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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73

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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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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