutination Titers of Homologous Organisms ........76
V. Fluorescent Antibody Titers of HomolgousOrganisms.........-..................................78
VI. FA Titers of Whole Antisera and Antiglobulinsas Determined by the Direct and Indirect Methodsof Staining.-----........................................81
VII. FA Titers of Fluorescein-Labelled Conjugates asDetermined by the Direct Method of Immuno-fluorescence.--.-........-.............................83
VIII. A Comparison of Agglutination Titers and FATiters Before and After Adsorption Techniques. ..... 86
IX. Stains of Streptococcus Faecalis Utilized inthis Immunofluorescent Study.......................89
X. Agglutination Titers of ATCC Strains ofStreptococcus Faecalis Reacted with theVarious Anti-Streptococcus Faecalis-Sera............90
XI. Fluorescent Antibody Titers of ATCC Strainsof Streptococcus Faecalis. Following Reactions withGroup-Specific Antisera------.-....................91
XII. Agglutination Titers of Streptococcus FaecalisAntisera with Heterologous Organisms-................93
XIII. Fluorescent Antibody Reactions of StreptococcusAntisera with Heterologous Organisms-................95
V
XIV. The Staining Titers of Isolates....................97
XV. Agglutination Titers of Fifteen ATCC Strainsof Eschericha Coli......................-....100
XVI. Fluorescent Antibody Reactions of ATCC Strainsof Escherichia Coli.......... "-...-- .-.-.-- ....--..... ---.....102
XVII. Amino Acid Analysis of Streptococcal Cell Wallsas Determined by Thin-Layer Chromatography ofHCL Hydrolyzates from Whole Cells........ .-... 107
XVIII. The Amino Acids Detected by Thin-LayerChromatography Following the Mild Hydrolysisof Streptococcal Cells in 6N HCL for Two Hoursat 37 C...........-..-.-.-.-.-.-.-.-.-.. . -... -....110
XIX. The N-Terminal Amino Acids Detected in the AcidHydrolyzates from Streptococcal Cell Walls asDetermined by Thin-Layer Chromatography, .... ..... ... 114
XX. Sugars Detected in HCL Hydrolyzates ofStreptococcal Cells by Thin-Layer Chromatography. .116
vi
LIST OF FIGURES
Figure Page
1. The Chemical Structure of Fluorescein-Isocyanate (FIC).............................11
2. The Chemical Structure of Fluorescein-Isothiocyanate (FITC)..................... 16
3. Reference Protein Curve as Determined withBovine Serum Albumin (BSA) by theBuiret Method................................44
4. Schematic Diagram of the Direct Method ofImmunofluorescence............................54
5. Schematic Diagram of the Indirect Methodof Immunofluorescence.......................55
6. Illustration of Immunofluorescence by theDirect Method of Staining................ 61
7.. Illustration of Immunofluorescence by theIndirect Method of Staining................62
8. Immunofluorescence of an Isolate as Demon-
strated by the Indirect Method ofStaining......................................63
9. A Fluorescent Reaction Obtained from CellsTaken from a Five-Hour Broth Culture,... 64
10. Identification Scheme for Group DEnterococci-----.--......................72
vii
11. Scheme for Hydrolyzing Cells to ObtainComponents for Analysis by Thin-
Layer Chromatographyr.......................... 113
viii
Figure Page
CHAPTER I
INTRODUCTION
The serum of an immunized animal will contain antibodies
referred to as agglutinins, precipitins, opsonins, bacterio-
lysins, or complement-fixing antibodies (Zinsser 1952).
The presence of such antibodies may be demonstrated in the
laboratory, the type of reaction depending on the circumstance
and the laboratory manipulation employed. Regardless of the
specific serolological method utilized, the manifestation
of the antigen-antibody reaction is the visible observation
that such a combination has occurred.
Fluorescent antibody (FA) is an additional immunological
reagent which provides brilliant and visible fluorescence
when homologous antigen and antibody have are reacted,
illuminated with an ultra-violet light of high intensity,
and observed through a microscope equipped with a dark-field
condenser. Goldman (1968) defined fluorescence as the
reversion of a molecule from an excited state to ground state,
or to some intermediate level of energy with a consequent
loss of energy. According to Beutner (1961), compounds which
2
emitted light in one wavelength range when illuminated by
light of a shorter wavelength were considered fluorescent.
Antiglobulins can be labelled with a fluorochrome,
such as fluorescein-isothiocyanate, without altering their
biological activity. Conjugates of this type will fluoresce
when excited by a stimulating beam of light, thus, enhancing
their application as a means of demonstrating antigen-antibody
reactions.
The development and refinement of FA has been adequately
investigated with major emphasis on pathogenic microorganisms.
The development of this technique has reduced both the
time and number of biochemical tests necessary to identify
a diversity of organisms. The organisms included are the
protozoans, as described by Goldman (1953 and 1957) and
by Ingram (1961), viruses, as reported by Liu (1955a) and
Burgdorfer and Lackman (1960a), pathogenic bacteria which
have been investigated by Moody, Goldman, and Thomason
(1956), Moody and Winter (1959), Deason, Falcone, and Harris
(1957) and Thomason, Cherry, and Moody (1957). Various
fungi have been studied with FA by Kaufman and Brandt (1964),
Kaufman and Kaplan (1961 and 1963) and Gordon (1958).
3
Therefore, due to the success of the fluorescent
antibody technique in many areas of microbiology in previous
investigations, the logical assumption was that immunofluor-
escence might be incorporated into an efficient system in
which a specific organism associated with fecal pollution,
such as S. faecalis, could be rapidly identified. Based
on this assumption, the feasibility of fluorescent antibody
techniques, using S. faecalis was investigated as a means of
rapid determination of bacterial pollution in water.
Although much progress has been achieved in the study
of cytochemical reactions by immunofluorescence, no attention
has been focused on the application of this method as a
determinative tool by which water contamination, due to the
presence of the enterococci, could be demonstrated.
Specifically, the purpose of the research reported in
this dissertation was to devise an applicable, valid, and
rapid method that could be employed in the detection and
identification of S. faecalis. The research included the
following phases:
(1) The staining ability and the specificity of' the
antisera had to be established. The procedure involved
slide tests using eighteen known strains of S. faecalis
4
which were obtained from the American Type Culture
Collection (ATCC) in addition to the agglutination
tests.
(2) Similar tests were performed with fifteen strains
of Escherichia coli. The purpose of this phase of the
study was to determine which of the organisms would serve
as the better indicator system with reference to FA.
(3) Methods were investigated that demonstrated a
substantial reduction in the time necessary for the
identification of S. faecalis.
(4) Approximately ' five-hundred unknown isolates
were used in this study. Standard bacteriological methods
and fluorescent antibody techniques were used to identify
these organisms in order to determine the reliability of
the FA method of identification.
(5) The cell wall components of various strains of
S. faecalis were extracted and analysed in an attempt to
determine differences in chemical composition. The purpose
of this phase of the investigation was to examine cells
which exhibited various degrees of staining.
Therefore, the experimental approach of this investigation
was an attempt to determine if fluorescent antibody can
5
be used in conjunction with S. faecalis as a bacteriological
method for the resolution of water potability.
Moody and Cherry (1965) stated, with reference to PA:
Many applications of immunofluorescence arerapid, sensitive, and reliable, but are of minorinterest due to the infrequency of the disease forwhich they are designed.
Substantial evidence from this study suggests that the
fluorescent antibody technique is applicable to areas of
microbiology other than disease diagnosis, specifically
that of determining water contamination due to the presence
of the enterococcus, S. faecalis.
CHAPTER II
REVIEW OF LITERATURE
Fluorescent Antibody
Early Work with Dye-Protein Conjugates
The conjugation of dyes to protein is not a novel
serological technique. Reiner (1930) prepared serologically'
active atoxyl-azo conjugates of pneumococcus Type II and
Type III antibodies. Haurowitz and Breinl (1932) used
chemically labelled antigens prepared from horse serum
diazotized with atoxyl and estimated the arsenic content of
various experimental animals organs. This study was broadened
by Haurowitz and Kraus (1936) when they used iodinated horse
serum as their antigen, as well as the atoxyl-horse serum
complex.
Heidelberger et al. (1933) conjugated the salt of
benzidine to egg albumin and injected rabbits with thisantigenic preparation. Spectrophotometric studies were
performed on antibody production and compared to the results
6
7
that were obtained by the precipitation test. Hopkins and
Wormall (1933) were the first to conjugate proteins to
aromatic isocyanates in an attempt to analyse at what point
linkage, between the protein and isocyanate, had occurred.
The study revealed that linkage was probably via an epsilon
amino group of lysine.
Marrack (1934) demonstrated that anti-typhoid or anti-
cholera serum conjugated to diazotized benzidine-azo-R-salt
stained homologous organisms pink. The colored group,
however, was not fluorescent.
Further immunological inquiries of the effect of diazo
compounds on the reactivity of antibodies were made by
Pauly (1904) ; Eagle and Vickers (1934) ; and Eagle, Smith,
and Vickers (1936). Pauly (1904) showed that diazo compounds
reacted with the imidazole ring of histidine and with the
phenyl ring of tyrosine. Eagle and Vickers (1934) demonstrated
that conjugation also occurred between the diazo compound and
the amino group of proline, the hydroxyl group of proline,
and the indole ring of tryptophane. Eagle, Smith, and Vickers(1936) concluded that the combination of antigen with antibody
was necessitated by one or all of these groups. The works
of these investigators demonstrated that diazo compounds
8
destroyed the reactivity of antibody. Also, the loss of
antibody reactivity, with reference to the length of time
necessary for antibody inactivation, varied with respect
to the particular antibody concerned.
Dyes were first employed in vivo investigations by
Sabin (1939) who formed an azoprotein by conjugating the
salt of benzidine to crystalline egg albumin in order to
examine, microscopically, the fixation of such antigen in
tissue cells known as macrophages. Smetana (1947) extended
these findings as he demonstrated that the proximal tubules
of the kidney also retained the dye, and traces of the dye
were present in the tissue twenty-eight days after injection.
The Beginning of Immunofluorescence
Creech and Jones (1941) showed that conjugates syn-
thesized.by the interaction of cyanates of polynuclear
aromatic hydrocarbons with several proteins were highly
fluorescent. Thus, interest was aroused in some researchers
as to the possibility of using such fluorescent compounds
in immunological studies.
The actual debut of fluorescent antibody (FA) was
marked by the work of Coons, Creech, and Jones (1941) asthis group of investigators was the first to label an
immune serum with a fluorescent dye. The major purpose
of this project was to investigate the possibility of
incorporating a tagged antibody, as a "tracer" into a
system which would lead them to an unmarked antigen.
This fact alone set apart Coons, and his associates,
objectives from those of others who had studied the
effects of various chemical radicals on the immunological
activities of antibodies (Goldman (1968). Coons referred
to earlier works and stated:
Previous investigations had been carried outto establish the protein nature of antibody molecules,or, to elucidate the influence of specific polargroups on antigen-antibody mechanisms,.
The conclusion of this work was that B-anthryl, isocyanate
conjugated to antipneumococcus Type III antiserum retained
its original immunological properties while rendering Type III
pneumococci specifically fluorescent in ultra-violet light.
Also, Type II pneumococcal cells did not fluoresce after
they were exposed to Type III specific antiserum. Thus,
fluorescent antibody was introduced as a specific immunological
method of detecting an antigen. The procedure became known
as the direct method of FA staining.
Problems were encountered in staining tissue sections.
The tissues emitted blue auto-fluorescence, the same color
10
emitted by anthracene-antibody conjugates reacted with
antigens. Coons et al. (1942) turned their attention from
anthracene derivatives to the fluorescein derivative,
fluorescein-isocyanate (FIc). FIC offered an excellent
fluorochrome because it fluoresced a brilliant green, and
the wavelength of its emitted light was between 510 mu and
540 myu, a wavelength to which the human retina is most
sensitive. The chemical structure of FIC is shown in
Figure 1.
Pneumococcal antigen was stained in this experiment
with FIC-antipneumococcus conjugates placed on tissues that
contained the antigen. The outstanding accomplishment of
the experiment was that of overcoming auto-fluorescence in
tissue sections with the use of a new fluorochrome, FIC.
World War II caused a lapse in the progress attained
in fluorescent antibody from 1941 until 1945. There were
no reports in this scientific area during this time.
Advances in Fluorescent Antibody
Coon et al. (1950 and 1951) published a series of five
papers which are now classic in immunofluorescence research.
Through their efforts fluorescent antibody was established,
11
OH .00
COOH
N =C==0
Fig. l---The chemical structure of fluorescein-iso-cyanate (FIC).
12
without doubt as an efficient scheme by which cytochemical
staining of a particular antigen with fluorescein-labelled
antiserum could be demonstrated. The integrated works of
these investigators displayed how appropriate controls were
established, and a method was found for the removal of non-
specific staining. The latter technique involved the
adsorption of conjugates with tissue powders which removed
many unwanted antibodies.
Improvements and refinements in the field were con-
tributed by numerous investigators following the classical
works of Coons et al. (1950 and 1951). Marshall (1951)
showed that foreign antigen could be differentiated from
native antigen. Marshall also made several other key
contributions to FA including the embedding of tissue in
paraffin, the use of a dark-field condenser for microscopic
examination of an FA preparation, and the storage of
fluorescein-isocyanate in acetone which prolonged its shelf-
li fe.
Marshall (1951) substantiated that labelled antiserum
was made more specific if it was adsorbed with an 'offending
organism. The result was the removal of undesired antibodies.
Subsequently, there was no loss of the staining ability of
13
of a conjugate treated in this manner.
Weller and Coons (1954) introduced.a revolutionary
achievement when they devised a new method which demonstrated
antigen-antibody complexes. The technique was called the
"sandwich" or indirect method. The antigen was overlayered
with unconjugated antiserum and this complex was made
fluorescent by addition of a species specific antiserum
labelled with FIC. The antiserum was produced in a rabbit
against human globulin. The introduction of this method
made possible the use of a single labelled-anti-antibody
which stained a variety of antigens. Hence, the groundwork
was laid which resulted in the broadening of the application
of fluorescent antibody.
FA drew widespread acclaim as many independent applications
were reported. The list of organisms that were identified
by this method increased which demonstrated it as an excellent
serological and taxonomic tool.
Goldman (1953) was the first to utilize the FA tech-
nique in a diagnostic manner. Species specific antiserum
was employed by him in order to differentiate Endamoeba
histolytica from Endamoeba ccli. Thus, Goldman showed
that fluorescent antibody was a versatile procedure as he
14
had applied it to protozoology for the first time.
Moody, Thomason, and Goldman (1956) performed studies
with Malleomyces pseudomallei which rendered unequivocal
evidence that immunofluorescence was a most sensitive
immunological test. The investigations of these workers
pointed out four pertinent factors of FA. The four factors
were: (1) the agglutination titers of antiglobulins were
decreased after they were conjugated to FIC, (2) as few as
220 cells per ml were detected and observed, (3) a much
smaller number of organisms were needed for detection by
FA as compared to the number required to visibly observe
agglutination, (4) and specific inhibition of fluorescence
was acquired by adsorption of the labelled-antibody with
homologous cells. Moody et al. (1956) described a similar
method for inhibition. This was performed by adding labelled
and unlabelled homologous antiglobulin.
Moody, Thomason, and Cherry (1957) used Salmonella
flphosa as a model organism and performed an FA study.
Various members of Salmonella were differentiated by this
technique. The study demonstrated and proved that particular
antigenic classes can be differentiated by the use of
specifically prepared and labelled antiserum. Also, these
15
results pointed out that specificity was altered by laboratory
manipulation.
Riggs et al. (1958) described the synthesis of two new
fluorochromes, fluorescein-isothiocyanate (FITC) and tetra-
ethylrhodamine-B. FITC has been used almost exclusively
as the dye of choice for the conjugation of antibody for
the FA technique, and it has not been paralleled to the
present time (Goldman, 1968).- The chemical structure for
FITC is shown in Figure 2.
FITC was superior to FIC because it was more stable,
and fluoresced with a greater brilliance and intensity.
The presentation of tetraethylrhodamine-B offered a compound
with an entirely different fluorescent color. Tetraethyl-
rhodamine-B emitted an orange color when exposed to ultra-
violet illumination, and conjugated to protein, it proved
to be an excellent counterstain. The use of this fluoro-
chrome eliminated the necessity of adsorption.
Chadwick, McEntegart, and Nairn (1958a and 1958b)
developed another superior counterstain, lissamine-rhodamine--
200, which was formed by the conjugation of protein to the
disulfonic acid derivative rhodamine-B. Cherry and Moody
16
HO 0.
COOH
Fig. 2--The chemical structure of fluorescein-iso-thiocyanate (FITc).
17
(1965) used this orange-fluorescing counterstain, and found
it a more satisfactory manner to alleviate cross-reactivity
than adsorption.
Marshall, Eveland, and Smith (1958) confirmed the
findings of Riggs et al. which offered substantial proof
that FITC was very superior to the original fluorochrome
of Coons, FIC. The isothiocyanate derivative was not only
shown to fluoresce more intensly, but it was also demonstrated
that proteins were not denatured when conjugated to FITC.
Riggs, Loh, and Eveland (1958) revealed a majority
of non-specific staining was eliminated when only the
globulin fraction of the antiserum was conjugated to the
fluorescent dye rather than the entire antiserum. Addition-
ally, these workers showed that the conjugate, if placed on
a DEAE cellulose ion exchange column, was recovered in
excellent condition due to the mild conditions offered by
that type material. Also, the column removed the unreacted
fluorescein. The sodium chloride concentration was found
to play an important role in the elution process. Non-
specific staining was observed if the sodium chloride' con-
centration exceeded 0.15 molar.
18
Spendlove (1966) found that pH and dye-protein ratios
played an important role in conjugational procedures. The
fluorescein-globulin ratio was determined to be twenty
milligrams of FITC per gram of protein. A pH of 9.5 facili-
tated a more rapid and uniform labelling of the globulin.
Thus, the earlier findings of Curtain (1958) were verified
as he reported protein molecules which carried the heaviest
load of fluorescein possessed the greatest amount of non-
specific activity. To this point, there were still some
obstacles encountered when FA was applied as a means of
immunochemical diagnosis,. but these were reduced appreciably
since the technique was originally introduced by Coons et
al. (1941).
Brooks (1964) showed that fluorescein-isothiocyanate
stained as much as sixteen-fold greater than the next best
dye, lissamine-rhodamine-B-200. Hiramoto et al. (1964)
confirmed these findings. Thus FITC was determined to be
the best dye to conjugate to immunoglobulins.
Application of FA to Various Areas
Immunofluorescence has been used to study protozoans,
viruses, pathogenic bacteria, and various fungi. The most
19
progress has been made in the last decade because of the
refinements of the FA microscope.
Protozoa
The initial studies on protozoans described by Goldman
(1953 and (1954). Other reports on the use of FA to identify
several different protozoans followed. Ingram et al. (1961)
utilized fluorescent antibody and detected antibodies against
Plasmodium sp., Sodeman. and Jefferey (1964), and Andrade
(1961) worked with and identified Shistosoma sp., while
Filho et al. (1965) demonstrated the presence of antibodies
against S. mansoni in infected mice. Fife (1959), and
Essenfeld and Fennell (1964), successfully diagnosed infections
due to Trypanosoma cruzi.
Viruses
Although viral studies have been complicated many times
by contaminating antigens, immunofluorescence has been used
rather extensively in the investigation of viral antigen and
antibodies. Liu and Coffin (1957) identified the causative
agent of canine distemper with FA. Liu (1956) and Coons
(1956) detected the influenza virus and used FA. Rous sarcoma,
a letal virus in chickens, was found by Mellors and Munroe
20
(1963). Chu et al. (1951) identified mumps, Goldwasser
(1958) detected the viral agent responsible for rabies, and
Enders (1951) demonstrated the measles virus with the
utilization of immunofluorescence.
Pathogenic Bacteria
Fluorescent antibody has played an important role in the
diagnosis and identification of some of the pathogenic bacteria.
Boothroyd and Georgala (1964) identified Clostridium botulinum,
Jaeger et al. (1961), and Moody and Winter (1959) detected
Pasteurella tularensis and P. estis, respectively. Haglund
et al. (1964) reported the detection of Salmonella thosa
in eggs and egg products. Other investigations on Salmonella
typhosa were recorded by Thomason (1965) and Thomason, Cherry,
and Moody (1957).
FA has been a most valuable immunological device in the
serodiagnosis of syphilis. A number of reports gave credit
for the rapid identification of antibodies produced against
the syphilis spirochaete to the technique of fluorescent
ant ibody.- De acon and Hunter (1962) , Deacon et _al . (195 7) ,and Deacon, Freeman, and Harris (1960) reported the. successful
utilization of FA in detecting antibodies against 2reponema
21
pallidum.
Moody et al. (1958) demonstrated the difference in
various streptococci and used conjugated antibodies that
were species specific. Kaplan (1958) showed the localization
of streptococcal antigen in mouse tissue with FA technique.
Staphylococcal organisms were examined by a number of
investigators. Komminos and Tomkins (1963) worked out a
method to eliminate cross-reactivity of staphylococcus.
Cohen et al. (1961) demonstrated that antibodies against
Staphylococcus aureus occurred as natural constituents in
the serum of 'non-immunized animals. Cohen and Oeding (1962)
developed several specific serological reagents for FA
investigations of S. aureus.
De Rapentigny and Frappier (1956) detected the surface
antigens of Hemophilus ertussis by means of FA. Moody
and Jones (1963) identified Corynebacterium diphtheriae
and used fluorescent antibody reagents. yoba cterium
tuberculosis was studied by the utilization of FA by
Shepard and Kirsh (1961) .
22
Various Funi
Padula and Vogel (1958) utilized the direct staining
method and detected antibodies of various pathogenic fungi.
Kaplan and Ivens (1960) showed that Sporotrichum schenckii,
a pathogenic fungus, was identified from cultures and clinical
materials by immunofluorescence. Al-Doory and Gordon (1963)
differentiated Cladosporium carrionii from Clad oium
bantianium by FA procedures.
Kaufman and Kaplan (1963). characterized the antigenic
relationships between yeast and mycelial forms of Histo-
plasma capsulatum and Blastomyces dermatitidis. Kaufman
and Brandt (1964) differentiated _istoplasma capsulatum
from morphologically similar fungi by the incorporation of
fluorescent antibody reagents. Kaufman and Blumer (1966)
performed a thorough study on the various serotypes among
the different strains of Histoplasma capsulatum. Species
specific antisera were employed in the experiment.
Streptococcus faecalis
Cultural Characteristics
S. faecalis is a member of Lancefield's group D entero-
cocci. The organism inhabits the human intestinal tract
23
and is therefore, invariably in the feces of man (Frobisher,
1962). The organism can be identified by the use of various
biochemical tests and by morphological and microscopic
examination.
Some biological characteristics of S. faecalis, as
cited in Bergy' s Manual of Determinative Bacteriology,
(ed. 7), are as follows: growth in brain-heart infusion
broth at 10 C and 45 C, growth in sodium chloride at a
concentration of 6.5 per cent, hydrolysis of arginine with
the liberation of ammonia, growth in brain-heart infusion
broth at a pH of 9.5, growth in methylene blue milk (0.1%),
is not lysed by bile salts (40%), and is tolerant to a
temperature of 60 C for thirty minutes.
Other characteristics that have .been reported by various
investigators are as follows: the organism ferments sorbitol,
it decarboxylates tyrosine (Collins, 1967), grows in
potassium tellurite at a concentration of 1:2500 (Papa-
vassiliou, 1962), human isolates fail to ferment raffinose,
reduces 2, 3, 5-triphenyltetrazolium chloride to triphenyl-
formazan (Slanetz and Bartley, 1960), and is resistant to
both penicillin and the sulfonamides (Zinsser, 1952).
24
Streptococcus faecalis-the Indicator system
Fecal streptococci have not been used as indicators
of pollution in the United States. However, these organisms
have been used routinely in bacteriological analysis of
water in Great Britain (Litsky, Mallmann, and Fifield, 1955).
The presence of S. faecalis has been shown to be
indicative of human pollution due to its apparent constant
inhabitance of contaminated water (Burrows et al., 1968).
According to Slanetz and Bartley (1960), S. faecalis has
been isolated only from excrement of humans. However, Mead
(1965) has claimed that this enterococcus has been found
occasionally in the feces of dogs. Mundt (1963) has stated
that the human harbors S. faecalis, and Frobisher (1962) _
has inferred that this organism has been found rarely in
any but human feces.
There is some disagreement among workers as to the
significance of the presence of fecal streptococci in water.
The exact identification of S. faecalis is complicated by
the existence of atypical forms, i.e. S. faecalis var.
2iquefaciens, and atypical strains which hydrolyse starch.
Such organisms may grow commensally on plants and reproduce
in soil (Mundt, 1962), and in periods of runoff, are washed
25
into water reservoirs (Geldreich and Kenner, 1969).
Investigators who favor the use of the coliforms as
the index organism contend that (1) the coliforms exist
in larger quantities in water than the enterococci, and
(2) the coliforms are less fastidious in their nutritional
requirements, and can therefore be isolated more easily
than the fecal streptococci. The latter contention is
refuted by the fact that several excellent media have been
developed for the isolation of the enterococci.
Factors favoring the employement of S. faecalis as
the indicator system are: (1) this organism does not repro-
duce in polluted water (Geldreich and Kenner, 1969), (2)
the organism does not reproduce in sewage effluents, or
in stored samples (Weaver and Morris, 1954), (3) recently
polluted water is more strikingly shown by the streptococci
than by the coliforms (Leninger and McClesky, 1953), (4)
the enterococci do not survive for a long period in soil
(Mallmann and Litsky, 1951), (5) the enterococci are short-
lived in open-waters, but they exist for a longer period of
time in heavily polluted waters (Frobisher, 1962) , and (6)
the coliforms are long-lived in all waters, their origin
being at least as doubtful as the streptococci.
26
The use of S. faecalis as an indicator of fecal pollution,
on the basis of the above discrepancies of source, can be
questioned. However, the fact that this organism, in contrast
to Escherichia coli, does not reproduce in polluted waters
or sewage effluent, provides a possibility of quantitation
in pollution studies which is not reliable in the use of
E. coli, like S. faecalis, is subject to variation in strains,
and the separation of fecal coliforms from non-fecal forms
has been the subject of a number of studies in the past
several years. The Standard Methods (12th ed., 1965) now
used to biochemically define and quantitate coliform pollution
in water require forty-eight to ninety-six hours to complete,
and quantitation value is questionable in light of possible
reproduction of coliforms in water sources.
A reliable method which will permit a reduction of
time required for testing, and which will. give greater
accuracy in terms of quantitation would be of considerable
value in the bacteriological analysis of water.
An accurate, sensitive method which offers great
specificity in the rapid identification of bacteria has
been thoroughly tested with a variety of organisms. The
use of fluorescent-tagged antibodies to identify and
27
quantitate bacteria, makes it possible to detect small
numbers of any bacterium for which specific antibody may
be prepared (Thomason, Moody, and Goldman, 1956; Al-Doory
and Gordon, 1963; Goldman, 1968). The use of this method
for bacterial identification has been largely confined to
clinical diagnosis, however, a membrane filter-fluorescent
antibody (MFFA) method for use in water studies involving
E. coli, was described by Guthrie and Reeder (1969).
From this background, the following study was undertaken
to test the feasibility of using fluorescent antibody methods
to provide rapid identification of S. faecalis with sufficient
specificity to provide detection of strains representing
fecal pollution, thus improving the accuracy of the test -
by separation of these organisms from saprophytes which may
be present in water supplies.
Cell Wall Analysis
Various strains of bacteria have been identified by
the FA technique, however, this technique has not been
applied in the specific identification of enterococcus,
S. faecalis.
Furthermore, there have been no previous reports
published concerning the FA staining ability of this bacterium
28
or of its related members of Lancefield's group D strepto-
cocci. Fluorescent antibody has not been used to detect
fecal pollution rapidly with the use of S. faecalis as the
indicator.
The investigation of the cell walls of various strains
of enterococci utilized in this study.was considered to be
a pertinent part of the research. It was decided that an
analysis of the. cell wall might reveal at least a partial
explanation of some of the results that were obtained in
the course of the experiment.
It was noted that two isolates biochemically identified
as S. faecalis failed to exhibit fluorescence after they were
treated with specific anti-S. faecalis serum. S. faecalis
var. licuefaciens, another member of group D, also failed
to stain with the same antiserum. Further biochemical
tests on the two isolates revealed that these strains were
atypical, i.e., they hydrolyzed starch.
The interesting and pertinent point inferred at this
point is that several organisms within the same group,
the group D streptocccci, were observed to manifest different
FA reactions. These differences were determined rapidly
by immunofluorescence. These same organisms, if determined
29
by conventional bacteriological tests, would cause .erroneous
levels of pollution to be reported.
The noted differences in staining reactions were
possibly due to differences in antigenic constituents located
in the cell wall. These cell wall components were shown by
several investigators to be composed of various amino acids,
sugars, and amino-sugars.
Cummins and Harris (1956) were the first to report the
use of cell-wall analysis as a possible taxonomic aid. They
determined the cell wall composition of Corynebacterium.
The technique has undergone little transition since that
time (Gibbs and Shapton, 1968).
Maxted (1948) showed that acid hydrolysis of the cell
walls of various streptococci yielded amino acids. McCarty
(1952) demonstrated that trypsin-treated cell walls, which
were enzymatically hydrolyzed by an extract from Streptoyces
albus, contained amino acids.
Salton (1953) used paper chromatographic methods with
the acid hydrolyzates from streptococcal cell walls, and
reported the presence of large amounts of alanine,: glutamate,
and glucosamine. Harris and Cummins (1956) obtained similar
results. In addition, they found lysine, glycine, and
30
diaminopimelic acid.
The presence of various sugars has been shown in the
cell walls of Streptococcus. The analysis of cell-wall
components has been performed mainly with the group A
hemolytic streptococci due to their ability to produce
disease. Analysis of the group D cell walls has been rather
limited.
Strange (1956) advanced a formulation for a new compound
that was isolated by Strange and Powell (1954). The compound,
an amino sugar, was called muramic acid. Cifonelli and
Dorfman (1957) claimed that they found muramic acid in
streptococcal cell walls.
Hayashi and Barkulis (1958) showed that muramic acid
was present in the cell walls of group A streptococci.
They also reported that the group specific polysaccharide
constituted 50-60 per cent of the dry weight of trypsin-
treated cell walls. This polysaccharide consisted of glutamic
acid, lysine, and alanine which were attached to a rham-
nose-hexosamine polymer.
Jones and Shattock (1960) demonstrated that the group
D specific polysaccharide was located in the cell membrane
fraction rather than the cell wall. The exact chemical
31
nature of the group D antigen was uncertain, but it was
shown to contain some protein and some carbohydrate. Elliot
(1959) showed, also, that the group specific polysaccharide
was .found in the cell and not the cell wall.
Slade and Slamp (1962) examined the acid hydrolyzates
of all groups of streptococci. They reported that seven
of eight group D enterococci examined, contained the sugar
galactose. Rhamnose and glucose were found in all of the
strains examined. Amino acids and amino-sugars from group
D strains were not analysed.
It was thus of interest to examine the cell walls of
several of the group D organisms utilized in this study in
an attempt to demonstrate the reason for the observed
differences in immunofluorescent reactions. The results
and explanations of this part of the research are given in
the chapter on results and discussion.
CHAPTER III
MATERIALS AND METHODS
Bacterial Strains Employed in the Study
The bacterial strains employed in this study were
obtained from the stock culture collection of North Texas
State University, Southwestern Medical School, and Baylor
School of Dentistry. The stock cultures at North Texas
Sate University have been accumulated from a variety of
sources, so an explanation of the nomenclature of the stock
organisms is necessary.
The following categories were used. ATCC denotes those
cultures that have been procured from the American Type
Culture Collection, Rockville, Maryland. The initials MCS
signify that the culture was acquired from the Midwest
Culture Service, Terre Haute, Indiana. MC stands for the
McBryde Collection, which contains cultures that have been
added to the stock culture collection by Dr. J.B. McBryde,
a former professor in the Biology.Department at North Texas
State University. NT appears.on some of the cultures that
have been obtained from diverse sources. Several additional
32
33
cultures have been obtained from Southwestern Medical School,
designated with the letters SW. Baylor School of Dentistry
cultures are labelled BD. The final group of cultures,
those denoted by the letters TR, are bacterial strains that
have been isolated from the Trinity River and biochemically
identified during this investigation. Approximately four-
hundred such isolates have been utilized in this study. The
organisms that were specifically dealt with are listed in
Table I.
The initial phase of this research utilized these
cultures in two approaches. First, several of the strains
of the fecal enterococci were made into vaccines and injected
into laboratory animals for the sole purpose of producing.
specific antisera. Second, these bacterial strains were
employed as known antigens in both tube agglutination tests
and fluorescent antibody reactions. The purpose of this
facet of the study was to determine the agglutinin and
immunofluorescent titers of each antiserum with the homologous
antigen. Also, the existence of any cross-reactivity between
the specific antisera and heterologous antigens had to be
determined. These factors needed to be defined prior to
further investigations.
34
TABLE I
STRAINS OF BACTERIA EMPLOYED IN THE STUDY
Strain Designation Source
Escherichia coli 11303" a "
Escherichia coli 128...... .Escherichia coli 10586-. ......-.-.-... AEscherlchia coi 4157.............Escherichia coli 11775.. ...... .----- ATFlavobacterium arborescens 4558 - . ATAerobacter aeroc enes 1. . . . .. ---Klebsiella nuemoniae 65....-..-..-..Pseudomonas aeruginosa 15442. - ATProteus vulgaris 13316-- ---. . . . ATSalmonella t yhosa............. .
h edla _dyenteriae."... . . ASarcina lutea. . .....--..-..-
...
Bacillus cereus 10876 .. ..-.-.-.-... ATBacillus meatarium 9885 - . . . - ATBacillus subtills 7 - -. - -. - - -Bacillus rycoides . . - . . * .a
Clostridium s oro ns ---!--.-a--a-a
Staphylococcs aureus 4774. . . . . - ATStaphylococcus epidermidis. . . a . - -Micrococcus luteus. . - -Gaffk tetragena . .*.*0. - - -Streptococcus e o nes 10782. .. AStreptococcus ajalactiae 6638.a.
treptococcus lactis 11454. . . . ATBacillus licheniformis. - - - - - --Strept coccus faecalis TRI-TR4 .
(Trinity River Isolates)
[CC
CC
[CC
[CC'CCNT
NT
CCCCMCCCMCCCCC
4CC'SCC'IC'CS'ICIC
MC
Cs
_ p? cuss facalisBDp--BD4-..,(Baylor Dental School Isolates)
S.tretococcus faecalis SWp-SW3 - .(Southwestern Medical School Isolates)
35
TABLE I -- Continued
Strain Designation Source
Streptococcus faecalis 349. . . . . ATCC(NT 145)Streptococcus faecalis 8043- - . . . . ATCC(NT 146)Streptococcus faecalis 10541. . . . ATCC
(NT 148)Streptococcus faecalis.. -.-.-.-... MCS
(NT 147)Streptococcus faecalis. . . . . . . . NI(local isolate)Streptococcus faecalis 11420- . . ATC.C
trept coccus faecalis 12984. - . . ATCCStreptococcus faecalis 14507. . . . ATCCStreptococcus faecalis 14508. . . . ATCCStreptococcus faecalis 19634. . . . ATCCStreptococcus faecalis 19953. . . . ATCCStreptococcus faecalis 19432. . . . . ATCCtetcoccus faecalis 14506-. . . ATCCstreptococcus faecalis 19433. . . . , ATCCStreptococcus faecalis 12952. . . . . ATCCtretococcus faecalis 6057 . . . . ATCC
Streptococcus faecalis 7080 . - - - - ATCCStreptococcus faecalis var. liquefaciens MCSStreptococcus faecalis var. zymogenes 6055 ATCCStreptococcusfaecium 14432 - . . - - ATC.Streptococcus bovis 15351 . . . . . ATCC
Streptococcus equinus 9812-.-0-10- - TC .Fusobacterium polymorphun 10953 - -
ATCS
SPhaeophorus necrophorus 12290 . N ATCCBacteriodes vulgatus 8482 - . - - - - ATCC
36
Preparation of Antigens
Five strains of Streptococcus faecalis, ATCC 349, ATCC
8043, ATCC 10541, MCS, and NI* were employed in the preparation
of antigens that were used in the research. These strains
were chosen at random.
Initially, these organisms were streaked from stock
sultures onto m-Enterococcus agar (Difco) plates and incubated
for twenty-four hours at 37 C. An isolated colony was
transferred to a tube containing Tryptic Soy Broth (Difco)
and 6.5 per cent sodium chloride with subsequent incubation
at 45 C for eighteen hours. Gram stains were performed as
a measure for verifying the purity of each culture. These
pure, individual cultures were used as stock cultures from
which 250 milliliter (ml) Erhlenmeyer flasks, containing
either Tryptic Soy Broth or Brain-Heart Infusion Broth (Difco)
in 100 ml quantities, could be incubated and ultimately
harvested and utilized as antigen.
The two different culture media were divided equally
into a set of three flasks each. One member of each set
was subjected to a different temperature after inoculation.
Then, the flasks were inoculated with the desired particular
strain of S. faecalis. The organisms were allowed to grow
37
at either room temperature, 37 C, or 45 C. The purpose of
this study was to see if various cultural media and temp-
erature had any influence on the antigenicity of these
enterococci that could be observed by serological testing.
Specifically of interest were tube agglutination tests and
FA tests.
Differences in opinion pertaining to the growth of
organisms in various culture media exist in the literature.
Some investigators have found that the antigenic response
produced in animals, seemingly, was no different when the
organism to be injected 'was grown on different cultural
media (Moody, et al., 1958). However, Thomason et al. (1957)
reported that the antigenic constitution of a cell can vary,
depending on the growth medium as well as other physiochemical
factors. It is of merit to mention that the latter work
consisted of preparing the various classes of antigens
found in Salmonella typhosa, and other member organisms of
this group. The antigenic components of this group have
been shown to be very complex.
All cultures were shaken on a rotary shaker at 100 rpm
(Eberbach Corporation, Ann Arbor, Michigan) for eighteen
hours at their respective temperatures. After the lapse of
38
the growth time, the cells were collected in sterile,
plastic centrifuge tubes by centrifugation in a Sorvall
refrigerated centrifuge (Ivan Sorvall, Incorporated;
Model RC2-B, Norwalk, Connecticut). Following centrifugation
for ten minutes at 8000 rpm, the harvested cells were
washed twice with physiological saline (0.85%). After the
second washing and decantation, the packed cells were covered
with twice their volume of formalinized saline (0.5%).
The cells and the formalinized saline were mixed by means
of a vortex junior mixer (Scientific Industries, Incorporated;
Model K-500 J, Queens Village, New York). Subsequently, the
cells were incubated at 37 C for twenty-four hours.
Following incubation, Gram stains were made of the
formalinized cultures. An inoculum of each culture was
transferred to Tryptic Soy Broth, or to a Tryptic Soy Agar
(Difco) plate, and to a tube of Thioglycollate medium (Difco).
This procedure was followed in order to check the viability
of the individual cultures and to be assured that no contaminat-
ing organism was present. Karawara et al. (1964) have
emphasized the importance of the purity of immunizing' antigens
in producing specific fluorescent antibody reagents.
The formalin-killed cultures were transferred to vaccine
39
bottles and stored in a refrigerator at 4 C until they were
diluted to the proper concentration of bacterial cells for
injection into test animals. Contamination of a finished
vaccine was encountered in one instance, in which case the
vaccine was discarded. No viable cells were present after
exposure to formalinized saline and incubation for twenty-four
hours at 37 C.
Immunization Schedule and Antisera Production
Rabbits, weighing approximately three to four kilograms,
were bled from the heart by means of cardiac puncture. A
small aliquot of the animal's serum was pre-titered for the
presence of antibodies against the particular strain of
S. faecalis that was to be injected into the animal in
question. If a titer of 1:8 or greater could be demonstrated
by tube agglutination, this animal was not used for sub-
sequent antiserum production. A total of fifteen test
animals (rabbits) were injected for the study.
The excess non-immune sera obtained from the pre-titer
bleeding that displayed no agglutination titer for the
definite test organisms was pooled and frozen so that it
could be used as a normal rabbit serum (NRS) for negative
controls in succeeding fluorescent antibody tests. Agglutinin
40
titers were also performed on all NRS for the presence of
antibodies against Staphylococcus aureus because such
antibodies are virtually always present in laboratory
animals, especially rabbits (Bergman et al., 1966; Moody
and Jones, 1963).
The injection schedule was essentially that of Campbell
et al. (1964). However, due to poor previous responses
in test animals to challenge dosages, it was decided that
it was necessary. to increase the dosage of antigen injected
in order to obtain a high titered antiserum.
In an earlier experiment, the somatic antigen preparations
had been subjected to a temperature of 100 C for a one hour
period and to two thirty-minute periods with intermittent
washings. This method was described by Edwards and Ewing
(1957) in the preparation of non-viable antigens for the
members of the family Enterobacteriaceae
The resultant poor response could have been due,
possibly to the denaturation of the specifically desired
antigenic protein resulting in the lowered agglutination
and FA titers that were observed.
In the earlier experiment, the animals were injected
with 9.0 x 10 cells per ml as determined comparatively
41
with #3 McFarland standard nephelometer tube. Agglutination
titers and FA titers were much lower than those acquired
in the following experiment.
In this investigation, formalin-killed vaccines were
utilized in lieu of heat-killed preparations, and the
injection dosages were increased to 1.8 x 109 cells per ml
as determined by comparison to a #6 McFarland standardized
tube. Dilutions to the appropriate density of cells were
made from the stored stock. Sterile saline was used as
the diluent.
Extraordinary increases in agglutinin titers were
noted in every fraction of the antisera tested. The whole
antiserum, the globulin and labelled antiglobulin, were
included in the tests.
Since the protocol for injection as described by
Campbell et al. (1964) was relatively unsuccessful, it wasdecided that the quantity of the dosages injected would be
increased in conjugation with the increase in the density ofcells -per ml. The injections were administered intravenouslyinto the marginal vein of the ear. The immunizing.schedule
and dosage of antigen injected were as follows: (1) injected
one ml on the first day, (2) injected two ml on the fifth
42
day, (3) injected three ml on the ninth day, (4) injected
four ml on the thirteenth day, (5) injected four ml on the
seventeenth day, (6) trial bled and titered on the twenty-
first day, (7) on the twenty-third day the animals were
sacrificed and blood obtained if titer was sufficient, and
(8) if an insufficient titer was demonstrated, a booster
injection was given.
It is of interest to note that Goldman (1968) has
stressed that agglutination titers of 1000, or greater,
must be obtained to insure strong fluorescence when using
immune sera for fluorescent antibody staining.
Bleeding of the Animals
The animals were anesthetized with ether and bled by
means of cardiac puncture. The blood was collected in
sterile test tubes which were stoppered and evacuated with
a vacuum pump (Precision Scientific Company, Model 25, Chicago,
Illinois). The blood was allowed to clot and stand at room
temperature for thirty minutes after which time the clot was
removed, and then the tubes were placed in a refrigerator
at 4 C overnight. The clots were removed and each antiserum
was carefully separated from it's cellular components by
centrifugation at 2500 rpm for ten minutes in a clinical
43
centrifuge (International Clinical Centrifuge, Model CL,
I.E.C., .Needham Heights, Massachusetts). The immune sera
were frozen at -20 C until their specificity could be
ascertained.
Protein Determinations
Protein determinations were performed on each anti-
serum by the Biuret method as described by Gornall et al.
(1949). Samples were compared to a reference curve determined
by the different concentrations of bovine serum albumin
(BSA). A reference curve is illustrated in Figure 3.
Protein concentrations varied from 53 mg per ml to 72
mg per ml on the various antisera obtained from the test
animals. The concentration of each individual antiserum
was standardized to 50 mg of protein per ml, and these data
were used to calculate the dilution ratio of a particular
antiserum. A protein concentration of 1 mg per ml was
sufficient to obtain maximum staining in the indirect method
of Weller and Coons (1954), and in the direct staining
procedure of Coons et al. (1950). However, a concentration
of a 2 mg per ml was employed uniformly throughout the study
to insure that a sufficient amount of antibody was present.
This density of protein accorded brilliant fluorescence
44
If)
E0@
0
z
w@
MILLIGRAMS OF PROTEIN PER MILLliTER
Fig. 3--Standard protein curve as determined wt oiserum albumin (BSA) by the biuret method .
45
(4+ reactions) repeatedly for the entirety of the testing.
Homologous and heterologous antigens were reacted with this
concentration of protein in all antisera investigated.
Each antiserum was categorized for comparative reaction
purposes. A fraction of each antiserum was used as whole
antiserum which was applied to the various antigens tested
in the indirect method. A portion was fractionated in order
to obtain only the globulin component which was also utilized
in the indirect staining procedure. The third category was
that of labelling the globulin fraction of the antiserum
with fluorescein-isothidcyanate as prescribed in the direct
test.
Preparation of the Globulin
The globulin fraction of a portion of each antiserum
was precipitated by two different methods in an attempt to
compare the recovery of the globulin and the retention of
immunochemical activity after having been subjected to the
process of fractionation.
One method of precipitation was by the addition offully saturated ammonium sulfate, i.e., 800 grams per liter
at a room temperature of 23 C as described by Campbell (1964
46
In this procedure, the pH of the ammonium sulfate is adjusted
to 7.5 with 1N sodium hydroxide prior to its addition to
the whole antiserum as suggested by Spendlove (1966). After
three precipitations, dissolution of the globulin was carried
out with physiological saline (0.85 per cent), using a
volume of saline equal to one-half that of the original
volume of the antiserum. The globulin was placed in a
dialysis bag, and dialysed against phosphate buffered saline
(PBS) having a pH of 7.2. This buffer was changed three
times daily for a period of two to three days, or until no
sulfate ion could be detected in the globulin. A small
aliquot of the globulin was made acidic with IN hydrochloric
acid, then a few drops of a 2 per cent solution of barium
chloride were added dropwise, dialysis being considered
complete if no precipitate was formed. All globulin fractions
had protein determinations run on them so that fluorescein-
isothiocyanate (FITC) to protein ratios could be resolved.
The chemical configuration of both the fluorochrome and theconjugate are given in Figures 1 and 2, as shown on pages
11 and 16.
A second method consisted of fractionation of theglobulin with 33 1/3 per cent ammonium sulfate, and the pH
47
was not adjusted until the salt had been added to the anti-
globulin. This procedure was employed for comparative
purposes.
Conjugation of Antiglobulin to FITC
The antiglobulins were conjugated to FITC (Nutritional
Biochemicals Corporation, Cleveland, Ohio, Control 2096) at
a rate of 20 mg FITC per gram protein, as recommended by
Spendlove (1966). The protein concentrations were adjusted
to 50 mg per ml; however, in some instances the protein wasless, in which situations the proteins were dehydrated and
concentrated by placing the globulin in a dialysis tube,
covering it with polyvinylpyrrolidinone K-30 (Matheson,
Coleman, and Bell, Cincinnati, Ohio), and placing it in arefrigerator at 5 C for approximately three hours.
Conjugation consisted of reacting the cold globulin
with a solution of FITC in 0.1 M dibasic sodium phosphate(Na 2 HiPO4 ) at a rate of 1.25 mg FITC per ml of Na2npo4 as
discussed by Collins (1967). The reaction was allowed toproceed for thirty minutes to an hour at room temperature
with gentle stirring by means of a magnetic stirrer.. (E. H.Sargent & Co., Dallas, Texas). According to Spendlove (1966),
48
this allowed sufficient time for reaction between the dye
and the antiglobulin. The protein was diluted to 25 mg
per ml as the pH was adjusted to 9.5 with 0.04N sodium
hydroxide (NaOH) and Na2 HPO4 .
The conjugate was placed on a Sephadex G-50 column
(Pharmcia, Uppsala, Sweden) with subsequent removal of the
extraneous dye that had not reacted with the antiglobulin.
Previous investigations have shown that heavily labelled
globulins exhibited much unwanted non-specific fluorescence
(Cherry et -al., 1960, and Goldstein et al., 1961, and Curtain,
1958). Hence, most of the non-specific staining was removed
by passage of the conjugate over a Sephadex column.
Collection of the conjugate, as it was eluted from
the column, was facilitated with a fraction collector
(Micro-Chemical Specialities Co., Berkeley, California,
Model 6550). The conjugate was collected in aliquots of
two ml and diluted to a protein density of 10 mg per ml.
The diluter conjugate was stored in a freezer at a temper-
ature of 20 C until it was needed in test procedures. These
aliquots aided conservation of the conjugate, since anydesired amount was diluted into a working solution. Any
remaining portion was re-frozen and used in subsequent tests.
49
Working solutions of the conjugates consisted of a
1:5 dilution of the stored protein-dye complex. Thus, the
working solutions consisted of 2 mg per ml protein, a
concentration that proved to be satisfactory for intense
fluorescence when reacted with it's homologous antigen. This
constituted a positive control, and one was set up each
time a staining scheme was executed. Working solutions were
prepared on the day they were to be utilized in staining
smears in all critical examinations and evaluations of
slides. However, such preparations were used on several
instances when they had 'been stored in the refrigerator
for a period of ten days. Homologous antigens were still
stained intensely after this storage period.
The fluorescent antibody staining titers of each
conjugate were derived by adding various dilutions of a
known protein concentration of the conjugate to a slide
prepared with the known homologous antigen. These titers
are shown in the chapter on results.
Agglutination titers were compiled concomitantly sothat possibly some correlation could be drawn between' thisserological technique and that of fluorescent antibody.
Various authors have contended that no correlation can be
50
made between agglutination titers and FA titers (Thomason
et al., 1965; Moody etal., 1958; Eldering et al., 1957).Eldering et al. (1957) hypothesized that different serum
components may be responsible for agglutination and FA
staining.
It should be noted at this point that a protein
concentration of 2 mg per ml was used in both the direct
and indirect methods of immunofluorescence. This density
of protein produced brilliant fluorescence and provided
a method of uniform protein to dye relationship. The
method of using a standard protein concentration aided
storage, conservation, and dilution of the stain as it was
used in staining slides. The uniform dye to protein ratio
facilitated the comparison of the results that were procured
from the direct and indirect methods.
Agglutination Reactions
Agglutination is one of. several serological methodsfor determining and observing antigen-antibody reactions.
It is the most simple serological test to perform, and italso has the widest range of usefulness (Mallen and Cuellar,1949). Agglutination tests were performed on all of theimmune and non--immune sera that were employed in this research.
51
It served as a monitoring device by which the presence
of various antibodies could be detected.
A two-fold dilution scheme was maintained throughout
the study as clear cut end points of titers were determined
more easily. The antisera were diluted in order to
conserve them. Dilutions were performed with saline
(0.85 per cent) and a constant amount of antigen was added
each time. The densities of the antigen were made and
compared with a number 3 McFarland nephelometer (9.0 x 108
cells per ml). Saline controls were set up simultaneously.
The titers were recorded as the reciprocal of the dilution
factor of the antiserum. These data are shown in a later
chapter.
Agglutination titers do not necessarily parallel PAtiters (Thomason et al., 1964; Moody et al, 1958--- ',,oy .- tt.- . , 95 ;Karawara,
1964). fHowever, it is a means by which antisera can beexamined to determine the presence, or absence, of certain
antibodies.
Preparation and Staining of Slides
The preliminary phase of this study involved invst-igation to determine if antiserum against S. faecalis couldbe produced and procured (from rabbits) that would contain
52
a high enough titer to stain it's homologous antigen and
emit intense fluorescence when excited by a stimulating
beam of light from a mercury arc bulb. In so doing, pure
known cultures of the five homologous antigens that had
been injected into test animals to produce specific anti-
sera had to be maintained in order to ascertain the
fluorescent staining abilities of these enterococci.
Cells which were stained, were grown in various media
at different temperatures in an attempt to see if such
variables would have any affect on the staining of the cells.
Each of the organisms were grown in Tryptic Soy Broth,
Brain Heart Infusion Broth, and Todd Hewitt Broth (Difco)
at 23 C, 37 C, and 45 C for varying lengths of time from
three hours to ninety-six hours. Various combinations of
these variables were set up and slides were made, stained,
and evaluated. The resultant responses of the organisms
to FA staining are discussed in the chapter on the results
obtained from the investigation.
The cultures were washed with phosphate buffered saline(pH 7.2), and the turbidity of each culture was adjusted
to compare to a number 3 McFarland tube. This gave an
ideal density of cells from which slides were made and
53
examined.
Fluoro-slides (Aloe Scientific, Chamblee, Georgia),
with marked areas for two smears, were the slides chosen
because these slides have etched markings to contain the
smear. This facilitated locating the organisms rapidly
with a microscope. One of the smears served as the test,
while the other smear served as the control. Prceeding the
fixation of the organisms onto the slides, the slides were rinsed
in acetone and dried with lint free napkins. Extraneous debris,
or lint, can lead to poorly prepared slides because anti-
factual and auto-fluorescence have been observed on un-
clean slides. The interpretation of results has been made
more difficult in many instances because of extraneous debris
being left on slides.
The direct method of Coons and Kaplan (1950), and
the indirect method of Weller and Coons (1954), were executed
to determine which of the tests would be better in
detecting S. faecalis by fluorescent antibody. Schematic
diagrams of these two methods of immunofluorescent staining
are shown in Figures 4 and 5.
54
c --
Culture of Antigen
Fluorescent Complex
Antigen-antibody Complex
Ant icgen
Vaccine
Production of antiserum
5-C &SH-N NNH
Conjugate
iAId M dnAntibodies
Figure 4,. Schematic diagram of the direct method ofimmunofluorescence 0
- - 9
_
M
.,.
5'-
55
Ant ign-
Fluorescent Complex
Antibody
Antigen-Antibody An t-jntibod
Complex
.Cgn gate(anti-antibody)
Figure 5. Schematic diagram of the indirect me-hodfimmunofluorescence
56
The Direct Method
In the direct test, the test smears were reacted with
conjugated antiglobulin, and the negative controls were
simultaneously treated with NRS. The tagged NRS had no
titer for any of the strains of S. faecalis used for
injection. This was demonstrated by tube agglutination
tests.
After placing conjugated antiglobulin on the test
antigen and labelled NRS on the negative control, the
slides were icubated at 37 C for thirty to fory-five
minutes in. humudity chamber made especially for this study.
This metal box measured 12" x 6" x 3". A piece of absorbent
styrofoam was cut to fit in the bottom of the chamber to
hold moisture. The styrofoam was saturated with water, thus
humid environment was produced for the incubating slides.
This humidity chamber enhanced the immunochemical reaction
between the antigen and the fluorescent antibody. Also,
a rather substantial quantity of slides were incubated
concurrently.
The stained slides were removed from the humidity
chamber, placed in a Coplin jar, and washed twice in abuffer solution. Two different buffers were utilized for
57
comparison; one a commercially prepared phosphate buffer
(Difco) with a pH of 7.2,. and the other a laboratory
preparation that has been suggested by Pital and Janowitz
(1963) which consisted of 0.5 M CO3 and 0.5 M HC03 mixed
in approximately a 1:3 ratio to give a pH of 9.5. Both
ot these buffers were studied in the FA tests and a
comparison between the two was made.
A final wash was carried out in distilled water.
After maintaining the wash for three minutes, the slides
were removed from the wash jars and partially dried, but
the smears were not allowed to dry. Mounting fluid was
added to each wet smear. A cover slip was placed carefully
on the mounting fluid, preventing air from becoming
entrapped between the slip and the slide.
A commercially prepared mounting fluid and one mixed
in the laboratory were utilized in the investigations. The
former was purchased from Difco, and the latter was
prepared according to Pital and Janowitz (1963). The
laboratory substance was made by mixing one part glycerol
with nine parts of a carbonate-bicarbonate (C0 3-HC03 ) (pH
9.5) buffer. The pH of the Difco buffer was 7.2. Some
difference was noted in the two buffers. These differences
58
are discussed in the results.
Tne Indirect Method
The initial steps of preparing slides to be stained
by the indirect method are identical to those followed in
the direct procedure.
The test antigens were first covered with unconjugated
antisera and the controls were layered with unconjugated NRS.
In both instances, the protein concentration was 2 mg per ml
and the reaction was considered complete after the slides
had been incubated for thirty to forty-five minutes in the
humidity chamber at 37 C.
Following incubation and washing in a carbonate-
bicarbonate buffer and distilled water, the slides were
allowed to air dry. Both the test and the control smears
were overlayered with goat-anti-rabbit globulin (Difco)
which had been conjugated to fluorescein-isothiocyanate
and diluted to contain a protein concentration of 2 mg
per ml. An additional incubation period of thirty to
forty-five minutes was performed.
Rinsing and mounting of slides in the direct test were
the same as those executed in the indirect staining procedure.
59
the prepared slides were then examined and evaluated
by fluorescent microscopy.
Evaluation Values Assigned to the Examined Slides
Fluorescent values were assigned to each examined
smear. These values ranged from negative (-) to four--
plus (4+). If no fluorescence was exhibited when the
smear (antigen) was exposed to ultra-violet light, then
the slide was graded as negative. A plus-minus (+)
gradation was assessed to those slides with very faint
fluorescence, the fluorescence being questionable. The
slides that exhibited a visible, but faint reaction, were
given a 2+ value. Cells that demonstrated a bright, but
not intense, peripheral cell wall fluorescence were
conferred a three-plus (3+) reading. Maximal fluorescence
with a brilliant emission of green fluorescing light was
considered to be four-plus (4+) reaction.
Although these values are rather subjective, with
practice in examining slides over an extended period of
time, one can become quite adapted to assigning values to
slides that correspond with another examiner. In other
words, with practice these grades become more significant
60
and meaningful.
Fluorescent Microscopy and Photomicroscopy
A Nikon SKE microscope (Nikon, Incorporated; Garden
City, New Jersey), equipped with a dark-field condenser, a
97 X oil immersion objective with a numerical aperature of
1.25, and 10 X oculars was utilized to examine the slides
and evaluate them. Illumination was by an Osram HB 200 W
mercury arc bulb (Osram, Berlin, West Germany) coordinated
with a Mercury Power Supply SP-200 (Bausch and Lomb,
Rochester, New York). Incorporated into the system was
a Corning 5-58 exciter filter and a Nikon T-2 barrier filter.
Photographs were taken with a 35 mm camera (Nikon)
using Tri-X-Pan film (Eastman Kodak Company, Rochester,
New York) with exposure times ranging from two to four
minutes. Some of the resultant photographs are shown in
Figures 5, 7, 8, and 9.
Results from this phase of the entire investigation
proved to be successful. It has been shown that S. faecalis
can be stained with specific antiserum using known cultures,
both pure and mixed. The enterococcus could be detected and
identified due to the presence of intense fluorescence, or
61
Fig. 6--Illustration of immunofluorescence by thedirect method
62
Fig. 7--Illustration of immunofluorescence by theindirect method of staining.
63
Fig. 8--Immunofluorescence of an isolate as
demonstrated by the indirect method of staining.
64
Fig. 9--A fluorescent-antibody reaction obtained
from cells taken from a five hour broth culture.
65
by the total absence of fluorescence after the employment
of various immunological manipulation. These included
adsorption and preinhibition techniques. The use of these
procedures have been used and cited by a number of workers
in the field (Bergman et al., 1963; Moody and Jones, 1965;
Moody et al., 1956; Goldman, 1956; Moody et al., 1958;
Eldering et al., 1957 and 1962).
The Adsorption Method of Removing Unwanted Antibodies
The presence of unwanted antibodies which result in
cross reactions between a specific antiserum and an antigen,
or a group of antigens in a mixed preparation, presents an
almost insurmountable problem in fluorescent antibody
research (Bergman et al., 1963). However, with the advent
of adsorptive techniques, this obstacle has been greatly
reduced.
The method of Moody et al. (1958) was employed for
adsorption. Bacterial cells, containing the cross-reacting
antigen were grown overnight in a broth culture (Brain
Heart Infusion, Tryptic Soy, or Todd-Hewitt) at 37 C. These
cells were treated with 0.5 per cent formalin solution to
render them non-viable. The formalin solution and cells
66
were incubated for twenty-four hours at 37 C and washed
three times with phosphate buffered saline (pH 7.4).
After the final wash, the resulting packed cells were
mixed with an equal quantity of antiserum and incubated
for one hour at 37 C.
The cellular-antiserum mixture was centrifuged at
2500 rpm in a clinical centrifuge for ten minutes. The
supernatant liquid contained the antiserum which had been
freed of unwanted antibodies. This fraction was decanted
and preserved by freezing at a temperature of -20 C until
further testing could be conducted, with subsequent disposal
of the cells and adsorbed antibodies.
After removal of the cross-reacting antibodies, tube
agglutination tests and fluorescent antibody tests were
carried out on the resultant antiserum to determine if
the troublesome antibodies had been removed.
Pre-inhibition tests were employed in instances where
fluorescence occurred due to the preserved common antigens.
These tests served as an auxiliary method for decreasing or
removing completely unwanted fluorescence. This is made
possible due to cross-reacting antibodies binding to
heterologous.antigens.
67
In this method, the smear (antigen) was initially
reacted with normal rabbit serum (NRS) (Goldman 1956, and
Moody et al., 1956. Another method for blocking cross-
reactivity was performed by adding a small amount of un-
tagged antiderum, which was against the offending organisms,
to the smear before the addition of the specific antiserum
(Redys et al., 1960 and 1963). This procedure tied up
unwanted sites on the antigen and prevented them from
reacting when FA was added,
Application of FA to Biochemically Defined Isolates
The attention of the investigation was next focused on
the attempt to prove the applicability and validity of
the immunofluorescent technique. These were two most out-
standing criteria for evaluating this method as an
efficient serological tool.
Earlier studies showed that seventeen ATCC cultures of
S. faecalis were stained with each of five different
specific antisera that was produced for this research problem.
Additionally, three patient isolates acquired from South-
western Medical School (Dallas, Texas) and four patient
isolate strains obtained from Baylor School of Dentistry
68
(Dallas, Texas), all fluoresced after having been reacted
with the specific anti-S. faecalis serum. These seven
strains had been serologically typed as group D by each
institution respectively. This seemed to provide ample
validity to the test.
Approximately 500 strains of enterococci were isolated
and studied in an attempt to ascertain the relaibility of
the technique of rapid identification by fluorescent anti-
body. Aliquots of samples, suspected of containing entero-
cocci, were inoculated into the selective medium azide-
dextrose broth (BBL). This medium was formulated by
Mallmann and Seligmann (1950) for the isolation of entero-
cocci, and the Gram-negative organisms were inhibited.
The ingredients of this medium are given in Table II.
Several other organisms have been grown in Azide-
Dextrose Broth (BBL); therefore, the necessity of confirm-
ation existed. The confirmatory medium that was employed in
the study, m-Enterococcus (BBL), was devised by Slanetz
and Bartley (1957). The formula for this medium is listed
in Table III.
The enterococci appeared as pink or red colored
colonies on the confirmatory medium. Various biochemical
69
TABLE II
AZIDE DEXTROSE BROTH MEDIUM*
Ingredient Amount**
Polypeptone Peptone,................ 15.0Beef Extract....-...-......-......... 4.5Dextrose...................-- -...... 7.5Sodium Chloride..-......-............. 7.5Sodium Azide......................0.2Brom-Cresol Purple .-.-.-.-.-...... 0.25
*This medium was used as a presumptive test forthe isolation of Streptococcus faecalis, The typicalreaction is acid formation and the medium turns frompurple to yellow.
**Formula in grams per liter.
70
TABLE III
M-ENTEROCOCCtJS AGAR*
Ingredient
Yeast Extract...-.....
Trypticase Peptone -.--Dextrose .-.-.-.-.-....-.-Phytone Peptone.-.-.....Potassium Phosphate. .--
Sodium Azide ..-..-.--. -Agar... . . . . . . . ..Tetrazolium Chloride .
*This medium was usedTypical colonies appear as
Amount**
- - . . 5.0
- .. . 15.0
. - . . 2.0- -. . 5.0
- . . . 4.0
P i 10.0. . . . . 0.1
in the confirmatory test.pink to brick red.
**Formula in grams per liter.
71
tests were necessary to identify S. faecalis and the other
members of the group D enterococci that were isolated.
The sheme that was used to specifically identify the isoltates
is shown in Figure 10.
Following the biochemical analysis of the isolated
organisms, fluorescent antibody methods were utilized to
identify all of the isolated strains. The results of the FA
analysis are given in the chapter on results in this
dissertation. The data presented was obtained by both
the direct and the indirect methods.
Thin-Layer Chromatography
The cell-wall constituents of various strains of the
enterococci included in the study were examined. These
components were catagorized into amino acids, sugars, and
amino-sugars.
Cell walls were prepared by sonication as described
by Jones and Lewis (1966). Washed whole cells were also
used in this study. This type of prepartation was also
used by Jones and Lewis (1966) in a previous study with
Corynebacterium diphtheria and some related strains.
The cells were initially hydrolyzed with 6N hydro-
chloric acid at 105 C for a period of two hours. The
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resultant solution was -filtered through a 0. 4 5y millipore--
filter (Millipore Filter Corporation, Bedford, Mass-
achusetts) and evaporated to dryness with a flash evaporator
(Buchler Instruments, Fort Lee, New Jersey).
The residue was dissolved in two ml of a 10 per cent
solution of isopropanol and neutralized with ammonia
(0.90 specific gravity). The solution was then stored at
5 C until the analysis of the components was made. All
cell-wall hydrolyzates included in the study were treated
in the same manner.
Thin-layer chromatography was utilized to detect
sugars, amino acids, and amino-sugars. The silica gel
plates (E. Merck, Darmstadt, Germany) were prepared as
described by Gibbs and Shapton (1968), and placed in a
drying cabinet (Boekel, Philadelphia, Pennsylvania) until
they were used. Immediately before use, the plates were
activated in an oven (Aloe Scientific, St. Louis, Missouri)
at 105 C for thirty minutes.
Controls were used in the identification of the various
amino acids, sugars, and amino-sugars. The amino acid
controls were made by dissolving'O.O0M of each of the
amino acids used in a 10 per cent solution of isopropanol.
74
The sugar and amino-sugar consisted of dissolving 100
milligrams (mg) of each of the sugars used in 10 ml of
water (1 per cent) as described by Clark (1964).
The solvent system used to separate the amino acids
was propanol and water (80:36 v/v) as discussed by Jones
and Lewis (1966). The chromatograms were left in the
solvent for approximately three hours, after which time
they were dried at 105 C for ten minutes. The chromato-
grams were then sprayed with ninhydrin (Nutritional Bio-
chemicals Corporation, Clevland, Ohio), and placed in an
oven for ten minutes at 105 C. A comparison was made
between the controls and the test spots.
The solvent for resolving the sugars was 65 ml of
ethyl acetate plus 35 ml of a mixture of two volumes of
isopropanol and one volume of water. The sugars were
located on the chromatograms by spraying with either
aniline diphenylamine or anisidine phthlate (Sigma
Chemical Company, St. Louis, Missouri).
The next phase of the experiment concerned itself
with the preparation of dinitrophenyl-amino acid derivatives
(DNP). The DNP-amino acids were prepared as described by
Clark (1966). The amino acids, amino-sugars, and sugars
75
that were incorporated into the study were obtained from
the Sigma Chemical Company, or from the Nutritional Bio-
chemical Company.
The DNP-labelled amino acids were resolved with
chloroform-amyl alcohol-acetic acid (70:30:3 v/v). These
derivatives were developed by reacting them with ninhydrin.
Comparisons were made between the DNP-amino acid controls
and the DNP-amino acids that were obtained from the
hydrolyzates of cells that were reacted with dinitrofluoro-
benzene (DNFB).
Jones and Lewis (1966) described a method by which the
N-terminal amino acid can be blocked with DNFB, hydrolyzed
with 6N hydrochloric acid, and determined by thin-layer
chromatography. This method, essentially, was the one
used in this procedure.
The organisms that were studied were grown for eighteen
hours at room temperature on a rotary shaker. These same
conditions were executed when cells, in the initial phase
of the study, were grown and made into antigen preparations.
At the end of the growth period, the cells were collected
by centrifugation (8000 rpm for ten minutes) and washed
three times with sterile physiological saline.
76
The clean, harvested cells were weighed out in a
0.5 g quantities and placed in a sterile screw cap test
tube. Four such tubes were incorporated for each organism
that was tested. The cells in each tube were suspended in
a 4 per cent solution of DNFB in ethyl alcohol, and an 8
per cent solution of sodium bicarbonate. Two volumes of
the DNFB solution was added to each tube while one volume
of the sodium bicarbonate was added.
The four tubes were used in two studies. First, the
cells were allowed to react with the DNFB-sodium bicarbonate
solution for twenty-four hours. A small aliquot of the
cells were taken from the tube with a pipette, placed in
a sterile test tube, and washed four times with PBS and
cold distilled water. The packed cells were then diluted
with PBS to coincide with a number 3 McFarland nephelo-
meter standard. FA slides were prepared from these diluted
solutions. The slides were stained and examined by both
the direct and indirect methods of fluorescent antibody.
Examination and evaluation of each slide was performed to
see if the fluorescent characteristics of the organisms
were altered in any way. The remaining portion of this
experiment was concerned with the mild hydrolysis of the
77
DNFB reacted cells.
The DNFB-labelled cells were subjected to mild
hydrolysis by 6N hydrochloric acid after they were thoroughly
washed. One tube of cells was placed in an incubator for
fifteen minutes at a temperature of 37 C. This temperature
afforded only mild hydrolytic conditions. Foloowing the
incubation period, the cells were washed three times with
PBS. The initial solution which contained the DNFB was
saved and analyzed for the presence of DNP-amino acids.
Controls of- DNP-amino acids were prepared by the method
described by Clark (1964). The Rf values of these controls
were compared with amino acids as a means of determing
which ones were present in the hydrolyzates.
Also, smears were prepared from the DNFB-labeled
cells after they were thoroughly washed. These smears were
stained with FA reagents and examined for fluorescence
microscopically. The Hydrolyzates were analyzed for DNP-
amino acid (s) by thin-layer chromatography.
The remaining three tubes of each set were subjected
to thirty-minute, forty-five minute, and one hour periodsof hydrolysis, respectively. The hydrolyzates were checkedfor the presence of DNP-amino acids, and the cells were
78
stained with FA to determine if any change had occurred
in their fluorescent qualities. This procedure was uniform
for all strains that were used. The results of these data
are given in the chapter on results and discussion.
CHAPTER IV
RESULTS
Specificity of Antisera
Hoolous Reactions
The specificity of each antiserum that was used in the
experiment was established following its production. Initially,
agglutination titers and antibody specifications were
determined by reacting homologous antigen-antibody pairs.
These serological tests were performed to demonstrate the
presence of specific antibodies in each antiserum.
Each of the five different strains of S.faecalis that
was used as an antigen, and injected into test animals,
was reacted with its homologous antiserum by means of tube
agglutination tests. The same reaction was observed by the
use of both the direct and the indirect FA methods. Comparisons
of these two serological methods were made.
The results obtained from these two different immuno-
logical methods can not be correlated in every instance
(Moody et al., 1958; Karawara et al., 1964) . However, the
79
80
data obtained from this phase of the study, as shown in
Tables IV and V, demonstrated that high agglutinin titers
and intense fluorescence were obtained in all of the homologous
reactions.
Some degree of difference in staining was noted after
intrastrain cross-reactions were made. It was observed
that when S. faecalis ATCC 8043 (NT 147) was reacted with
antiserum produced against S. faecalis ATCC 10541 (NT 148)
a rather low agglutination titer was demonstrable. The
agglutination titer was 1:128. However, a high fluorescent
antibody titer of four-plus (4+) was observed.
The reverse of the above was also noted. In two
instances, a reasonably high agglutination titer was observed
with a corresponding lower FA titer. For example, S. faecalis
ATCC 349 contained a high agglutination titer for the
antiserum against S. feacalis NI. The same reaction with
fluorescent antibody demonstrated a three-plus (3+) reaction.
These findings were in agreement with previous invest-
igators (Eldering et al., 1962). As a rule, the higher the
agglutination titer of an antiserum, the more intense the
FA reaction. Also, the higher the agglutination titer and
FA intensity, the higher the, antiserum had to be diluted
81
TABLE IV
AGGLUTINATION TITERS OF HOMOLOGOUS ORGANISMS
Agglutination Titers Antiserum*Organism
145 146 147 148 NI
145 2048 256 64 512 1024
146 256 2048 128 512 512
147 64 32 8192 128 128
148 1024 1024 256 4046 512
NI 1024 1024 32 1024 4096
S. faecalis var.liguyefaciens 40 0 0: 0
S. faecalis;TR .0 8 2 0 0
S. faecalis TR7 2 0 4
TRH 64 128 128 256 512
TR2 64 64 32 256 128
TR3 256 128 256 512 512
TR4 256 256 512 256 512
82
TABLE IV ---Continued
Agglutination Titers AntiserumOrganism
145 146 147 148 .NI
T R5 128. 64 128 512 512
TR6 128 256 512 512 512
TR4 6 128 32 32 16 8
TR8 0 64 16 8 16 32
TR7 3 256 512 256 512 256
128 128 1024 512
f d lgutiration titers are expressed as reciprclsof dilutions of the various antis era.
83
TABLE V
FLUORESCENT ANTIBODY TITERS OF HOMOLOGOUS ORGANISMS
FA ReactOrganism
145 146
145 4+ 2+
146 2+ 4+
147 3+ 3+
148 4 3+
NI 3+ 4+
S. faecal isvar. li -faciens -- -.
S. faecalisTR~ - +
S. faecalis
TR1 4 4+
TR2 3+ 3+
TR3 4+ 3+
TR4 3+ 3+
ion Intensity Antiserum*
147 148 NI Pool
4+ 4+ 4+ 4+
3+ 4+ 4+ 4+
4+ 4+ . 4+ 4+
3+ 4+ 4+ 4+
2+ 4+ 4+ 4+
4+
4+
4+
4+
+ +... +..
4+ 4+ 4
4+ 4+ 4+
4+ 4+ 4+
4+ 4+ 4+
84
Table V -- Continued
FA Reaction Intensity Antiserumorganism ___
145 146 147 148 NI Pool
TR5 3+ 2+ 4+ 4+ 4+ 4+
TR6 3+ 4+ 4+ 4+ 4+ 4+
TR4 6 + 1+ - + 1+ +
TR8 0 + 1+ 1+ 1+
TR7 3 3+ 4+ 4+ 4+ 4+ 4+
TR7 5 4+ 4+ 4+ 3 4 4+
*FA reactions were obtained by the indirect methodwith the addition of antiserum (2 mg/ml protein) followedby fluorescein-labelled goat anti-rabbit antiglobulin(1 mg/ml protein) .
85
before a decrease in the FA titer became obvious.
Once it was determined that the specific antibodies
would stain their homologous antigen, the fluorescent anti-
body titers were resolved. The FA titers were determined
in a two-fold manner. First, the protein concentration of
each antiserum was demonstrated by the Biuret method as
described by Gornallet al. (1949). Secondly, two-fold
serial dilutions were made on each individual antiserum.
FA slides were prepared with the homologous antigen. An
aliquot of each dilution was placed on a different antigenic
smear, and the resultant FA reactions were observed. The
fluorescent intensity of each reaction was recorded. The
results are shown in Tables VI and VII. The FA titers were
expressed as the reciprocal of the highest dilution which
gave a four-plus reaction.
Each antiserum was diluted until the FA titer was
extinct, however, these higher dilutions were not used in
testing the staining ability of an organism in question.
The point emphasized was that only trace amounts of high-
titered, specific antisera stained organisms. This demonstrated
that FA reagents were conserved, and only a small amount
was needed to demonstrate an immunological reaction. Both
86
TABLE VI
FA TITERS OF WHOLE ANTISERA AND ANTIGLOBULINS ASDETERMINED BY THE DIRECT AND INDIRECTMETHOD OF STAINING
Dilution of Antiserum (whole)*Organism
1:20 1:4d 1:80 1:160 1:320 1:640 1:1280
145 4+ 4+ 4+ 4+ 2+
146 4+ 4+ 4+ 4+ 2+ 2+
147 4+ 4+ 4 4+ 3+ 2+
148 4+ 4+ 4+ 4+ 3+ 2+
NI 4+ 4+ 4+ 4+ 3+ 1+
B
Dilution of Antiglobulin**Organism~
St 1:5 1:10 1:20 1:40 1:80
145 4+ 4+ 4+ 4+ 3 1+
146 4+ 4+ 4+ 4+ 3+.
147 4+ 4+ 4+ 4+ 2+
148 4+ 4+ 4 4+ 4 2
NI 4+ 44+4+ 4 2
87
TABLE VI -- Continued
*The whole antisera were adjusted to 50 mg/ml proteinconcentration and varying dilutions were made from thesestandardized solutions. After reacting the antigens withspecific antisera, the complexes were overlayered withgoat anti-rabbit globulin (labelled with fluorescein iso-thiocyanate) which was diluted to 1 mg/ml.
**The globulin fractions were adjusted to 20 mg/mlprior to dilution.
88
TABLE VII
FA TITERS OF THE FLUORESCEIN-LABELLED CONJUGATESAS DETERMINED BY THE DIRECT METHOD
OF IMMUNOFLUORESCENCE*
Dilutions of the ConjugatesOrganism
1:10 2 140 180
145 4+ 3+ 2+
146 4+ 3+
147 4+ 3+ 1+
148 4+ 4+ 2+ 1+
NI 4+ 4+ 2+
*The conjugates were stored in aliquots at a proteinconcentration of 10 mg/ml. Dilutions of all the antiserawere made from this concentration. The conjugates werediluted 1:10 to give a protein concentration of 1 mg perml protein before they were added to the antigen (smear).
89
agglutination and precipitation tests required more anti-
serum. This enhanced the use of FA.
Reverting to protein determination again, it is
probably necessary to state briefly the reason for this
procedure. The protein quantity in each antiserum was
needed so that dye to protein ratios could be calculated
for conjugational procedures for the direct test. Also,
this information was necessary for dilution schemes that
were used in the indirect test. Hence, the same known
protein concentration was used in both methods, and comparisons
of the two techniques were made.
In many instances, the indirect method was superior
to the direct method because the peripheral fluorescence
was more intense in the former procedure. This finding
has been reported previously.
Specificity determination of an antiserum was also
shown by adsorptive techniques, as well as by the methods
that were previously mentioned. This consisted of reacting
homologous antigen and antiserum for a period of time inan attempt to remove specific antibodies. Tube agglutinatio ntests followed this procedure.
90
Pure broth cultures of the antigen were grown for
eighteen hours, harvested by centrifugation, and washed
four times with PBS. The cells were inactivated with a
0.5 per cent solution of formalin in physiological saline.
The cells were added to a homologous antiserum and placed
in a refrigerator for periods of one hour, three hours, six
hours, and twelve hours. Following refrigeration, the cells
were removed by centrifugation, and the antiserum was diluted.
Agglutination titers were determined on the antiserum.
A decrease was noted in most of the agglutination titers.
An antiserum that had been adsorbed for six hours demonstrated
an appreciably lower titer than one that had been adsorbed
for only one hour. Reactions that were allowed to continue
for twelve hours did not show any difference. In several
instances, adsorption was performed three times before the
titer was totally abolished. This was probably due to the
presence of a large quantity of antibody.
The adsorbed antisera were serially diluted in two-fold
steps with saline. Several variations were employed in the
tests. Living cells were added to the antisera in sc'me
cases. Also, the tubes were incubated at 37 C for two
hours part of the time, and in other instances, the tubes
91
were set in the refrigerator for twelve hours. No significant
differences were observed due to these varied conditions.
Table VIII contains the results of these adsorption tests.
Reactions of ATCC Cultures
Sixteen cultures of S. faecalis that were obtained
from ATCC, and some other members of group D, were grown
in pure cultures. These organisms were stained with FAreagents and examined. These organisms, and their FA and
agglutination titers, are given in Tables IX, X, and XI.
All of the strains of S. feaalis stained with the
various antisera. Intense fluorescence was noted in allthese strains, and rather high agglutination titers were
exhibited by these organisms when they were reacted with
specific antisera.
Since it was shown that the antisera would stain otherstrains of S. faecalis, the next approach was to determineif the antisera would stain any heterologous organism. Suchknowledge was needed to validate the test.
Heterologous Reactions
Cross-reactivity with heterologous organisms has beenan insurmountable problem in some studies that utilized FA.
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)
N- C
'P
CC)N -
CN
10
CNS0
ON
0
'P H
'PN00HA
CoN-
Lfl'CN O q
000
N
IQ
0l.LONl
coN-
CC)'lPr -
0H-
00
co
'P4CN
H
0
43040,
02
4-4
4,
HHH
121
-1
00 00 AQJ
4t
V
-r4
4
o0)
4
43
:3CQ3 -4J
4)
4
021
43J
00
rd
U)
92
0.434
0co
0
4-
Q)
4
H
041
a)0
N-
H
02
04C
H
"-1
1)
^1
-i
t
l
M
to
4
I
"
i
I
I I i I II I I II
+1+1 +11 +11 r +1
I I I I +11 -- l]I
I I
I I
4- ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++ ++
++ +++ + + + +
+ ++ ++4 + ++
C'r-4 r-
+ ++ + + ++
coqz;J H
N
'k0N
CO
Ld
U- -4 i
Cd4N 'r
4U
*C CdN
LnN
OCD
H
co
N~
0-H-
0U)
0
4-)
.p
-H
4-)
93
0
ro
4-
0)
4J
0,..,
4)-p
z
4
N
>1
0
Or-
4
0Co
04)
0
04
>1
r a
a
4)
Iii
a-a
r
CO
0
r4
+
U)f)
-H4-,
E
94
TABLE IX
STRAINS OF STREPTOCOCCUS FAECALIS UTILIZED IN THISIMMUNOFLUORESCENT STUDY
Strain
Streptococcus faecalisStreptococcus faecalisStreptococcus faecalis
trpococcus faecalisStreptococcus faecalisStreptococcus faecalisStreptococcus faecal isStreptococcus faecalisStreptococcus faecalisStreptococcus faecalisStrptoccUs faecalisStreptococcus faecalisStreptococcus faecalis
rpt2coccus faecalisStreptococcus faecalisStreptococcus faecalisStreptococcus faecalis
Source
ATCC*ATCCATCCATCCATCCATCCATCCATCCATCC
ATCC
ATCCATCCATCCATCC
ATCCMCSNI
Culture Number
349 (NT 145)8043 (NT 146)10541 (NT 148)1142012984145071450819634199531943214506194336057708012952(NT 147)NI (local isolate)
These strains of S. faecalis were employed to determinethe staining abilities of fifteen different antisera. Fivestrains, NT 145, NT 146, NT 147, NT 148, and NI were preparedinto antigens. These antigens were injected into rabbitswith subsequent production of antibody.
95
TABLE X
AGGLUTINATION TITERS OF ATCC STRAINS OF S. FAECALISREACTED WITH THE VARIOUS ANTI-S. FAECALIS-SERA*
AntiserumStrain
145 146 .. 1471 148 N Pool**
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
11420
12984
14507
14508
19634
19953
19432
14506
19433
6057
7080
12952
1024
256:
512
1024
512'
256.
1024
2048
512-
512'
1024
512
512
512
1024
512
1024
512
64
128
256
1024
64
256
256
512
1024,
1024
512
32.
512
1024
512
516
512
512
256
1028
512
1024
1024
1024
2048
2048
1024
256
1024
1024
512.
512
1024
2048
64
1024
2048
4096
2048
1024
2048
2048
1024
2048
4096
2048
1024
1024
2048
4096
2048
4096
4096
2048
*The titers are expressed as the reciprocal of thehighest dilution in which agglutination was clearly visible.
**Represents a mixture of an equal volume of all fiveantisera.
96
TABLE XI
FLUORESCENT ANTIBODY TITERS OF ATCC STRAINS OFS.GFAECALIS FOLLOWING REACTIONS WIT
GROUP SPECIFIC ANTISERA*
Strain
ATCC 11420
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
12984
14507
14508
19634
19953
19432
14506
19433
6057
Antiserum
145 146 4 4 NIPoo
3 4 2+ 4+ 2 4+
4+
4+
4+
4+
4+
4+
3+
3+
4+
4+
4+
4+
4+
4+
4+
4+
4+
3+
4+
2+
4+
4
4+
4+
3+
4+
3+
4+
4+
4+
4+
4+
4+
4+
3+
4+
2+
4+
4+
4+
4+
4+
3+
2+
4+
4+
4+
4+
4+
4+
4+
4+
3+
3+
ATCC 7080 4+ 4+ 3+4+ 4+
ATCC 12952 3+ 3+ 4+ 4 4+ 4+
*Both the direct and indirect methods of FA were utilizedA protein concentration of 2mg per ml was used in theseprocedures.
97
This has been shown to be due to the presence of unwanted
antibodies in the antiserum in question. These antibodies
have been, in most instances, removed by the process of
adsorption. Jones and Lewis (1966) have reported that
this technique did not alleviate their problems with cross-
reactions in a study on Corynebacterium,. Another technique
which has been used successfully by Thomason et al. (1965),
is that of counter-staining.
Cross-reactivity was encountered only infrequently
in this FA experiment. Tube agglutination tests and FA
tests were performed on a variety of heterologous organisms
as an additional means of showing the specificity of the
antisera that were used in the study. This group included
both Gram-positive and Gram-negative organisms, aerobic
and anaerobic species. The reason for the use of these
bacteria was to see if any organism, that might be found
as a member of an indigenous microbiota with S. faecalis,
would stain with any of the antisera.
It is of interest to note that Stajhylococcus aureus
was stained in two instances. The adsorption of these
particular antisera with the offending .S tahylococcus
removed the unwanted fluorescence. Some dim fluorescence
98/99
was observed with two other species of Streptococcus.
However, these reactions lacked the intense, billiant,
peripheral fluorescence observed when S. faecalis was stained.
Some of these streptococci did not grow in azide-dextrose
broth; thus, they were lost on primary isolation. Azide-
Dextrose Broth was used to inoculate samples suspected of
containing the enterococci, namely S. faecalis. The
agglutination and staining results of the heterologous
organisms are given in Tables XII and XIII.
S. faecalis var. liuefaciens was not stained by any
of the antisera. This was an important finding because
this organism has been shown to be of no sanitary significance.
This organism grew in azide-dextrose broth, and on m-Entero-
coccus agar. So, by conventional bacteriological methods,
an erroneous quantitation of the enterococci can be reported.
The use of FA, in lieu of the standard procedure of
identification, at least in this case, was favored.
Since the staining specificity was determined adequately,
the attention of the investigation was then focused on the
application of the technique to isolated strains that were
identified as S. faecalis by biochemical tests. Four-
hundred and eighty-seven strains were examined by the FA
100
TABLE XII
AGGLUTINATION TITERS OF S. FAECALIS ANTISERAWITH HETEROLOGOUS ORGANISMS
Organism
E. coli ATCC 11303
E. coli ATCC 128
E. coil ATCC 10586
E. coi ATCC 4157
E. coil ATCC 11775
E. arborescens ATCC 4558
A. aerogenes NT 1
K. J?.eumniae NT 65
P. aeruinosa ATCC 15442
P. vulgaris ATCC 13316
S. tahosa MC
Sh. ysenteriae ATCC
S. lutea MC
B. cereus ATCC 10876
B. mom-tarium ATCC 9885
Aggutination Titers Antiserum*14 M " 1 46 147 148"jNl
. ...
8
8.
32
16
16
128
0
8
0
4
0
64
16
64
64
32
0
0
16
32
32
0
4
16
16
32
32
16
0
4
32
8
8
2
2
0 4 2 0 0
16 8 32 8 4
64 0 32 8 4
F
2
2
4
4
2
0
4
32
2
2
2
4
0
8.
8
2
0"
0
0
8
8
0
4
2 8
101
TABLE XII --Continued
Organism
B. subtilis NT 7
B. mycoides MC
Cl. sporogenes MCS
S. aureus ATCC 4774
S. epidermidis MC
M. luteus MCS
G. tetagena MC
S. pyogenes ATCC 10782
S. agalactiae ATCC 6638
S. lactis ATCC 11454
B. licheniformis MC
F. Polymorphum ATCC 10953
2. necrophorus ATCC 8482
*Agglutinatinttrof the highest dilution o:observed.
Agglutination Titers Antiserum
145 146 147 f.148 NI
8
8
.0
64
32
2
32
0
32'
16
16
o
32
2
128
64
8
32
0
16
32
8
16
8
0
512
128
0
64
.0
32
8
8
8
16:
0
256
64
8
32
0
16
16
4
4
8
4
64
128
8
64
8
32
32
8
NT** NT NT NT NT
NT' NT NT NT NT
s were expressed as the reciprocalf antiserum in which clumping a
**NT= No tube agglutination test.
102
TABLE XIII
FLUORESCENT ANTIBODY REACTIONS OF S. FAECALISANTISERA WITH HETEROLOGOUS ORGANISMS
FA Reaction Intensity Antiserum*Organism
145 146 147 148 NI
E. col ATCC 11303 _ -
E. coli ATCC 128
E. coli ATCC 10586
E. coli ATCC 4157
E. coil ATCC 11775
E. arborescens ATCC 4558
A. aerogenes NT 1
K. neumniae NT 65
P. aeruginosa ATCC 15442
P.vulgaris ATCC 13316
S. typhosa MC
Sh. cysenteriae ATCC
S. Iutea MC
B. cereus ATCC 10876
B. megatarium ATCC 9885
.........
__
103
TABLE XIII --Continued
FA Reaction Intensity AntiserumOrganism
145 146 4
_B . subtilis NT 7__ _o ,,7.
B. mycoides MC
CI. sporogones MCS
S. aureus ATCC 4774
S. idermidis MC
M. luteus MCS
G. tetagena MC
S. yogenes ATCC 10782
S. agalactiae ATCC 6638
S. lactis ATCC 11454
B. licheniformis MC
F. polymorphum ATCC 10953
.. nnecrpphorus ATCC 8482
2+
+
+
2+
1+
3+
1+
2+
*Fluorescent antibody reactions were ---ermied .ts ollows4 , brilliant peripheral fluorescence; 3+, bright peripheralfluorescence; 2+, moderately bright peripheral fluorescence-
+, dulufluresseen-ceflSdull fluorescence; , fluorescence; -, negative fluore-scence.
+
2+
wo
vs-w
104
method using the specific antisera that were produced especially
for this study.
A large majority of the isolated group of organisms
exhibited three-plus and four-plus titers. These results are
listed in Table XIV.
Two isolates were identified as atypical strains because
they hydrolyzed starch. These strains, like S. faecalis var.
liguefaciens, are of no sanitary significance. After the
application of specific antisera to these strains, no
fluorescence was observed. Also, some of each individual
antiserum was adsorbed with these organisms. Following
this procedure, each antiserum still failed to stain the
atypical strains. However, the same antiserum stained the
homologous organism that had engendered its production.
Also, none of the previously stained isolates were rendered
negative following application of an adsorbed antiserum.
This was significant because evidently the antigenic constituents
of the atypical strains differed from S. faecalis
Tube agglutination tests were performed and these three
strains were used as antigens. All three organisms demonstrated
very low, or negative, titers.
105
TABLE XIV
THE STAINING TITERS OF ISOLATES*
Reaction Intensity Number of Isolates
1+
2+
3+
4+
I
2.5
9. 2
2
12
45
98 .3
*Four-hundred eighty-seven isolates that were bio-chemically defined as S. faecalis were employed in the study.
**The two organisms that exhibited negative fluorescencewere atypical strains.
Percent
1 4.281 .. .
106
An FA Study on Escherichia coli
E. coli, one of the coliforms, has been used as the
index organism in standard bacteriological procedures for
quite some time. This organism, or rather a particular
strain of the organism, has been the subject of an appreciable
amount of research. The reason was because this strain was
shown to be enteropathogenic. Cherry et al. (1961) used the
FA technique to rapidly identify the enteropathogenic strain
of E. coli in fecal materials from suspected cases.
Specific antisera were produced in rabbits against
fifteen ATCC strains of E. coli. The antisera were used
in both tube agglutination tests and in immunofluorescent
tests. The same procedures of growth, identification, and
antigenic preparations were followed as with the strepto-
cocci.
This part of the study was incorporated to determine
the. feasibility of using E. coli as the index organism in
the fluorescent antibody technique. Hence, after completion
of the study, a comparison between the coliform and the
streptococcus was made.
The conclusion of the study was that the diversity
of antigenic components in E. coli was too varied and their
107
identification by FA was hampered by this fact. Therefore,
the detection and identification of this organism by
immunofluorescence was considered to be of much less value
than the use of the streptococcus, S. faecalis. The
agglutination titers of all the cross-reactions between the
various strains are shown in Table XV, and the FA reaction
results are given in Table XVI.
The remainder of the study was concerned with the
analysis of the cell-wall components of some of the strepto-
coccal strains that were included in the experiment. This
analysis consisted of studying the cell-wall hydrolyzates
by thin layer chromatography.
Whole cells, which had been thoroughly washed, were
hydrolyzed with 6N hydrochloric acid. The cells were
hydrolyzed for a period of two hours at 105 C. The amino
acid content of each hydrolyzate was detected by spraing the
chromatogram with ninhydrin. Amino-sugars, such as glucos-
amine, galactosamine, N-acetyl-glucosamine, and N-acetyl-
galactosamine were analyzed for in the same manner. Theamino acids appeared as purple spqts on the chromatogramswith the exception of aspartate, glycine, and diaminpimelic
acid. Aspartate appeared gray, while both glycine and diamino-
108
TABLE XV
AGGLUTINATION TITERS OF FIFTEEN ATCC STRAINSOF ESCHERICHIA COLT
AntiserumStrain ___.-__.
Ii J L 4 5 ___ 6 78ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
4157
15224
11775
11303'
15223.
12696
10536
8739
10586
9723E
9723H
4350
128
11143
ATCC 8677
1024
8
512
256-
256,
512
64
512.
32
128
1024
256
512
32
64
64
2048
128
64:
512
256
512
512
64
128
512
64
256
128
32
256
32
2048
512
64
2
256
256
64
8
64
16
256
64
256
517
256.
128
1024
128
64
512.
64
512,
0
32
32
2048
128
128
256
64
128
256
4096
512
512
256
512
256
128
512
64
256
512
128
32
256
512
64
1024
256
32
256
128
1024
256
0
128
256
256
128
512
1024
128
256
2048
256
8
64
64
1024
512
64
8
64
512
128
256
256
32
512
1024
64
16
128
256
512
128
256
0
109
TABLE XV --Continued
Antiserum
Strain910 11 1III ~ 22 13 1 1
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
4157
15224
11775
11303
15223
12696
10536
8739
10586
9723E
9723H
4350
128
11143
8677
256
1024
64
256
32
128.
64
512.
512
256,
16
512
1024
128
1024
16
256
256
128
128
128
256
256
256
2048
32
8
256
256
512
32
256
256
64,
512
0
1024
256
64
64
1024
64
16
256
64.
512
64
256
256
256
256
512
2
32
128
128
4096
1024
256
256
64
128.
1024
1024
512
128
256
128
1024
64
256
256
4096
64
512
0
256
64
256
512
128
512
128
128.
16
128
256
64
1024
256
128
32
256
64
1048
64
512
1024
128
512
64
512
512
256
4096
Titers were expressed as the reciprocal of the highestdilutions that gave visible agglutination. ..Many cross-reactionswere observed to occur with a strain of A . aerogenes, K.pneumoniae, B. cereus, and P. aeruginosa.
. ,...... ..... ,.. ,.,.....,,.. w, ... ,............... . .
110
TABLE XVI
FLUORESCENT ANTIBODY REACTIONS OF ATCC STRAINSOF ESCHERICHIA COLI
AntiserumStrain
1 21 3 1415 16 17_ _ _ _ _n M w l a w+ " fL~W r " -- - i ~ r r .."i I f ., w , / M f ..t+. - -- - I I _ _ _ I I I I _ __ra+w.wf. - .« r~wrr~n
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
AT CC
4157
15224
11775
11303
15223
12696
10536
8739
10586
9723E
9723H
4350
128
11143
AT CC 8677
4+
+
2+
3+
1+
3+
1+1+
4+
1+
3+
2+.
4+
2+
M-
3+
2+
2+
3+
2+
2+
2+
-
1+
2+
1+
4+
3+
2+
1+
1+
2+
2+
1+
3+
2+
4+
1+
2+
3+
2+
1+
4+
2+
2+
4-
2+
3+
4+
3+
2+
1+
2+
2+
2+
3+
2+
1+
3+
1+
1+
3+
4+
1+
4+
3+
1+
4+
1+
+
+
1+
4+
4+
1+
2+
4+
2+
2+
+
4+
3+
1+
1+
4+
1+
3+
2+
+
3+
4
2+
1+
2+
4+
2
2
111
TABLE XVI -- Continued
AntiserumStrain ____4___
.. l . _ . . .. ..11 . . , , .3. . . . . , .
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
4157
15224
11775
11303
15223
12696
10536
8739
10586,
9723E
9723H
4350
128
4+
24
2+2+
1+
1+
3+
4+
2+
3+
3+
4
3+
2+
3+
1+
2+
1+
3+.
2+
4+
1+
1-
1+
2+
1+
3+
3+
2+
4+
1+
1+
4+
2+
2+
2+
2+
2+
3+
3+
1+
3+
1+
4+
4+
3+
1+
4+
1+
4+
3+
2+
+
3+
2+
3+
3+
4+
2+.
3+
2+
4+
1+
3+
1+
1+
2+
2+
1+:
1+
+
3+
2+
4+
+
4+
4+
1+
3+
3+
ATCC 11143 1+ 2+ + 1+ 2+ 4+ 1+ATCC 8677.4+ 3+ 2+ 3+ + 4+
The FA titers represent the fluorescence observed followingthe addition of specific-labelled antisera. A protein con-centration of 2 mg per ml was used in both the direct andindirect technique.
112
pimelic acid turned a brownish color after development. The
scheme that was used for the assay of these components is
shown in Figure 11.
Variations were noted in the amino acids that were
detected in the different test organisms. The amino acids
aspartate, glutamate, and lysine appeared to be quite common
in a large majority of the strains that were tested. Alanine
was also found in a relative high number of the strains.
Serine and diaminopimelic acid were found in only three of
the strains, S. faecalis var. liuefaciens and two atypical
strains. Glycine was detected in two of the hydrolyzates.
According to Baird-Parker and Woodroffe (1967), the
cell walls of Gram-positive bacteria contained appreciable
amounts of only four or five amino acids. Also, aromatic
and sulfur-containing amino acids were characteristically
absent. Controls of cysteine and phenylalanine were employed
to see if either of these amino acids were present. Neither
phenylalanine nor cysteine was detected in any of the test
organisms.
The results of the amino acid analysis of the: various
strains are listed in Table XVI. Three members of the
group, S. faecalis var. liguefaciens and two atypical strains,
-PUt)C)43
-H
-d
EH
~)to
0
-o
r-- 4
43- 4Cd
-- ---- N - -- I - ;
'e I -HC)N
0
Fr- 1V
0
too Cd Z
__C 0 >1N-->1 ru -H
ol 0
1 U H
C)43
04Cdn
c
0
0
-H C
--- ) 0---o o
0 0U)U)U)" -H
C)N
-H
5-443
C)iz
41-
Ui
-r
C)
U)
0
0
U)ro-HUCr
0
-H
Cd
IV-Ho qC)
Cd
- s^.V
-H
-ri
0C0
0
43 0col) -H ECd 0.t b
r- -I 0
0ri Co -
.r
113
)
4J U)r r4
: r-I
U .C
o U)O D
Cov-I
0)
C
0
r-
U)(s
C
0
430
0
-4
rH
(C)
1
d
d
rr M
Nr-4
0
vC)
r4 Z
4
A
C)W -H
-tn -HD U)
-2 Ur0~C
HU -H
0 U
~C)C
43
0rt
-H
"-4
ru.V
N
-H
rs
114
TABLE XVII
AMINO ACID ANALYSIS OF STREPTOCOCCAL CELL WALLS ASDETERMINED BY THIN LAYER CHROMATOGRAPHY OF
HCL HYDROLYZATES FROM WHOLE CELLS
Strain ~r, 4r-1 -r "r
ATCC 11420ATCC 12984ATCC 10541ATCC 349
ATCC 8043MCS (147).NIATCC 14508ATCC 19432ATCC 14507S. faecalisvar. tq.
faciensTR1TR2TR3
TR7 3TR
-.
-,
-
-.
.-.
-
+..
-
+,T
+
+
+
+1+
++
+++
TR 7 4 + + -+ -TR76 + + +S. aureus
ATCC 4776
0
C.
1"r
T
I
+
+
-
4-
+
Q.
r-4:
0)
T
-T
-+
-
-
-
~ r
>- 1 -)- -I
-1 C
4+
- 4+(1) 0
+4+
-4+
4+
-4+
-4+
-4+
-4+- 4+
4+-4+
-4+
4+
4+
.. 4
+ +4
- 4-f
-..
_t
I
115
failed to exhibit fluorescence after being stained with
specific antisera. It was noted, upon analysis, that both
serine and diaminopimelic acid were present in the hydrolyzates
of these three strains. Also, neither of these amino acids
was detected in the other organisms. It was speculated that
the antigenic binding site was perhaps covered by these
amino acids as part of a chain, or, possibly these amino
acids were incompatible with the reactive site of the antibody.
Ingram and Salton (1957) indicated that alanine was an
important N-terminal amino acid in DNFB-labeled cells of
several Gram-positive bacteria, namely Micrococcus
lysodeikticus and Sarcina lutea. Jones and Lewis (1966)
suggested that alanine was a very important amino acid in
the antigen-antibody combining site.
Mild hydrolytic conditions with 6N hydrochloric acid
were employed in this phase of the study in order to compare
the intensity of fluorescence before and after hydrolysis.
Bacterial cells were placed in screw cap tubes, mixed with
the acid, and placed in an incubator at 37 C. These mixtures
were removed at fifteen minute intervals, their hydrolyzates
were checked for the presence of amino acids by thin-layer
chromatography. The maximum incubation time was sixty minutes.
116
The cells were stained before hydrolysis in order to
determine their FA intensity. Aliquots of the cells were
removed following each hydrolysis period, and the FA intensity
of each was determined. Mild hydrolysis seemed inadequate
in the initial fifteen minute period as fluorescence was not
altered, and no amino acid was detected in the hydrolyzate.
Lysine was detected in one instance, but the FA titer was
unchanged following a fifteen minute hydrolytic period.
The thirty minute hydrolyzates, for the most part,
contained alanine and lysine. The fluorescent antibody
titers were noted to decrease markedly when these cells
were stained and examined. Negative fluorescence was
observed following sixty minutes of hydrolysis in all of -
the strains examined. The extinction of FA titer was due,
probably, to the destruction of the tertiary structure of
the amino acids in the cell walls. The majority of the
cells were totally lysed as noted by dark-field microscopy.
Alanine, as described by the cited investigators, seemed to benecessary for the binding of the antibody to the antigen.
Several other strains of group A, C, and G streptococci
were examined, and their cell wall components were compared
to the enterococci. Alanine was also found in the hydrolyzates
117
of these groups. The cells were not stained with specific
antisera due to not having any group specific antisera for
any of these groups. These cells, however, did not stain
with anti-s. feacalis-serum before or after they were
hydrolyzed. Hence, the only comparison made was that the
walls of these three groups contained alanine which possibly
implicated this amino acid as part of the antigenic site in
these cells. The results are listed in Table XVIII.
Jones and Lewis (1966) utilized l-fluoro-2, 4-dinitro-
benzene (DNFB) in a study on Corynebacterium dihtherae
and three groups of streptococci. DNFB was reacted with
cells in an attempt to obtain the N-terminal dinitrophenyl
(DNP) amino acids. A similar study was included in this
investigation.
Washed cells were reacted with a 5 per cent solution of
DNFB in ethanol at room temperature overnight. An 8 per
cent solution of sodium bicarbonate was added to the mixture.
FA staining titers were determined following the reaction
with DNFB. The staining titers of the organisms were not
altered due to this reaction.
DNP-amino acids were prepared as described by Clark(1964). The -DNP-amino acids were used as controls in thin-
118
TABLE XVIII
THE AMINO ACIDS DETECTED BY THIN-LAYER CHROMATOGRAPHYFOLLOWING THE MILD HYDROLYSIS OF STREPTOCOCCAL CELLS
IN 6N HCL FOR TWO HOURS AT 37 C
Hydrolytic Periods (minutes)Strainmiue)
15 30456
145 lysine lysine lysinealanine alanine alanine
146 alanine alanine alanine
147 - lysine alanine lysinelysine alanine
148 lysine lysine lysine lysinealanine alanine alanine
NI alanine
14508 alanine alanine alanine
glutamate
TR 7 3 alanine alanine alanineTR7 8 lysine lysine lysine
aspartate
TR7 4 aspartate aspartateTR76 - alanine alanine alanineS. faecalis
alaninevar. lique-. alaninefaciens
119
TABLE XVIII -- Continued
FA REACTIONS OF CELLS BEFORE AND FOLLOWING HYDROLYSIS
Strain
145
146
147
148
NI
14508
TR73
TR7 8
TR 7 4
TR7 6
faecalisvar. liqcue-
faciens
*Cells wmicroscopy.
FA Reactions
0 15 30 45 60*
4+ 4+ 2+ 1+ -
4+ 4+ 1+ - -
4 3 - -
4+ 4+ 2+ - -
4+ 3+ 1 -
4+ 3+ 2+ - -
4+ 4+ 2+ 1+ -
4+ 3+ 2+- -
+ ~ ~.-
ere mostly lysed as determined by dark- field
120
layer chromatographic procedures in an attempt to determine
the N-terminal amino acids of the various organisms that
were analysed.
The cells, after being reacted with DNFB overnight,
were hydrolyzed for two hours at 105 C. The hydrolyzates
were filtered, evaporated to dryness, redissolved in 10
per cent isopropanol, and neutralized with ammonia (0.90
specific gravity). The resultant mixture was again
evaporated to dryness, and redissolved in isopropanol (0.25
ml).
Chromatograms were employed to detect the DNP--amino
acids. The Rf values of the controls were compared to
those of the samples following the developement of the
chromatograms with ninhydrin. DNP-alanine was noted in
several samples. DNP-lysine was also detected in the
hydrolyzates of three organisms.
The results of the DNP-amino acid analysis is shown
in Table XIX. Definite conclusions were not made due to
the inadequate data that was obtained. DNFB reacted with
lysine in one case, but this was not substantial evidence
that lysine was an N-terminal due to the reaction of its
epsilon aminq group with this compound.
121
TABLE XIX
THE N-TERMINAL DNP-AMINO ACIDS DETECTED IN THE ACIDHYDROLYZATES FROM STREPTOCOCCAL CELL WALLS AS
DETERMINED BY THIN-LAYER CHROMATOGRAPHY
ci) 0" r
Strain* C ei j . 1W ac -- r -1 - - - -- 3+
4U-- -Hr- -f >0-rA -i) 3+re >1 U)W :5 U1~ Ur-4 r--1 > r r ~ H~ ) , r-4 >,
145 - - + - - - - - - 4+
146 - - + - - - - - 3+147 - - -. . -- +148 - + + - - - - - 4+NI +.. ... - :. - 414508 +' .. . .,. - .-. ... ... 4+T1.Vr 3 +.w. - -, w. w. " 4+iTR7 5 - - -4+TR7 4 - -TR7 6 - - -
S. faecalis --
var. lique-faciens
*Each strain was stained with FA following its reactionwith 1,2, 4 -dinitrofluorobenzene (DNFB). The reactions wereunaltered by this treatment. The hydrolyzates were analyzedby thin-layer chromatography for the presence of dinitro-phenyl amino acids.
122
The hydrolyzates were analysed for the presence of
sugars. The analysis was performed by thin-layer chromatography.
It has been shown that the cell walls of group A-beta hemolytic
streptococci contained a rather limited variety of sugars.
Hayashi and Barkulis (1958) suggested that rhamnose-hexosamine
polymers, with recurring units of glutamic acid, lysine, and
alanine, constituted the group specific C polysaccharide in
the group A streptococci.
Collins (1967) stated that Gram-positive bacteria
contain one or more of the following sugars: arabinose,
galsctose, glucose, mannose, or rhamnose. Slade and Slamp
(1962) reported that sugars varied considerably in their
distribution among the groups of streptococci. These
workers claimed that the presence or absence of the various
sugars did not aid in the identification and differentiation
of these bacteria.
The analysis of the sugars in the cell-wall hydrolyzates
was undertaken to attempt and show a difference between the
cells that fluoresced versus those strains that exhibited
negative fluorescence. Rhamnose was found in the most of the
hydrolyzates. Glucose and galactose were detected in some
of the strains, but no definite pattern was interpreted.
123
The results are shown in Table xx.
The amino-sugars were detected in only a very small
quantity of the strains examined. The same problem existed
as with the sugars, no definite correlation between
fluorescence and the presence or absence of a particular
amino-sugar was made. N--acetyl""-glucosamie was detected
in only one of the test organisms, an organism that was
fluorescent in the presence of anti-S. faecalis-serum.
N-acetyl-galactosamine was not detected in any of the
hydrolyzate.s. The results of the amino-sugars were given
with the amino acids.
The conclusion to the analysis of the cell-wall
components of the enterococci was that some insight as to
the role of these components was attained. The detectionof the two amino acids, serine and diaminopimelic acid, in
the walls of the organims that did not fluoresce showed
that these organisms were different structurally. The
importance of this finding can only be assumed.
124
TABLE XX
SUGARS DETECTED IN HCL HYDROLYZATES OF STREPTOCOCCAL
CELLS BY THIN-LAYER CHROMATOGRAPHY
Organism 0QU-}-
CJ C c<C
ATCC 11420 + --
ATCC 12984 + + --
ATCC 10541 + - - - -
ATCC 349 + - - - ~
ATCC 8043 + - + - -
MCS (147) - - - -
NI + + - - -
ATCC 14508 - - + - --
ATCC 19432 + - - -
ATCC 14507 + - - - -
S. faecalis + - -- -
var.lique-
faciens
TRI + - - - -
TR2 + + - -
TR3 - - - -
TR7 3 + - - -- --
TR 7 8 - - - - - -
TR 7 4 + - + - -
TR7 6+ - - -- -
CHAPTER V
DISCUSSION
The use of FA, in conjunction with S. faecalis
as the indicator organism, has not been applied as a
technique by which water pollution can be determined.
Hence, this approach in determining pollution in water,
due to the presence of the specific bacterium S. faecalis,
was novel. The research reported in this investigation
clearly showed that S. faecalis can be utilized in the
rapid detection of fecally polluted water.
Specific, high-titered antisera were used to
rapidly detect and identify S. faecalis. The results
that were obtained from the fluorescent antibody tests
were compared to standard biochemical methods of identi-
fication such as fermentation and MPN tests. The
comparison demonstrated that this test was a valid and
reliable method for the detection and the identification
of S. faecalis.
125
126
The specificities of the antisera were determined
by reacting the antisera with their homologous antigens.
Adsorption of the antisera with their homolgous antigens
resulted in negative fluorescence. Heterologous strains
of bacteria failed to stain with these FA reagents.
S. faecalis var. liquefaciens and two atypical
strains of S. faecalis were employed in the test. These
three strains grew in Azide Dextrose Broth, a selective
medium used in the initial isolation procedure for S.
faecalis. The three strains of enterococci also grew
on M-Enterococcus Agar, a medium utilized in the confirm-
ation of S. faecalis. These two factors made the differ-
entiation between S. faecalis and S. faecalis var. lique-
faciens impossible on the basis of macroscopic, cultural
evaluation alone. However, S. faecalis var. liquefaciens
and the atypical strains failed to stain with specific
antisera. The use of fluorescent antibody to identify
S. faecalis, and to differentiate it from S. faecalis
var. liquefaciens and the atypical strains rapidly, was
a most important and novel finding.
The atypical strains and S. faecalis var. liquefaciens
127
are of no sanitary significance because they can live
commensally on vegetation. Hence, it was shown that FA
was a sensitive and discriminating serological test in
detecting and identifying S. faecalis. The degree of
sensitivity, or the staining ability of S. faecalis
has not been reported.
E. coli has never been used as the indicator organism
with FA. However, FA has been used to differentiate
the enteropathogenic strains, but the organism has not
been used to determine fecal pollution by FA.
A minimum of one-hundred different antigenic strains
of E. coli have been reported. This fact complicated
the production of a specific antiserum. Also, this
accounted in part for the variety of titers that were
obtained in FA and agglutination tests in this investigation.
The results acquired in the tests demonstrated that E. coli
was not as satisfactory as S. faecalis when employed as
the indicator organism for fluorescent antibody.
The presence of the amino acids serine and diamino-
pimelic acid has not been reported in the atypical
strains, or in S. faecalis var. liquefaciens. These
12b
amino acids were detected in acid hydrolyzates from cells
by thin-layer chromatography.
The observed differences in the FA staining of
S. faecalis and the three biotypes were interpreted to
be due to antigenic differences. The antigenic sites of
S. faecalis, compared to the antigenic sites of the
three strains that did not fluoresce, might have been
a different entity. Shattock and Jones (1960) showed
that the group specific C polysaccharide of the group
Drenterococci was located in the cytoplasmic-cell
membrane component of the cell. Thus, the antigenic
sites could have been masked, and its combination with
the antibody was prevented. Moody et al. (1957) have
reported difficulties with masking in studies on Sal-
monella.
The possibility existed that serine and diamino-
pimelic acid were part of the antigenic determinate
group and caused the antigen to be incompatible with
the antibody. Elliott (1959) stated that group D biotypes
vary chemically in their cell walls, and that these
differences may be assoicated with noted serological.
differences. Hence, group D organisms, including S.
faecalis, were not always identified by the more
classical serological techniques such as -agglutination
and precipitation. Such difficulty was not experienced
with the application of FA.
Standard Methods presently require forty-eight to
ninety-two hours to identify S. faecalis. The organism
has been identified in five hours by this FA technique
which utilized a selective medium and an elevated
temperature of 45 C. A reduction in the time necessary
to identify S. faecalis was adequately shown. This
fact could make this FA technique a vital part in
monitoring sewage effluents and determining the bacterial
quality of water for re-use. S. faecalis was identified
in two hours by filtering a two liter quantity of sewage
effluent through several 0.45u millipore filters. These
filters were washed with sterile saline and concentrated
by centrifugation. The cells were detected by both
direct and indirect immunofluorescence.
The scope of some previous studies has been limited
due to the peculiar staining characteristics of some
.LJU
particular organisms. Carter and Leise (1958) reported
that seventy-two hour cultures of Pasteurella pests,
Brucella suis, and Vibrio comma failed to stain with PA.Thomason et al. (1957) stated that Salmonella did not
stain following initial incubation, but did stain after
a longer incubation.
S. faecalis stained after five hours of incubation,
and stained up to ninety-two hours following 'incubation.
No change in FA intensity was observed. This demonstrated
that the antigen of S. faecalis was unaltered after a
rather extensive incubation period, and that the antigen
was evidently quite stable.
The intense staining of S. faecalis made it an
excellent index organism for FA. This procedure may be
incorporated into a standard method due to its applicability.
CHAPTER VI
SUMMARY
This study has been concerned with the integration
of the fluorescent antibody technique with an index organism
of pollution, S. faecalis, in an attempt to rapidly detect
fecal pollution in water. The study had five specific phases
that were investigated.
1. The staining ability and the specificity of
specific antisera were resolved. Sixteen strains of S.
faecalis, which were obtained from ATCC, were incorporated
into the study and stained with anti-S. faecalis--serum.
This initial phase of the study demonstrated that S. faecalis
can be stained with high-'titered specific antiserum.
2. Fifteen strains of E. coi, which were obtained
from ATCC also, were stained and cross-reactions were performed
to determine the staining ability of these organisms. It
was concluded that many cross-reactions resulted in low FAtiters in many instances, and because of this fact, S. faecalis
was shown to be a better index organism than E. coli.
131
132
3. Methods were investigated that demonstrated a
way in which the enterococcus, S. 'faecalis, could be rapidlydetected. This consisted of inoculating a sample into azide-
dextrose broth at an elevated temperature of 45 C for a period
of five hours. This represented a substantial reduction in
the identification time when compared to identifying the
organism by conventional bacteriological methods.
4. The validity of the test was determined by taking
isolates that were randomly chosen and identifying these
strains rapidly and accurately by fluorescent antibody.
Four-hundred and eighty-seven isolates were determined by
this method as well as by standard procedures. The percentage
of agreement between the two methods demonstrated that-this
test was valid and applicable.
5. The cell walls of some of the various strains that
were studied, were shown to contain two different amino acids.
These amino acids were not present in the cells that exhibitd
fluorescence, but they were present in three strains that didnot fluoresce. This finding could be ignificant in the basicreaction involved in fluorescent antibodies of S. faecalis.
The possibility of using this method of identification
in conjunction with standard methods was discussed.
133
The conslusion drawn from this study was that valuable
information, pertaining to fluorescent antibody, was in-
deed obtained. Consideration must be given to the fact,
however, that the scope of the research was limited with
reference to the number of organisms studied. Although
the number of organisms employed was limited, the initial
approach to the problem defined several objectives that
were considered. These objectives, for the most part,
have been resolved. Therefore, the problem was considered
to be successful.
134
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