■ e 2 9

'LEGIONNAIRES''

the disease, the bacterium
and methodology

U.S. DEPARTMENT OF

HEALTH, EDUCATION, AND WELFARE

PUBLIC HEALTH SERVICE

CENTER FOR DISEASE CONTROL

ATLANTA, GEORGIA 30333

MBL/WHOI

Library

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LEGIONNAIRES'

the disease, the bacterium
and methodology

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Edited by

Gilda L. Jones

Bacteriology Training Branch

Laboratory Training and Consultation Division

and

G. Ann Hebert

Immunofluorescence Section

Analytical Bacteriology Branch

Bacteriology Division

May 1979

U.S. Department of Health, Education, and Welfare

Public Health Service

Center for Disease Control

Bureau of Laboratories

Atlanta, Georgia 30333

HEW Publication No. (CDC) 79 8375

Developmental, May 1978

Revised Developmental. October 1978

First Edition. May 1979

Use of trade names is for identification only and does not constitute endorsement by the Public
Health Service or by the U.S. Department of Health, Education, and Welfare.

Foreword

Legionnaires' disease and its causative agent are subjects of worldwide
concern. Tin's is evident from tfie response to an iiiness wliose cause
could not initially be defined and from the efforts of the clinical, medical,
and general health surveillance communities around the world to treat
patients with Legionnaires' disease effectively in order to minimize the
threat posed to the public health.

The staff at the Center for Disease Control (CDC) responded quickly
and continues to respond to the need for more complete information on
Legionnaires' disease, the bacterium which causes it, methods for confirm-
ing a presumptive diagnosis, and the most appropriate therapy.

The Legionnaires' disease bacterium is special-it does not follow the
usual rules. It is a bacterium but remains invisible with traditional bacteri-
ological techniques; it does not grow on the usual bacterial media, and it
does not stain with the usual bacterial stains. It grows both inside and out-
side cells; it mimics some of the laboratory characteristics of rickettsiae, and
the clinical characteristics of viruses. This organism does not even seem to
follow the usual rules in causing illness; it causes pneumonia under some
conditions but not under others, and when it does cause pneumonia, it may
mimic viral pneumonia but at the same time leave a few small tantalizing
bacterial clues such as an increase in the white blood count.

We had not identified an important new human bacterial pathogen since
the I950's, but now we must ask how many others there might be that do
not follow the rules-a real challenge to all microbiologists and clinicians to
reevaluate techniques.

This manual is an effort to disseminate knowledge gained at CDC in the
hope that it will be used by the health community for rapid identification
of the bacterium and prompt treatment as well as prevention of the illness.
It is also hoped that others in academic and research settings will build on
the information presented here to further improve diagnostic tools.

William H. Foege, M. D.
Director, Center for Disease Control

Acknowledgements

We are indebted to Dr. John H. Krickel, Director, Laboratory Training and Consultation
Division, Bureau of Laboratories, whose guidance, support, and encouragement made tiiis manual

a reality.

We gratefully acknowledge the assistance of our colleague Elliott Churchill, Writer-Editor,
formerly with the Bureau of Laboratories and currently with the Bureau of Epidemiology. Her
editorial expertise, patience, and sense of humor were invaluable.

We are further indebted to Dr. Roslyn Q. Robinson, Director, Bureau of Laboratories,
Dr. Philip S. Brachman, Director, Bureau of Epidemiology, and many staff members from both
Bureaus whose cooperation and support were vital to the development of this manual.

IV

Contributors

WILLIAM B.BAINE.M.D.

Medical Epidemiologist, Hospital Infections Branch. Bacterial Diseases Division, Bureau of Epidemiology

ALBERT BALOWS, Ph.D.

Director, Bacteriology Division, Bureau of Laboratories

JOHN V.BENNETT, M.D.

Director, Bacterial Diseases Division, Bureau of Epidemiology

JOHN A. BLACKMON. M.D.

Staff Pathologist, Pathology Division, Bureau of Laboratories

PHILIP S. BRACHMAN, M.D.

Director, Bureau of Epidemiology

DON J. BRENNER, Ph.D.

Chief, Enteric Section, Enterobacteriology Branch, Bacteriology Division, Bureau of Laboratories

CAREY S. CALLAWAY. B.S.

Biologist, Pathology Division, Bureau of Laboratories

FRANCIS W. CHANDLER, D.V.M., Ph.D.

Chief. Histopathology Laboratory, Pathology Division, Bureau of Laboratories

WILLIAM B. CHERRY, Ph.D.

Scientist Director, Analytical Bacteriology Branch. Bacteriology Division. Bureau of Laboratories

R. ELLIOTT CHURCHILL, M.A.

Writer-Editor, Editorial and Graphic Services. Bureau of Epidemiology

ROGER M. COLE, Ph.D., M.D.

Chief, Laboratory of Streptococcal Diseases, National Institute of Allergies and Infectious Diseases,
National Institutes of Health

DONNA D.CRUCE, M.S.

Research Microbiologist, Bacterial Immunology Branch. Bacteriology Division, Bureau of Laboratories

SALLY B. DEES, M.S.

Research Microbiologist, Analytical Bacteriology Branch, Bacteriology Division, Bureau of Laboratories

WALTER R. DOWDLE, Ph.D.

Director, Virology Division, Bureau of Laboratories

Contributors

CAROL E. FARSHY. B.S.

Research Microbiologist. Bacterial Immunology Branch, Bacteriology Division, Bureau of Laboratories

JAMES C.FEELEY. Ph.D.

Chief. Special Pathogens Laboratory Section. Epidemiologic Investigations Laboratory Branch. Bacterial
Diseases Division. Bureau of Epidemiology

JOHN C.FEELEY, Ph.D.

Chief. Bacterial Immunology Branch. Bacteriology Division, Bureau of Laboratories

BONNIE J. FIKES. B.S.. M.T. (ASCP)

Medical Technologist. Bacterial Immunology Branch, Bacteriology Division. Bureau of Laboratories

WILLIAM H. FOEGE, M.D.

Director. Center for Disease Control

DAVID ERASER, M.D.

Chief. Special Pathogens Branch. Bacterial Diseases Division, Bureau of Epidemiology

J. RICHARD GEORGE. M.S.

Research Microbiologist, Bacterial and Fungal Products Branch. Biological Products Division. Bureau of
Laboratories

ROBERT J.GIBSON, M.S.

Microbiologist, Special Pathogens Laboratory Section, Epidemiologic Investigations Laboratory Branch,
Bacterial Diseases Division, Bureau of Epidemiology

GEORGE W. GORMAN. B.S.

Microbiologist, Epidemiologic Investigations Laboratory Branch, Bacterial Diseases Division, Bureau of
Epidemiology

PATRICIA W. GREER, A.B., M.T. (ASCP)

Chief Technologist, Control Tissue Repository. Pathology Division, Bureau of Laboratories

W. KNOX HARRELL. Ph.D.

Chief, Bacterial and Fungal Products Branch. Biological Products Division. Bureau of Laboratories

G. ANN HEBERT, B.S., M.T. (ASCP)

Research Biologist, Analytical Bacteriology Branch, Bacteriology Division, Bureau of Laboratories

MARTIN D. HICKLIN, M.D.

Director. Pathology Division, Bureau of Laboratories

GILDA L. JONES, Dr.P.H.

Microbiologist, Bacteriology Training Branch, Laboratory Training and Consultation Division, Bureau
of Laboratories

LINDA A. KIRVEN, M.S.

Research Microbiologist. Clinical Bacteriology Branch. Bacteriology Division. Bureau of Laboratories

VI

Contributors

JOHNH.K.RICKEL.Ed.D.

Director, Laboratory Training and Consultation Division, Bureau of Laboratories

JOSEPH E. McDADE, Ph.D.

Research Microbiologist, Leprosy and Rickettsia Branch, Virology Division, Bureau of Laboratories

ROGER M. McKINNEY, Ph.D.

Chief, Immunofluorescence Section, Analytical Bacteriology Branch, Bacteriology Division, Bureau of
Laboratories

GEORGE K, MORRIS, Ph.D.

Chief, Epidemiologic Investigations Laboratory Branch, Bacterial Diseases Division, Bureau of Epidemiology

C.WAYNE MOSS, Ph.D.

Chief, Analytical Bacteriology Branch, Bacteriology Division, Bureau of Laboratories

CHARLOTTE M. PATTON, M.S.

Chief, Bacterial Zoonoses Laboratory Section, Epidemiologic Investigations Laboratory Branch, Bacterial
Diseases Division, Bureau of Epidemiology

LEO PINE, Ph.D.

Chief, Products Development Branch, Biological Products Division, Bureau of Laboratories

M.W.REEVES, Ph.D.

Research Microbiologist, Products Development Branch, Biological Products Division, Bureau of Lab-
oratories

ROSLYN Q. ROBINSON, Ph.D.

Director, Bureau of Laboratories

PETER SKALIY, Ph.D.

Chief, Microbiological Control Section, Epidemiologic Investigations Laboratory Branch, Bacterial Diseases
Division, Bureau of Epidemiology

ARNOLD G. STEIGERWALT, B.S.

Research Microbiologist, Enterobacteriology Branch, Bacteriology Division, Bureau of Laboratories

W. DANIEL SUDIA, Ph.D.

Deputy Director, Laboratory Training and Consultation Division, Bureau of Laboratories

MORRIS T. SUGGS, Dr.P.H.

Director, Biological Products Division, Bureau of Laboratories

BERENICE M. THOMASON. B.S.

Researcii Microbiologist, Analytical Bacteriology Branch, Bacteriology Division, Bureau of Laboratories

CLYDE THORNSBERRY, Ph.D.

Chief, Antimicrobics Investigations Section, Clinical Bacteriology Branch, Bacteriology Division, Bureau
of Laboratories

vu

Contributors

AVIS VAN ORDEN

Histopathology Technician. Pathology Division, Bureau of" Laboratories

ROBERT E. WEAVER, M.D.. Ph.D.

Cliief, Special Bacteriology Section. Clinical Bacteriology Branch. Bacteriology Divisioti. Bureau of Lab-
oratories

HAZEL W. WILKINSON. Ph.D.

Chief". Special Immunology Laboratory. Bacterial Immunology Bratich. Bacteriology Division, Bureau
ot~ Laboratories

LYNNP. YEALY. M.S.

Medical Technologist, Bacterial Immunology Branch, Bacteriology Division, Bureau of Laboratories

vui

Preface

This laboratory manual was designed to be used in training activities related to Legionnaires'
disease methodology and as such is intended to be a working model. It represents a concerted
effort to compile useful information for the medical community on the progress which has been
made at the Center for Disease Control (CDC) in defining a disease entity and its causative agent.

The history and nature of Legionnaires' disease are discussed on the basis of evidence
gathered through examination of the clinical and pathological features of the disease.

All of the known characteristics of the Legionnaires' disease bacterium (LDB), including
cellular fatty acid composition, antimicrobial susceptibility, and deoxyribonucleic acid (DNA)
hybridization, are described in a series of chapters. Brenner et al. proposed the classification
Legionella pueiuuophilu sp. nov. for the LDB after evaluating the results of their extensive DNA
relatedness studies with LDB strains from serogroups 1, 2, and 3. The recently described sero-
group 4, as well as some future LDB isolates, may or may not be closely related to these first
three serogroups; therefore, until proven to be L. piieuinopliila, they should be designated by the
broader term LDB. Since a clear-cut distinction is not always made between serogroups 1-3
(Z.. pneuniopliila) and serogroup 4, the editors and authors have used the more general and
familiar designation LDB when discussing the causative organism of Legionnaires' disease.

Specific procedures are detailed for animal and egg inoculation, media preparation and use,
direct and indirect immunotluorescence, and various other methods for safely testing clinical
and environmental material. Although modifications in technique will continue to appear as
research progresses, the procedures outlined in this manual have proven reliable and can be used
diagnostically.

Another section of the manual catalogs most aspects of the CDC assistance program pertain-
ing to Legionnaires" disease. Included are pohcies regarding technical support and consultation and
procedures to follow in obtaining materials and services beyond the scope of many institutions.

The final section of the manual contains a reading bibliography as a general reference. We
realize that the rapidity with which new material on Legionnaires' disease is being published
makes any such listing incomplete. but it is offered to the reader as a reference base.

Although the material presented in this manual reflects the "state of the science" relative
to the LDB at CDC, the editors wish to stress the fact that this is not an all-inclusive publication.
Valuable work is being reported from other institutions, and we urge the reader to utilize these
resources as they become available.

In addition to the contributors listed elsewhere, this manual could not have been produced
without the assistance of Glenda S. Cowart and Thena M. Durham of the Biological Products
Division. Bureau of Laboratories; the staff of Publications Management Branch, and Louise
Lewis, CDC Library, Office of the Center Director; Fay Neal, Gerri Stedman, and Lois Jennings
of the Laboratory Training and Consultation Division, and Claudia Lewis, Effie Spencer, Donna
Mills, and Barbara Gary of the Word Processing Activity, Bureau of Laboratories.

Gilchi L Jones
G. Ann Hebert
Editors

IX

Contents

Page

Foreword iii

Acknowledgements iv

Contributors v

Preface ix

PART ONE
The Disease

Clinical Manifestations of Legionnaires' Disease and Recommended Therapy 3

William B. Baiiie

Pathologic Features of Legionnaires' Disease 9

Joivi A. Blacknioii. Francis W. Chandler, and Martin D. Hicklin

The Epidemiology of Legionnaires' Disease 13

William B. Baine

PART TWO
The Bacterium

Cultural and Biochemical Characterization of the Legionnaires' Disease

Bacterium 19

Robert E. Weaver and Janies C. Feeley

Physiology: Characteristics of the Legionnaires" Disease Bacterium in

Semisynthetic and Chemically Defined Liquid Media 27

Leo Pine, J. Richard George, M. W. Reeves, and W. Knox Harrell

Electron Microscopy of the Legionnaires' Disease Bacterium 41

F.W. Chandler, J. A. Blackmon, M.D. Hicklin, R.M. Cole, and CS. Callaway

Cellular Fatty Acid Composition of the Legionnaires' Disease Bacterium 47

C Wayne Moss. R.E. Weaver, S.B. Dees, and W.B. Cherry

Antimicrobial Susceptibility of the Legionnaires' Disease Bacterium 55

Clyde Thornsberry and Linda A. Kirven

Legionella pneumophila sp. nov.: The Legionnaires' Disease Bacterium 59

Don J. Brenner, Arnold G. Steigerwalt, G. Ann Hebert. and Joseph E. McDade

X

PART THREE
Methodology

Primary Isolation Using Guinea Pigs and Embryonated Eggs 69

Joseph E. McDade

Primary Isolation Media and Methods 77

James C. Feeley. George W. Gorman, and Robert J. Gibson

Method for Isolating Legionnaires' Disease Bacterium from Soil and

Water Samples 85

George K. Morris. Peter Skaliy. Charlotte M. Patton. and Janies C. Feeley

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by

Direct Immunofluorescence 91

William B. Cherry and Roger M. Mc Kinney

Demonstration of the Bacterium in Tissue by a Modification of the Dieterle

Silver Impregnation Stain 105

A.E. Van Orden and P.W. Greer

Indirect Immunofluorescence Test for Legionnaires' Disease Ill

Hazel W. Wilkinson. Donna D. Cruce. Bonnie J. Fikes. Lynn P. Yealy,
and Carol E. Farshy

Analysis of Cellular Fatty Acids of Bacteria by Gas-Liquid Chromatography 117

C Wayne Moss

ELISA for Legionnaires' Disease - Antibody and Antigen Detection Systems:

An Interim Report 123

Carol E. Farshy cvid John C. Feeley

PART FOUR
Policies, Resources and Services

I. Laboratory Safety: Precautions and Practices 134

II. Laboratory Services

A. Pohcy 137

B. Reference Material 137

C. Reference Diagnostic Services 142

D. Submitting Reference Specimens 144

III. Epidemiologic Services 1 49

A. Policy 149

B. Epidemiologic Consultation and Assistance 149

C. Clinical Consultation 149

xi

PART FIVE
Appendix

I. Chromogenic Cephalosporin Test foriS-Lactamase Production 154

II. Cultivation of tiie Legionnaires' Disease Bacterium in Yolk Sac

of Embryonated Hens' Eggs 156

III. Detection of tlie Legionnaires' Disease Bacterium in Clinical

Specimens (tlovv chart) 157

IV. Blood-Freeze Storage of Bacteria 158

V. Immunonuorescence Buffers and Mounting Fluid 159

VI. Normal Chicken Yolk Sac (NYS) 160

VII. McFarland Nepiielometer Density Standards 160

PART SIX
Bibliography

XII

PART ONE

The Disease

Clinical Manifestations of

Legionnaires' Disease

and Recommended Therapy

William B. Baine

Hospital Infections Branch

Bacterial Diseases Division

Bureau of Epidemiology

Clinical Manifestations of Legionnaires' Disease
and Recommended Therapy

William B. Baine

Legionnaires' disease (LD) has most commonly been recognized as a form of pneumonia.
Symptoms of this syndrome usually become apparent 2 to 10 days after known or presumed
exposure to airborne Legionnaires" disease bacteria (LDB). The earliest symptoms are malaise,
myalgia, and mild headache. A non productive cough is common, but sputum production is
sometimes associated with the disease. Within less than a day the patient may experience rapidly
rising fever and the onset of chills. Although physical examination of the patient may reveal rales
on auscultation, fever to 39°- 41°C (102°-105°F), and relative bradycardia, no physical findings
are specific to this disease. Associated manifestations may include confusion, chest pain, abdom-
inal pain, imparled renal function, and diarrhea.

Routine laboratory tests commonly reveal a moderate leukocytosis with a shift to the left in
the granulocyte series. Proteinuria, hyponatremia, hypophosphatemia, azotemia, elevated amino-
transferase levels, and a high erythrocyte sedimentation rate are often seen in various combina-
tions. Radiographs of the chest reveal patchy infiltrates that may progress to extensive consolida-
tion. Cavitary pneumonia is uncharacteristic, but some examples have been reported. A case-
fatality rate of approximately 15% for sporadic cases has been reported in the United States.

Two LD case histories are presented below:

Case History #1.

A 59-year-old construction worker was hospitalized July 5 in Missouri with a 5-day
history of malaise, myalgia, chills, and fever to 105°F after a 2'/2 week vacation trip to
Canada, the western United States, and Mexico. On this trip he had gone boating: visited a
sawmill, a zoo, and the engine room of a ship: fed birds in a public park: and repaired some
malfunctioning air conditioners in a hotel. He did not smoke, and his past medical history
was unremarkable except for two episodes of pneumonia in childhood and a case of malaria
while serving in the South Pacific during World War II.

The physical examination was unremarkable except for the presence of fever and of
occasional rales in the left lung.

Admission urinalysis revealed a specific gravity of 1.033, pH 6, 1+ protein, 6-8 white
cells, and 3-4 red cells. The admission hematocrit was 44% with a hemoglobin of 14.2 g/dl,
and 13,300 white cells/nl. The white cell differential was 72% neutrophils (including 3%
bands), 4% monocytes, and 24% lymphocytes with an adequate number of platelets. Blood
chemistries were generally within noimal limits. The chest radiograph on admission revealed
an infiltrate at the left base.

He was treated with cephalexin, penicillin, and glucocorticoids and continued to have
hectic fevers. Within 3 days the infiltrate at the left base had partially resolved, but new
infiltrates were present in the left mid-lung field, the left infraclavicular area, and the right
mid-lung field (Figure 1). A lung biopsy perfonned on the I 1 th hospital day revealed
consolidation and a severe intlammatory reaction with a significant polymorj^honuclear
component. After 8 days in the hospital, the patient became afebrile. Routine attempts to

Cliiiic;il M;init'cstations of Legionnaires' Disease and Recommended Therapy

Figure 1. Case No. 1. Postero-
anterior radiograph of chest
on 4th hospital day, showing
infiltrates at the left base, at
the left and right mid-lung
fields, and at the left infra-
clavicular area.

Clinical Manilcstations of Legionnaires' Disease and Recommended Therapy

define a microbial etiology were unsuccesst'ul. The patient's condition improved greatly, and
he was discharged on the 1 5th hospital day with a diagnosis of pneumonia of undetermined
etiology, possibly caused by a virus or the LDB. From July 8 to July 18 his antibody titer to
the LDB (serogroup 1) by indirect immunonuorescence rose from 32 to 256.

Case History #2.

A 52-year-old British tourist experienced malaise and fever on September 15 while
vacationing in Spain. He returned to England on September 20. Three days later he develop-
ed cough, shortness of breath, and diarrhea. On admission to a hospital on September 24, he
was confused and had a temperature of 40°C and signs of right-sided pneumonia. His chest
radiograph showed infiltrates out of proportion to the clinical findings, and his leukocyte
count was nomial; he was therefore treated for primary atypical pneumonia with erythro-
mycin. His fever lysed within 3 days, but he remained confused, and a lumbar puncture and
an encephalogram were done because encephalitis was suspected. No abnormalities were
found, and by October 7 the patient was asymptomatic except for residual asthenia. Rou-
tine microbiological diagnostic tests were unrevealing. Indirect immunotluorescence was
used to document the patient's seroconversion to the LDB (serogroup 1 ) antigen.

Legionnaires' disease may also take the form of a self-limited illness with fever, myalgia,
malaise, and headache, but with few or no respiratory manifestations and no pneumonia. This
syndrome, originally temied "Pontiac fever," is remarkable for the absence of associated fatal-
ities. The reason for the difference between the pneumonic and Pontiac fever syndromes of LD
remains conjectural.

Symptomatic therapy suffices for patients with the Pontiac fever syndrome. Specific treat-
ment for pneumonia caused by the LDB is not based on controlled clinical trials of human illness,
but available evidence suggests the eiythromycin reduces the risk of fatality among patients with
pulmonic LD. Doses of up to 1 gram every 6 h of erythromycin gluceptate or lactobionate can be
given intravenously as the initial therapy for seriously ill patients. Note that the intravenous
formulations of erythromycin must be reconstituted in Sterile Water tor Injection without bacte-
riostatic preservatives. The dose can be lowered, and the medication can be given orally as the
patient's condition stabilizes. The optimal duration of treatment remains unclear; some clinicians
experienced in treating patients with LD continue antibiotic therapy for 3 wk or longer, but this
is not a universal practice. The dosage of erythromycin must be adjusted for patients with severe
hepatic disease or renal failure. An alternative to erythromycin is a tetracycline. Rifampin is also
effective against the LDB in several laboratory models, but it should only be prescribed for LD
patients as a supplement to erythromycin if the patients have not responded well to a single
antibiotic. It could also be used in conducting properly organized trials to compare erythromycin
therapy with erythromycin-rifampin therapy. Treatment with penicillins, cephalosporins, or
aminoglycosides does not appear to benefit patients with LD.

Supportive therapy is as important as antibiotic treatment for patients with LD. Those with
pneumonia should have their arterial blood gases monitored, and oxygen therapy and assisted
ventilation should be provided as indicated. Attention to fluid and electrolyte balance is manda-
tory—particularly for patients with high fever, diarrhea, or disturbances of consciousness. Patients
who have hypotension or shock may need to have carefully monitored urine output, central
venous pressure, or pulmonary capillary wedge pressure, and intra-arterial blood pressure to
provide a guide for intravenous fluid administration and vasopressor therapy. Renal function
should be followed carefully during the acute illness; an occasional patient requires dialysis until
renal function is restored. Stuporous and comatose patients require special nursing care to pre-
vent conjunctival desiccation, parotitis, aspiration of gastric contents, urinary retention, fecal
impaction, and pressure sores.

Clinical Manifestations of Legionnaires' Disease and Recommended Therapy

SELECTED REFERENCES

1. Beaty, H.N.. A.A. Miller, C.V. Broome, S. Goings, and C.A. Phillips. 1978. Legionnaires" disease in Vermont,
May to October 1977. JAMA 240: 127-131.

2. Center for Disease Control. 1978. Legionnaires" disease: Diagnosis and management. Ann Intern Med 88:
363-365.

3. Fraser, D.W., I.K. Wachsmulh, C. Bopp, J.C. Feeley and T.F. Tsai. 1978. Antibiotic treatment of guinea-pigs
infected with agent of Legionnaires" disease. Lancet i: 175-1 78.

4. Friedman, H.M. 1978. Legionnaires" disease in non-Legionnaires: A report of tlve cases. Ann. Intern. Med.
88:294-302.

5. Gregory, D.W.,W. Schaffner, R.H. Alford, A.B. Kaiser, and Z.A. McGee. 1979. Sporadic cases of Legionnaires'
disease: Tl;ie expanding clinical spectrum. Ann. Intern. Med. 90:518-521.

6. Helms. C. J. Viner. R. Strum. E. Renner. and W. Johnson. 1979. Comparative features of pneumococcal,
mycoplasmal, and Legionnaires' disease pneumonias. Ann. Intern. Med. 90:543-547.

7. Kirby, B.D., K.M. Snyder, R.D. Meyer, and S.M. Finegold. 1978. Legionnaires" disease: Clinical features of
24 cases. Ann. Intern. Med. 89:297-309.

8. Saravolatz, L.. K. Burch, E. Fisher, T. Madhavan, D. Kiani, T. Neblett, and E. Quinn. 1979. The compromised
host and Legionnaires' disease. Ann. Intern. Med. 90:533-537.

9. Tsai, T.F., D.R. Finn, B.D. Plikaytis, W. McCauley, S.M. Martin, and D.W. Fraser. 1979. Legionnaires' disease:
Clinical features of the epidemic in Philadelphia. Ann. Intern. Med. 90:509-517.

10. Tsai, T.F. and D.W. Fraser. 1978. The diagnosis of Legionnaires" disease. Ann. Intern. Med. 89:41 34 14.

Pathologic Features of
Legionnaires' Disease

John A. Blackmon

Francis W. Chandler

Martin D. HickHn

Pathology Division
Bureau of Laboratories

Pathologic Features of Legionnaires' Disease

John A Blackitioii. Fnincis W. Cliaiuller. and Manui D Ilicklin

Both autopsy and biopsy specimens from patients with Legionnaires' disease (LD) have been
examined in the Pathology Division laboratory at the Center for Disease Control. The only con-
sistent pathologic findings in autopsies of patients dying with acute LD were in the lungs where
a pneumonia was present. This pneumonia was frequently contTuent. often involving one or more
lobes in their entirety. In lung specimens the diseased tissue has appeared gray and granular.
Fibrinous pleuritis has frequently been present.

Microscopically, the histologic pattern of tissues examined at autopsy from patients with
LD has been principally that of an acute fibrinopurulent pneumonia which resembles the hepati-
zation stages of lobar pneumonia. Of particular note is the exudation of neutrophils, macro-
phages, and large amounts of fibrin into the alveolar spaces (Figs. 1 and 2). Often this material
can be seen passing through pores of Kohn. In some cases there is extensive necrosis of the in-
flammatory exudate. The extent of inflammatory cell necrosis varies, and the observer should
not be surprised to find areas and sometimes entire lungs in which the cells are quite intact. The
underlying lung structure remains relatively undisturbed in acute pneumonias caused by the
Legionnaires' disease bacterium (LDB). However, focal interstitial necrosis, which is virtually
nonexistent in uncomplicated pneumococcal lobar pneumonia, is occasionally seen in LD. The
degree to which red blood cells extravasate into the alveoli varies. Most sections contain few or
no intra-alveolar erythrocytes, but areas of moderate to severe hemorrhage are occasionally
present. In LD pneumonia, the inflammatory process has not been noted to involve blood vessel
walls or large bronchi. Interstitial infiltration during the acute stage of the disease is minimal.

The staining quahties of the LDB are unusual. It does not stain with hematoxylin and eosin
and stains only rarely with standard tissue stains for bacteria, such as the Brown-Brenn, Brown-
Hopps, and MacCallum-Goodpasture procedures. However, the organism can be demonstrated
consistently with the Dieterle silver impregnation procedure (Fig. 3). a fact which makes it the
staining method of choice for the LDB in our laboratory. Other investigators have reported to us
that the Warthin-Starry and other silver impregnation techniques are also effective. Some institu-
tions have used various modifications of the Giemsa stain with some success in demonstrating
the LDB in paraffin sections, but in our hands this procedure has not been satisfactory. Even
when the organisms can be stained with this method, it is difficult to obtain adequate color
differentiation between the bacterium and the inflammatory background. The Gimenez stain
is useful for impression smears and frozen sections but is unsatisfactory for paraffin-embedded
tissue.

In tissue sections stained by the Dieterle selver impregnation procedure the LDB's appear
as short, pleomorphic rods (Fig. 4) measuring 2 to 4 microns in length and up to 1 micron in
diameter. Some of these rods appear bipolar, and beaded forms are noted. Organisms are dif-
fusely distributed throughout the areas affected by the acute pneumonia, but concentrations
vary from patient to patient. The degree of necrosis of the inflammatory exudate is directly
related to the number of organisms that can be demonstrated. Clusters of organisms are commonly
observed within macrophages in Dieterle-stained sections. In some cases, by focusing up and

10

Pathologic [■eatures of Legionnaires' Disease

Z77!?

Figure 1. Intia-alveolar exudate filling alveolar space. Note
necrosis of inflammatory cells (hematoxylin-eosin. original
magnification X 100).

Figure 2. Detail of inflammatory e.xudate. Note mixture of
polymorphonuclear leukocytes, macrophages, and fibrin
(hematoxylin-eosin, original magnification X 200).

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Figure 3. Legionnaires" disease bacteria in area of consolida-
tion. (Dieterle silver impregnation, original magnification
X 200).

Figure 4. Legronnaires' disease bacteria. Note that they
appear as short, blunt rods (Dieterle silver impregnation,
original magnification X 500).

11

Pathologic Features of Legionnaires' Disease

down through individual cells under oil immersion or by examining tissue with the electron
microscope, 50 or more organisms can be observed in individual macrophages. Many extra-
cellular fonns can also be seen.

We have examined a number of surgical specimens from patients who had serologically
confirmed LD. In some, the histologic pattern was identical to that of the acute fibrinopurulent
pneumonia previously described for autopsy specimens. In other cases an organizing pneumonia
with varying degrees of interstitial fibrosis has been present. We have been unsuccessful in our
efforts to demonstrate organisms in these organizing pneumonias using silver impregnation or
immunofluorescent staining, and in these cases the diagnosis of LD has been confirmed retro-
spectively by seroconversion. Organization may occur late in the course of the disease in an
unknown percentage of patients and may result in serious pulmonary functional impairment.

The pathologic features described here cannot serve as the sole basis for diagnosing LD.
However, such histologic patterns in lung tissue certainly indicate the need to rule out or confirm
a diagnosis of this disease. A definitive diagnosis of LD must be obtained on the basis of results
from the more specific laboratory methods described in other sections of this manual.

REFERENCES

L Blackmon, J. A., R.A. Harley, M.D. HickJin, and F.W. Chandler. 1979. Pulmonary sequelae of acute
Legionnaires' disease pneumonia. Ann. Intern. Med. 90:552-554.

2. Blackmon, J. A., M.D. HickJin, F.W. Chandler, and the Special Expert Pathology Panel. 1978. Legion-
naires' disease: Pathologic and historical aspects of a "new" disease. Arch. Pathol. Lab. Med. 102:
337-343.

3. Chandler, F.W., M.D. Hicklin, and J. A. Blackmon. 1977. Demonstration of the agent of Legionnaires'
disease in tissue. N. Engl. J. Med. 297:1218-1220.

4. Gimenez, D.F. 1964. Staining rickettsiae in yolk-sac cultures. Stain Technol. 39:135-140.

5. Luna, L.G. 1968. Manual of Histologic and Special Stairung Technics of the Armed Forces Institute
of Pathology, ed. 3. New York, McGraw-Hill Book Co., Inc.

6. Van Order, A.E., and P.W. Greer. 1977. Modification of the Dieterle spirochete stain. J. Histotechnol.
1:51-53.

7. Winn, W.C, F.L. Glavin, D.P. Perl, J.L. Keller, T.L. Andres, T.M. Brown, CM. Coffin, J.E. Sensecqua,
L.N. Roman, and J.E. Craigliead. 1978. The pathology of Legionnaires' disease. Arch. Pathol. Lab.
Med. 102:344-350.

The Epidemiology of
Legionnaires' Disease

William B. Baine

Hospital Infections Branch

Bacterial Diseases Division

Bureau of Epidemiology

13

The Epidemiology of Legionnaires' Disease

William B. Baiiie

The temi "Legionnaires' disease" (LD) is currently used to refer to illness caused by the
bacterium that caused the 1976 outbreak of respiratory illness in Philadelphia or by another
strain of the same organism. Legionnaires' disease can occur in large common-source outbreaks,
as a series of cases in areas of apparently high endemicity, or as sporadic cases without apparent
temporal or geographic clustering.

The earliest documented outbreak of LD occurred in 1965 at St. Elizabeth's Hospital, a
large psychiatric facility in Washington, D.C. Eighty -one patients became ill, and 14 died. Epi-
demiologic evidence suggested a link between infection and wind-blown dust from excavations on
the hospital grounds. In 1968 at least 144 cases of self-limited Olness occurred in employees or
visitors who had entered a health department building in Pontiac, Michigan. Investigation at that
time demonstrated that the etiologic agent was present in water in the evaporative condenser of a
malfunctioning air-conditioning system. The Pontiac agent is now known to have been the
Legionnaires' disease bacterium (LDB). In July 1973, an estimated eight Scottish vacationers
contracted LD while vacationing in Benidorm, Spain; three died. In 1974 at least 20 persons
attending an Oddfellows Convention at the Bellevue Stratford Hotel in Philadelphia developed
pneumonia, and two died. This outbreak has since been shown to have been a cluster of cases of
LD.

The existence of the LDB was first recognized in laboratory investigations of a large out-
break of pneumonia among persons associated with Philadelphia's Bellevue Stratford Hotel in
1976. Of those who were known to have been inside the hotel at some point during an American
Legion Convention, 182 became ill, and 29 died. An additional 39 cases, of which 5 were fatal,
were reported among persons who had been near the hotel in that period. Subsequently, in 1977
and 1978, there were clusters of documented LD cases from Dallas, Texas; and in Columbus,
Ohio; Burlington, Vermont; Kingsport, Tennessee; Nottingham, England; Los Angeles, California;
Bloomington, Indiana; Atlanta, Georgia; New York, New York: Memphis, Tennessee; and Nor-
walk, Connecticut.

As methodology for the diagnosis of Legionnaires" disease has developed, it has become
possible to detect clusters of cases that might otherwise have gone unrecognized. In some in-
stances, LD has continued to affect people in localized areas without evidence of the temporal
clustering that would suggest a point-source epidemic. A special instance of such areas of high
endemicity is seen in several clusters of nosocomial LD. Although the LDB can infect persons
who have no serious underlying illness, LD has frequently been associated with patients who have
impaired immunologic defenses. Thus, cases may be associated with a given hospital only because
the institution is a focal point for patients with renal heterografts or advanced malignancy. In
some instances, though, hospitals, like other public buildings appear to have been the site of
acquisition of infection with the LDB.

A characteristic of the epidemiology of LD is an association between infection and reser-
voirs of the LDB in the inanimate environment. The bacterium has been isolated from water from
cooling towers or evaporative condensers in air-conditioning systems in Atlanta, Bloomington,

14

The I:pidcniicilogy of Legionnaires' Disease

Columbus, Dallas. Los Angeles, Memphis, New York, Norvvalk. and Pontiac, and from water and
soil from streams in Atlanta and Bloomington. Success in recovering the organism from environ-
mental sites does not implicate such sites in transmission of disease, but in some instances
epidemiologic evidence uiiplicating an environmental source has been supported by bacteriologic
results.

More than 500 sporadic cases of LD have been reported to the Center for Disease Control
(CDC) from at least 43 states and the District of Columbia (Figure 1). Approximately \97o of the
reported cases were fatal, but this figure may overestimate the true proportion of fatal cases. The
distribution of reported cases indicates a 2.4:1 ratio of males to females, and 907r of reported
cases occur in people over 30 years of age. Cigarette smoking and alcohol abuse are clearly risk
factors in acquiring LD, and a significant minority of patients report recent travel or exposure to
excavation sites. Outbreaks and sporadic cases in the United States appear to be most common in
the summer.

The Conference of State and Territorial Epidemiologists has collaborated with CDC in
establishing a reporting system for this disease in the United States. Outside the United States,
indigenous or imported cases of LD have been reported from Australia, Austria, Canada, Den-
mark, England, Israel, Italy, the Netherlands, Scotland, Spain, and Sweden. Cases have also been
associated with travel to other European countries.

For some outbreaks and most sporadic cases, the mode of transmission of LD has not been
documented, but in several outbreaks there is strong evidence for airborne spread of the agent,
and it is possible that the airborne route is the only significant mode of transmission. Person-to-
person spread of LD has not been conclusively demonstrated and must occur rarely if at all.

The need to isolate hospitalized patients with LD is not established, and superfluous iso-
lation of patients is detrimental to their care. On the other hand, the presence of the LDB has
been demonstrated in respiratory secretions from some infected persons; patients with LD may

0 - 2.5
0.5-1.0

n<.5

CASES PER 1,000,000 POPULATION

C2>

PUERTO BICO

^l

O.

VIRGIN ISLANDS

FIGURE 1. Sporadic cases of Legionnaires' disease in the United States, May 1973 through October 1978.
Numbers within states indicate total reported cases per state.

15

The Epidemiology of Legionnaires' Disease

receive assisted ventilation, witli the attendant generation of aerosols; and other hospitalized
patients may be particularly susceptible to infection with the LDB. Until more data are available
to assess the risk of interpersonal spread of LD in the hospital, CDC recommends that patients
with documented cases of LD be placed in respiratory isolation until they respond to antibiotic
therapy. CDC further recommends that the nursing care for any patient with bacterial or viral
pneumonia include oral secretion precautions. Isolation of patients with pneumonia should be
limited to instances in which there is clinical or laboratory evidence of infection with certain
transmissible respiratory pathogens (see Isolation Techniques for Use in Hospitals, 2nd ed.,
1975, HEW Pubhcation No. [CDC] 78-8314).

In practice, most cases of LD are diagnosed retrospectively, and the issue of isolation does
not arise.

SELECTED REFERENCES

1. Broome. C.V., S.A.J. Goings, S.B. Thacker, R.L. Vogt, H.N. Beaty, and D.W. Fraser. 1979 The Vermont
Epidemic of Legionnaires" disease. Ann. Intern. Med. 90:573-577.

2. Dondero, T.J., Jr., H.W. Clegg II, T.F. Tsai, R.M. Weeks, E. Duncan. J. Strickler, C. Chapman, G.F. Mallison,
B.D. Politi, M.E. Potter, and W. Schaffner 11. 1 979. Legionnaires' disease in Kingsport, Tennessee. Ann. Intern.
Med. 90:569-573.

3. Fraser, D.W., T.F. Tsai, W. Orenstein. W.E. Parkin, H.J. Beecham, R.G. Sharrar, J. Harris, G.F. Mallison, S.M.
Martin, J.E. McDade, (^ £ . Shepard, P.S. Brachmaii, and the Field Investigation Team. 1977. Legionnaires"
disease: Description of an epidemic of pneumonia. N. Engl. J. Med. 297:1 189-1 196.

4. Glick. T.H., M.B. Gregg, B. Berman, G. Mallison, W.W. Rhodes, Jr., and I. Kassanoff. 1978. Pontiac fever. An
epidemic of unknown etiology in a health department. I. Clinical and epidemiological aspects. Am. J.
Epidemiol. 107:149-160,

5. Haley, C.E., M.L. Cohen, J. Halter, and R.D. Meyer. 1979. Nosocomial Legionnaires' disease: A continuing
common-source epidemic at Wadsworth Medical Center. Ann. Intern. Med. 90:583-586.

6. Marks, J.S., T.F. Tsai, W. Martone, R. Baron, J. Kennicott, F. Holtzhauer, I. Baird, D. Faye, J. Feeley, G.
Mallison, D. Fraser, and T.J. Halpin. 1979. Nosocomial Legionnaires" disease in Columbus, Ohio. Ann.
Intern. Med. 90:565-569.

7. Politi. B.D., D.W. Fraser, G.F. Mallison, J. Mohatt, G.K, Morris, CM. Patton, J.C. Feeley, R.D. Telle, and
J.V. Bennett. 1979. A major focus of Legionnaires' disease in Bloomington, Indiana. Ann. Intern. Med. 90:
587-591.

8. Storch, G., W.B. Baine, D.W. Fraser, C.V. Broome, H.W. Clegg, II, M.L. Cohen, S.A.J. Goings, B.D. Politi,
W.A. Terranova, T.F. Tsai, B.D. Plikaytis, C.C. Shepard, and J.V. Bennett. 1979. Sporadic community-
acquired Legionnaires' disease in the United States: A case-control study. Ann. Intern. Med. 90:596-600.

9. Terranova, W., M.L. Cohen, and D.W. Fraser. 1978. 1974 outbreak of Legionnaires" disease diagnosed in
1977: Clinical and epidemiological features. Lancet 2:122-124.

10. Thacker, S.B., J.V. Bennett, T.F. Tsai, D.W. Fraser, J.E. McDade, Q.C. Shepard, K.H. WUliams. Jr., W.H.
Stuart, H.B. Dull, and T.C. Eickhoff. 1978. An outbreak in 1965 of severe respiraton' illness caused by the
Legionnaires' disease bacterium. J. Infect. Dis. 138:512-519.

16

PART TWO

The Bacterium

Cultural and Biochemical Characterization
of the Legionnaires' Disease Bacterium

Robert E. Weaver
James C. Feeley*

Clinical Bacteriology Branch

Bacteriology Division

Bureau of Laboratories

and

Epidemiologic Investigations Laboratory Branch''

Bacterial Diseases Division

Bureau of Epidemiology

19

Cultural and Biochemical Characterization
of the Legionnaires' Disease Bacterium

Robert E. Weaver and James C. Feeley

Although few cultural and biochemical characteristics have been defined, they are suffi-
ciently distinct to enable a clinical microbiologist to differentiate the Legionnaires' disease bac-
terium (LDB) from other bacteria. Over 50 strains of LDB have been isolated and identified with
the tests described here.

CULTURAL CHARACTERIZATION

Inoculate the suspected isolate of LDB on charcoal yeast extract (CYE), Feeley-Gorman
(F-G), and blood agar (BA) media (4). In addition, the medium on which the LDB first grew,
Mueller-Hinton agar supplemented with \9r hemoglobin and 1% IsoVitaleX (MH-IH), may be
inoculated (optional). (The chapter by Feeley et al. in this manual describes these media and
their fomiulation.) Streak the inoculum on each medium to obtain well-isolated and discrete
colonies.

The LDB grows best under aerobic conditions or in air plus 2.57c COj , depending on the
medium used. It does not grow anaerobically. Incubate all media except CYE at 35°C either in
air plus 2.5% COj or in a candle extinction jar. Incubate CYE agar at 35°C aerobically in a moist
chamber. Examine all plates daily for 2 wk.

A. Colonial Morphology

On the MH-IH agar, the LDB grows slowly. The colonies of the various strains which have
not been transferred more than a few times on the agar are pinpoint in size at 3 days. By 5-7
days, well-isolated colonies may reach a diameter of 3 or 4 mm. The colonies are convex, circular,
and have an entire edge. They are gray and glistening. In contluent areas the growth appears to be
slightly moist.

On CYE agar growth of the LDB is apparent in 48 h or less in confluent areas at 35°C and
42°C. More growth occurs at 35°C than at 42°C. At 25°C. growth is apparent in 4-7 days.
Further description of growth on F-G and CYE agars is in the chapter by Feeley et al.

B. Microscopic Morphology

Remove a portion of the growth from the CYE agar with a bacteriologic needle. Emulsify
the growth in approximately 1.0 ml sterile distilled water to make a slightly turbid cell sus-
pension. Transfer a small aliquot of this suspension to each of two microscope slides, spread into
a film, let air dry, and then heat fix. Stain the smears using the Gram and fat stain procedures
below.

I. Gram stain

Flood smears with crystal violet solution for 1 min; rinse with water. Add Gram's
iodine for 1 min; rinse and decolorize with 95% ethyl alcohol. Counterstain either with

20

Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium

.

^

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Figure 1. Gram stain using carbol tuciisin instead of safranin Figure 2. Gram stain using carbol luchsin counterstaiii (LDB

for the counterstain (LDB strain Piiiladeiphia 1 from CYE strain Knoxville 1 from CYE agar).

agar).

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Figure 3. Fat stain of LDB.

Figure 4. Gime'nez-stained smear of an LDB-infected egg
yolk sac, courtesy of Dr. Joseph E. McDade.

Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium

safranin or carbol fuchsin for 1 min; rinse and air dry. Examine microscopically using oil
immersion.

The LDB is a gram-negative bacillus having an appro-ximate width of 0.5-0.7 /jm and a
length that varies from 2 to 20 nm or more. The cells appear red when counterstained with
carbol fuchsin (Figs. 1 and 2) and light pink when counterstained with safranin. LDB cells
on initial culture and passage on MH-IH medium may appear slightly swollen in regions
having vacuoles that are stainable with the Sudan black B fat stain.

Prepare the carbol fuchsin counterstain by mixing the two following solutions.

Solution A;

Solution B:

Basic fuchsin

0.3 gm

Phenol (melted crystals)

5 ml

Ethyl alcohol

10.0 ml

Distilled water

95 ml

2. Fat stain

Flood heat-fixed smear with Sudan black B solution and allow to stand 5 to 15 min.
Drain slide and blot dry. Apply xylene to smear several times either drop by drop or by
immersing the slide into a staining jar containing xylene. Blot the smear dry and counter-
stain with safranin for 60 sec. Rinse with water and let air dry. Examine using oil immersion
(Fig. 3).

Cells should appear pink and contain either blue-black or blue-gray droplets in areas
that were vacuolated in the Gram stain.

Prepare the Sudan black B solution by dissolving 0.3 gm of Sudan black B in 100 ml of
70% ethyl alcohol and letting the solution stand overnight at room temperature.

3. Gimenez stain

The LDB can be demonstrated in an infected egg yolk sac with the Gimenez stain: the
LDB stains red, and the yolk sac tissue appears as blue-green background cells (Fig. 4). The
Gimenez reagents and procedure are described in the chapter by McDade. This stain can also
be used for impression smears and frozen sections (see chapter by Blackmon et al. in this
manual).

4. Flagella stain

Recently, tlagella have been observed (Berenice M. Thomason, personal com-
munication) on several strains of the LDB by using a modified Leifson tlagella stain (i) or a
simplified silver-plating stain for tlagella (y).

C. Preliminary Identification

Organisms that have the three characteristics described below and have a cut-glass appear-
ance on F-G agar should be strongly suspected of being LDB, and should be further examined by
procedures listed under Definitive Identification.

1. Requirement for L-cysteine-HCl

All strains of LDB examined to date require L-cysteine-HCI for growth. Therefore
organisms that grow on BA medium, which does not contain L-cysteine-HCl, are unlikely to
be LDB. Gram-negative bacilli that grow only on L-cyesteine-HCl-containing media such as
CYE, F-G, and MH-IH agars are possibly LDB. Examine these further for brown pigment
and yellow fluorescence production.

22

Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium

2. Brown pigment

Inoculate either an F-G agar plate or slant, and incubate at 35°C in air plus 2.5% CO2 .
Pigment should be observable in areas of heavy confluent growth after 2-3 days of incub-
ation. Browning may also be produced with other media enriched with L-tyrosine at a
concentration equivalent to that found in Mueller-Hinton agar (1). Browning is not observa-
ble with CYE agar.

3. Yellow fluorescence

Inoculate a tube of F-G broth heavily with the suspected organism. Incubate at 35°C in
air plus 2.5%' COj . Examine daily in the dark with a 366-nm ultraviolet light. Broth should
tluoresce bright yellow within 3-4 days of incubation and continue to do so with additional
incubation. F-G broth has the same formulation as F-G agar except that it lacks agar-agar,
and the concentration of soluble ferric pyrophosphate (wt/vol) is 100 mg/liter. Tube broth
in 2-ml aliquots in screw-capped tubes (13 x 100 ml).

D. Definitive Identification

The LDB characteristically gives negative reactions for most biochemical tests with the
exceptions of catalase. oxidase, and j3-lactamase. Therefore, an isolate can only be definitively
identified as LDB with results from the direct fluorescent antibody (FA) test, gas liquid chroma-
tography (GLC), and deoxyribonucleic acid (DNA) hybridization.

1. Direct FA test

Prepare smears of the candidate organism, and stain it with all available conjugates
according to the methods described by Cheriy and McKinney in this manual.

2. Gas liquid chromatography

Prepare extracts of the candidate organism by the methods described by Moss et al. in
this manual, and examine for cellular fatty acids.

3. Deoxyribonucleic acid (DNA) hybridization

Brenner et al. have used this test veiy effectively for studying the LDB. Their proce-
dure is described in this manual. However, because this test is very specialized and time-con-
suming, it should be reserved for isolates that ( 1 ) have a compatible cellular fatty acid
profile by GLC and do not stain with direct FA conjugates prepared against all established
serogroups or (2) are considered to be LDB but have some unusual characteristic.

BIOCHEMICAL CHARACTERIZATION

This section is for those investigators who wish to further characterize an isolate as the LDB.

A. Carbohydrate Utilization

The LDB will not grow in the commonly used carbohydrate test media. For this reason,
carbohydrates were incorporated into especially prepared basal media to demonstrate utilization
by the LDB. All 15 strains tested grew but did not produce acid (Riley, Weaver, and Feeley,
unpublished data). Similar results were obtained with "rapid sugar" techinques (2). One test that
can be easily perfomied is Starch Utilization. Heavily inoculate an area of an F-G agar plate and
incubate for 3-5 days at 35°C in air plus 2.5%' CO2 . Flood plate with Gram's iodine and observe.
Most of the agar should stain blue because of the reaction of the starch contained in F-G agar
with the added iodine. There should be a clear zone void of blue color extending about 4 mm

23

Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium

beyond the edge of the bacterial growtli. Tliis reaction is considered to be a positive test and
suggests that starch has been utilized. The LDB is positive by this starch utilization test.

B. Catalase

Inoculate an F-G or CYE agar slant heavily; incubate at 35°C for 48-72 h. Examine the
growth for catalase activity by adding 2 to 3 ml of 3% hydrogen peroxide to the agar slant.
Bubbles should appear vv'ithin 30 sec. The LDB catalase reaction is positive and can be speeded up
if the growth is "broken up" slightly with a sterile capillary pipette. The "sticky" consistency of
the growth apparently reduces contact between the peroxide and the LDB cells.

C. Gekitinase

This test can be performed with a photographic film or a gelatin cupule. Prepare a turbid
suspension in broth (Mueller-Hinton or heart infusion) by emulsifying some growth from an F-G
agar plate.

1 . Fihii method

Place a strip (0.5 x 1.5 cm) of photographic film (Kodak, plus-X) into 0.5 ml of the
turbid cell suspension in a screw-capped tube (13 x 100 mm). Incubate at 35°C in air plus
2.5% CO, . The emulsion on the film should be digested within 48-72 h if the isolate is the
LDB.

2. Cupule method

Inoculate an API (Analytab Products Inc.) gelatin cupule with the turbid cell suspen-
sion. Incubate at 35°C in air plus 2.5% CO, . Gelatin should be digested within 48-72 h if
the isolate is the LDB.

D. (3-Lactamase

Inoculate the test organism heavily on the medium developed by Martin and Lewis (5) to
detect penicillinase-producing A'fme/va ^o/7on7;ofat'. Incubate at 35°C in air plus 2.5% CO2 for 7
days. Although the LDB will not grow well on this medium, it will inactivate the penicillin
contained in the medium and allow growth of the Sarcina lutea that is also in the medium.
Growth of the S. lutea indicates lactamase activity by the test organism. All LDB identified to
date produce jS-lactamase. In addition, /3-lactamase is a consistent characteristic of all LDB (7)
examined with the chromogenic cephalosporin test (see Appendix).

E. Nitrate

Potassium nitrate (0.2%) is incoiporated into agar slants in which Fikles" enrichment is
substituted for hemoglobin. (Reduction of nitrate by Pseudoinoiuis aeruginosa and Yersinia
enterocolitica can be readily demonstrated with the medium). The slants are inoculated heavily
and are incubated for 7 days. All LDB isolated to date are nitrate negative.

F. Oxidase

Wet a piece of filter paper with a 0.5'? solution of oxidase reagent ('')) (tetramethyl-
p-phenylenediamine dihydrochloride) which has been freshly prepared or refrigerated tor less
than a week. Test the suspect isolate for oxidase activity by transferring some growth from an
F-G agar plate with a platinum loop; rub the growth into the previously prepared filter paper. A
dark blue color should appear in 9-10 sec, indicating a weakly positive test if the isolate is the
LDB.

24

Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium

G. Urease

Inoculate a Christensen's urea agar (S) slant heavily with the suspected isolate. Incubate at
35°C in air plus 2.57r CO, for 24 h. The slant should not change color if the isolate is the LDB,
since all LDB isolated to date are urease negative by this test. The LDB does not grow on this
agar; however, if it were urease positive, the heavy inoculum would contain a sulTicient amount
of the enzyme to cause a positive reaction.

REFERENCES

1. Buine. W.B., J.K. Rasheed, J.C. Feeley, G.W. Gorman, L.E. Casida, Jr. 1978. Effect of supplemental L-tyrosine
on pigment production in culture of the Legionnaires" disease bacterium. Current Microbiology 1 :93-94.

2. Brown, W.J. 1974. Moditlcation of the rapid fermentation test for Neisseria gonorrhoeae. Appl. Micro. 27:
1027-1030.

3. Clark, W.A. 1976. A simplified Leifson flagella stain. J. Clin. Miciobiol. 3;632-634.

4. Feeley, J.C, G.W. Gorman, R.E. Weaver. D.C. Mackel, H.W. Sinith. 1978. Primary isolation media for Legion-
naires" disease bacterium. J. Clin. Microbiol. 8:320-325.

5. Martin, J.E., and J.S. Lewis. 1977. Selective culture screening for peniciUinase-producing Neisseria gonor-
rhoeae. The Lancet U: 605-606.

6. Paik, G., and M.T. Suggs. 1974. Reagents, stains, and miscellaneous test procedures. In Manual of Clinical
Microbiology. E.H. Lennette, E.H. Spaulding, and J.P. Truant (Eds.). 2nd Ed. 930-950. American Society for
Microbiology, Washington, D.C.

7. Thornsberry, C, and L.A. Kirven. 1978. /3-lactamase of the Legionnaires" bacterium. Curr. Microbiol. L51-54.

8. Vera, H.D., and M. DumotT. 1974. Culture media.//; Manual of Clinical Microbiology. E.H. Lennette, E.H.
Spaulding, and J.P. Truant (Eds.). 2nd Ed. 881-929. American Society for Microbiology, Washington, D.C.

9. West, Marcia, N.M. Burdash, and Frank Friemuth. U)77. Simplified silver-plating stain for flagella. J. Clin.
Microbiol. 6:414-419.

25

Physiology: Characteristics of

the Legionnaires' Disease Bacterium

in Semisynthetic and Chemically

Defined Liquid Media

L. Pine
J.R. George*
M.W. Reeves
W.K. Harrell*

Products Development Branch

and

Bacterial and Fungal Products Branch*

Biological Products Division

Bureau of Laboratories

27

Physiology: Characteristics of the Legionnaires' Disease Bacterium
in Semisynthetic and Chemically Defined Liquid Media

L. Pine. JR. George. M.W. Reeves, and W'.K. HarreU

INTRODUCTION

Recently, semisynthetic and cliemicaliy defined liquid media were described (6) for growing
the Legionnaires' disease bacterium (LDB). These media have allowed certain new obsei-vations
concerning growth characteristics of the organism and verification of characteristics observed
using the more complex Mueller-Hinton medium supplemented with hemoglobin and IsoVitaleX.
Feeley-Gonnan (F-G) agar, and charcoal yeast extract agar (CYE) (7. 2). In this chapter, we
discuss the media, the methods used for detemTining growth responses, certain idiosyncrasies
related to the use of the media, and some nutritional requirements and physiological aspects of
the LDB.

METHODS

A. Media

The basic fomiula for the semisynthetic medium for growing the LDB is in Table 1 : that for
the chemically defined medium is in Table 2. A 2X concentrated broth medium was prepared by
combining double-strength solutions, adjusting the pH, and sterilizing by filtration (0.45 micron,
Millipore Corp.). The other solutions, which contained starch and oleic acid or agar, or both,
were prepared, autoclaved for sterilization, and added to the 2X broth medium while they were
hot to produce either the complete medium or a medium of 5/4 or greater concentration. Liquid
media were used within 24 h. Complete agar media were tubed at 10 or 15 ml; 8 and 1 2 ml were
used if they were at a 5/4 or greater concentration. These tubes or other basal agar media were
stored at 5°C for 1 to 2 wk. Before they were used, agar media were melted by being steamed for
5 min: if necessary, test materials were added to the melted agar. The melted media were then
poured into plates, which were always used within 16 h. Similarly, culture tubes (18 X 150 mm)
were prepared by adding 1 ml of water or chemical solutions to 4 ml of 5/4-strength basal
medium.

B. Standard Inocula

Most of our early studies (6) were done with LDB strain Philadelphia 1. which was isolated
from a patient who died in the 1976 epidemic of Legionnaires" disease (LD) in Philadelphia (3).
We later included other strains in all stages of media evaluation. Inocula for various experiments
were prepared as follows: cultures were grown on F-G plates or slants: cells were washed off the
agar surface with sterile distilled water and stored as thick suspensions (approximately 9.0 mg dry
wt of cells/ml) at -70°C. Media were inoculated with a freshly thawed cell suspension by placing
1-3 drops (0.05 to 0.15 mg dr\' wt cells) on agar plates containing 10 or 15 ml of medium or in
tubes (18 X 150 mm) containing 5 to 15 ml of broth. In broth, this inoculum was sufficient to
raise the zero time suspensions to an absorbance (A) of 0.05 (1 drop or 0.05 mg) to 0.20 (3 drops
or 0.15 mg) at 660 nm. Relationship of A to cellular dry weight was detemiined by suspending

28

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29

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

washed cells in 1*7 Formalin, measuring the A, diying aliquots for 48 h at 75°C, and recording
the dry weight (Fig. 1). All A's were measured in a model B Beckman spectrophotometer at 660
nm.

C. Incubation

For maintenance, cultures were incubated in candle jars or in 959^ air-5% CO2 . Experi-
mental agar cultures were incubated in air in moist chambers, in moist candle jars, in tiglitly
sealed chambers dried and depleted of COj by sandwiching the agar plates between open petri
dishes filled with NaOH pellets, in dessicators from which CO2 was depleted by the presence of
concentrated solutions of NaOH, or in tighdy sealed containers with an atmosphere containing
approximately 4.8?r CO, . The CO2 in sealed containers was generated by mixing 2 ml of 10%
Na2C03 /liter of internal volume with an excess of 2.5 N sulfuric acid. Growth on agar plates was
measured by adding 5 ml of distilled water or 1% Formalin to each plate and removing the cells
from the surface of the closed agar plate with a 1-cm wire brad cut from a paper clip. The wire
brad was placed on the agar and rotated by placing the plate on a magnetic stirring device. The

Table 2

Composition of a chemically defined medium (CDLp) for the

Legionnaires' disease bacterium

Amino acid-organic acid solution

Add 5 g each of serine, alpha-ketoglutaric acid, and sodium pyruvate to 500 ml of water. Add 0.5
g of each of the 1 8 amino acids listed below and bring the flnal solution to 1 liter.

L-alanine, L-aspartic acid, L-asparagine (monohydrate), L-arginine (monohydrochloride), L-histi-
dine HCl (monohydrate), L-isoleucine, L-glutamic acid, L-glutamine, glycine, L-leucine, L-lysine
(monoliydrochloride), L-methionine, L-phenylalanine, L-proline, L-threonine (allo-free), L-trypto-
phane, L-tyrosine, and L-valine.

Basal medium

1. Combme the following ingredients in the order shown:
250 ml of the 4X salts solution. Table 1 .

200 mi of the amino acid-organic acid solution described above.

10 ml of the vitamin solution. Table 1 .

0.1 ml of the thioctic acid solution. Table 1.

0.1 ml of the coenzyme A solution. Table 1.

0.5 g L-cysteine HCl.

0.5 g glutathione (reduced).

1 0 ml of the minor element solution. Table 1 .

2. Adjust pH to 6.3 with 20% KOH.

3. Add 1 ml of the hemin solution. Table I.

4. Adjust the pH to 6.5 with 20% KOH, bring the volume to 500 ml with distilled water, and sterilize
this 2X concentrated medium by filtering through a 0.45-;um membrane tllter.

Final medium

Bring the sterile 2X concentrated medium to final volume with sterile distilled water. For general
use, the final broth medium should contain 0.05%- soluble starch (Eastman, Mallinckrodt, or Baker), and
an agar medium should contain 1%. "Ion Agar" No. 2 (oxoid) and 1% soluble starch.

30

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

worker is safe from aerosols when removing cells with the spinning wire brad, and the procedure
does not release agar into the cell suspension (4). The suspensions were drawn off and diluted to
absorbance readings of 1.0 or less at 660 nm.

Anaerobic, aerobic, aerobic without COt , or aerobic + increased CO, cultural environments
were produced in test tubes with absorbent cotton plugs and combinations of saturated pyro-
gallol, 207f KOH, 10% NajCOj, and 1 M KH, PO4 ; rubber stoppers were then inserted in the
tops of the tubes (9). Virtually all of the broth cultures were incubated with Na^ CO, -KHj PO4
(CO2 ) seals. In general, air with increased CO2 tension was created by adding 0.3 ml each of 10%
Na2C03 and 1 M KHj PO4 to the cotton plug. The test tubes were placed on a rotary shaker at
70 rpm at 37°C; A's were read at chosen intei^vals. Other commonly used bacteriological, serolog-
ical, or chemical methods have been described (6).

RESULTS

A. Detemiination of basal growth conditions and development of a basal medium.

In the initial "probing" experiments, we examined culture conditions and media fonnula-
tions which would support consistent growth of small inocula and provide maximal cell density.
The experience of others and our own experimental findings showed that the major factors which
affect growth of the LDB are size and condition of the inoculum and the presence of air. carbon
dioxide, pyruvate, alpha-ketoglutaric acid, and cysteine. Certain of these results are shown below.

2.0-

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Figure 1. Relationship of dry weiglit to absorbance (660 nm) of the inoculum of Legionnaires" disease bacterium
strain Philadelphia 1. grown on the F-G medium. Cells used were obtained from CYE agar and were singles or
doublets of small, cigar-shaped bacilli.

31

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

The semisynthetic medium (Table 1) generally supported excellent growth of the standard
inocula described, although inocula with A's less than 0. 10 very often did not grow when initially
transferred to this medium. However, cells harvested from the primary passage to the semisyn-
thetic medium grew when transferred at lower cell densities. Growth on the chemically defined
medium occurred readily on the first transfer, but with subsequent passages growth occurred
sooner and reached higher densities on the chemically defined medium than on the semisynthetic
medium.

In an experiment with the semisynthetic broth in tlasks, in shaking tubes containing various
volumes of liquid (5 to 15 ml), and under different conditions of aerobiosis induced by incuba-
tion with or without shaking, growth was obtained in tubes which were incubated with shaking
and had COj seals. No growth was observed in tubes incubated anaerobically. Tubes incubated
stationary with CO, seals did not contain significant growth until placed on the shaker, after
which their A's increased from 0.19 to 0.77. Tubes incubated stationary without COj seals had
no visible growth for several days but had final A's ranging from 0.48 to 0.61 after COj seals
were added and the tubes were again incubated with shaking. These results suggested that the
LDB was a strict aerobe, which required CO2 and ready access to oxygen, and that it was
potentially sensitive to excessive aeration.

Adding agar to the chemically defined medium strongly inhibited growth of low concentra-
tions of inocula. Dilutions of inocula ranging from 10^' to 10"' grew in 7 to 10 days when
"plated" in broth in petri dishes and incubated in a 95% air-5% CO, atmosphere. Similar plates
containing Difco agar had no apparent growth at dilutions of more than 10^- . Others with 1% Ion
Agar No. 2 supported growth at 10"'', but when 1'? soluble or insoluble purified potato starch or
0.2% charcoal (Norit A) was added, the effect of the agar was completely reversed. The semisyn-
thetic agar and the F-G agar under moist, aerobic conditions supported comparable levels of
growth and cell yields, although the lag period on the semisynthetic medium was shorter. In air,
with a pH above 6.8, the growth rates (but not final cell yields) were definitely lower than those
obtained in candle jars. However, 0.1% alpha-ketoglutaric acid strongly increased the growth rate,
and cell yields were substantially higher at pH's above 6.8. Higher cysteine concentrations had no
apparent effect. The conditions required for growth raised many questions about the role of pH
and CO2 , the depletion of oxygen in a candle jar, the use of alpha-ketoglutaric acid as a source of
CO, , and the effect of pH on cysteine in the medium.

These data did not clearly delineate the functions of the factors tested. More complete
experiments were conducted in air, air with COj added, and air with CO, removed, using media
containing low concentrations of casein hydrolysate. The effects of pyruvate and alpha-ketoglu-
tarate were also determined (Fig. 2). The results obtained in "air" were between the extremes
obtained with "added" and "removed" CO2 and are not shown in Fig. 2. At pH 7.2, no apparent
growth occurred in air or in air depleted of CO2 when neither pyruvate nor alpha-ketoglutaric
acid was added. The results also showed that at pH 7.2 without CO, , alpha-ketoglutaric acid and
pyruvate stimulated rates of growth and cell yields; however, the resulting cell yields were not
greater than 33'v-66% of that obtained when only CO2 was added. Carbon dioxide was clearly
required for growth at pH 7.2 and organic acids could not entirely replace it. Further, at this pH,
the growth rates and cell yields were significantly higher in the presence of pyruvate than in that
of alpha-ketoglutarate.

However, at pH 6.5 with no organic acids added, CO, improved but was not required for
growth (Fig. 2). When the organic acids were present, cell yields in the absence of CO2 were only
66% of those obtained when CO2 was present. In the presence of 5%' CO; for an extended
growth period, the cell yields without added organic acids were only 85% of those obtained with
added pyruvate or alpha-ketoglutarate. These results indicate at least two separate roles for CO2
and the organic acids and emphasize that both are required for maximal growth.

32

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33

Physiology; Characteristics in Semisynthetic and Chemically Defined Liquid Media

In tlie liquid semisyntlietic or chemically defined media, the standard inoculum will grow in
dilutions of ICT'' incubated 7 to 10 days in shallow layers in a CO, -enriched atmosphere. At a
dilution of 10"^ or 2 X 10"' (final A = 0.05), this inoculum will grow slowly in slanted tubes but
will not produce the maximal cell yields obtained in control shake cultures. However, inocula of
lower concentrations do not grow with shaking. Even when we steamed the broths for 5 min to
lower the redox potential (as might occur with large inocula), growth of low concentrations of
inocula was not stimulated by shaking. Without CO, seals, there was less growth, and heavy
brown pigments were formed which were associated with bacterium's stationary phase (Table 3).
This effect of air curbing growth was directly correlated with a pH rise from 6.5 to 7.9 during
incubation in air, whereas growth under the COj seal barely increased the pH from 6.35 to 6.45
(Table 3). Control studies showed that the CO, seals decreased the initial pH from 6.5 to 6.2
within 48 h. These results show that in part the CO, atmosphere functioned as a buffer to
maintain the initial pH in agar media. CO, may have also regulated metabolism.

Using reagent-saturated filter paper discs on the surface of semisynthetic agar, we obsei-ved
no effects with succinate, fumarate, lactate, acetate, oxalate, gluconolactone, glucuronic acid,
glycerol, gelatin, and cobalamin. Yeast extract, pyruvate, and alpha-ketoglutaric acid stimulated
growth, whereas citrate strongly inhibited it.

B. Detemiiiiation of substrate

Having detennined conditions of growth and developed a basal semisynthetic medium which
satisfactorily supported growth, we examined the medium to detect components which acted as
substrate sources of energy.

An examination of the semisynthetic medium for constituents which affect growth showed
that cell yields were proportional to the concentration of casein hydrolysate (Fig. 3). Of the
amino acids tested, the presence of histidine, threonine, tiyptophane, tyrosine, or serine gave
substantially increased cell yields, with serine causing the greatest increase (Fig. 4). Cell yield
were slightly higher in the presence of glutamine. lysine, or proline; cell yields were not appar-
ently affected by adding alanine, arginine, aspartic acid, cystine, glutamic acid, isoleucine, leu-
cine, methionine, and/or valine to the casein hydrolysate. Chromatographic experiments did not
show that any one amino acid disappeared during growth in 0.4% casein hydrolysate. These
results show that the above specified amino acids were major sources of energy for growth.

Table 3

Effect of air and air + carbon dioxide on growth and final

pH in shake culture*

s

Initial A (inocuhim)

Condition

Final A

Final pH

6.35

Description

0.05

air + CO,

1.16

no color

0.10

air + CO2

1.13

6.40

no color

0.15

air + CO2

1.12

6.40

no color

0.05

air

.75

7.65

higli melanin

0.10

air

.74

7.70

high melanin

0.15

air

.75

7.75

high melanin

* Medium was the semisynthetic (SSLp) broth with 1.0% casein hydrolysate and an initial pH of 6.5. Cultures were incubated for
65 hat 37°C and shaken at 70 rpm. Carbon dioxide (CO, ) was generated with 0.3 ml of ]0';i Na^COj plus 0.3 ml 2.5 N HjSO,
per tube.

34

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35

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

On the other liaiui, the presence or absence of glucose in the semisyntlietic medium iuid no
effect upon growth. Although 1% glucose caused lactate to fomi in 0.8 to 0.^)-A/M/nil amounts,
no lactate was formed in the absence of glucose. No sugars other than glucose were tested. The
presence of amylose, amylopectin. dextrin, glycogen, yeast extract, or bovine albumin did not
affect cell yields.

In the presence of 0.1% alpha-ketoglutaric acid, some acids, including 0.4% pyruvate, fuma-
rate, and succinate, had no effect on growth, but others such as citrate, acetate, and malate
totally inhibited growth; lactate slightly inhibited growth. The concentrations of alpha-ketoglu-
taric acid, pyruvate, and casein hydrolysate could be varied somewhat without affecting growth.
Nevertheless, during several experiments in which growth was marginal because of limited casein
hydrolysate or, in a medium which by itself did not support growth, 0.1% pyruvate stimulated
growth. In experiments with the chemically defined medium, very small but definite increases in
growth were observed when at least 0.1% of pymvate was added.

The results with glucose, polysaccharides, and organic acids show that the bacterium does
not use these products as catabolic substrates for energy and carbon. The effects of alpha-keto-
glutaric acid and pyruvate are complex, although there is little question that they are beneficial
to growth. However, there is little evidence that they provide energy. It remains to be detennined
whether they are involved in buffering, transamination reactions, or are intermediaries or regula-
tors of some metabolic process. When the media were being prepared, we noted that both
alpha-ketoglutaric acid and pyruvate function as chelators because precipitates that fomied as the
pH was adjusted were reduced or eliminated.

C. Nutritional requirements

Cysteine was required for growth as previously reported (]0), and growth was directly
proportional to cysteine concentrations between 0.002% and 0.01% (Fig. 5); higher concentra-
tions did not increase cell yields and were slightly inhibitory. Cystine did not substitute for
cysteine, but glutathione did substitute for cysteine on an equimolar basis. Although the initial
growth was delayed with unadapted cells, cells adapted to utilizing glutathione had equal growth
rates and cell yields (Fig. 6). Under favorable growth conditions, the presence of 0.12 M dithio-
threitol, 2-mercaptoethanol, thioglycolate, or methionine moderately to completely inhibited
growth in medium containing only 0.02% cysteine. These and other results suggest that cysteine
functions as a required amino acid and is not required as a reducing agent or chelator. Indepen-
dent studies by one of us (JRG) showed that serine, methionine, arginine, valine, leucine, isoleu-
cine. and threonine are also required for growth of at least 10 LDB strains representing four
serogroups.

When all the vitamins (Part I, steps 3-5, Table 1), starch, and oleic acid were removed, growth
was delayed. However, all further attempts with several strains to demonstrate a vitamin require-
ment have failed, although one of us (MWR) used these same media to demonstrate thiamine and
biotin requirements of Escliericlua coli. Similarly, others have been unable to demonstrate any
vitamin requirements in liquid synthetic media (W. J. Warren and R. D. Miller, personal commun-
ication).

In addition to the -SH compounds above which inhibited growth, 1 /ig/nil of oleic acid, in
the absence of starch, completely inhibited growth; in the presence of 0.05% starch, 10 /jg/ml of
oleic acid did not inhibit growth. Whether the fatty acids of agars are inhibitors is not yet known;
nevertheless, it is known that 1% starch was optimal for agar media. The presence of 0.05%
ether-purified Tween 80 instead of starch totally inhibited growth, as did ethylenediaminetetra-
acetic acid. Inhibition by the latter compound plus the observations of inhibition by citrate,
acetate, and malate suggested that trace elements played a major role in metabolism. One of us
(MWR) demonstrated a requirement for iron in the defined medium with alpha-alpha dipyridyl;
other results indicate requirements for calcium and magnesium.

36

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37

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

The chemically defined broth was compared to the semisynthetic broth for maintaining
growth in five sequential transfers of 10 strains of the LDB: Philadelphia 1. Philadelphia 2,
Philadelphia 3, Philadelphia 4, Pontiac, Flint 1, Bellingham. Miami Beach. Detroit 1, and Togus 1.
The defined broth (with no starch) supported growth of the tested strains equally well. Indeed, in
one or more passages, the cell yields were higher than those observed in the casein hydrolysate
broth -suggesting that some amino acids of the defined mixture were at levels preferable to those
in the casein hydrolysate broth. The various strains of the organism generally adapted readily to
the two media. Although results obtained with the different strains varied to some degree, in
general we found that the defined medium containing 0.05'"' starch was superior lo the casein
hydrolysate medium.

D. Physiology

In most of the experiments with the various solid and liquid media, cells were examined for
morphology, catalase production, and hydrolysis of starch. Cells were also stained with the
serogroup-specific fluorescent antibody (FA) rabbit-anti LDB conjugate, and the presence of
surface antigen was compared with that of cells grown on the F-G medium. Catalase production
was extremely limited or absent, and no hydrolysis of starch was observed using any of these
media. In all cases, cells grown on either the semisynthetic or chemically defined medium stained
by direct FA as well as or better than control cells grown on F-G medium.

Morphology of the LDB varies widely under different growth conditions (Fig. 7). In general,
growth covers the entire surface of an agar plate because of the liquid present; growth apparently
starts sooner and is more rapid in higli humidity. However, we did not observe spreading or
crawling motility. When plates were incubated between NaOH pellets (to absorb CO, ). the pellets
also functioned as dessicants, and the surfaces were dried-producing discrete colonies with entire
edges; results were similar when plates were incubated 7 to 10 days, and the colonies were typical
of those grown on CYE medium. The slower growth observed under these conditions may have
been the result of depleted CO2 or a dried surface or both. Cellular morphology changed progres-
sively in broth, with large masses of filaments or chains of bacilli in the logarithmic phase
breaking into shorter filaments and ultimately forming single and double cigar-shaped cells. With
prolonged incubation or limited substrate, cells became coccal shaped (Fig. 7). The organism
grew at 25°C, 30°C, and 37°C. The cellular morphology was the same at 30°C and 37°C, but at
25° C cells appeared as tine, small bacilli (Fig. 7). We did not observe refractile cells or cells which
miglit suggest spores or microcysts but did see swollen cell terminals which were probably
spheroplasts.

SUMMARY AND DISCUSSION

In reviewing the characteristics of the LDB, we see a set of apparently contlicting growth
requirements and physiological responses, which, according to the limited data presently avail-
able, may be more apparent than real. We have an aerobic organism which sometimes seems
sensitive to excesses of oxygen, which can grow under the microaerophilic condition of the
candle jar, which produces limited amounts of-if any— catalase, and which has a specific require-
ment of cysteine as an -SH amino acid. With the lability and extreme reactivity of the -SH of
cysteine, particularly in alkaline solutions and in the presence of metals, it would not be difficult
to visualize this as a factor limiting growth in nature or in laboratory media. Thus the limited
aerobic requirements are supportive and not in conflict with this cysteine requirement. Further-
more, a preference for acid pH further supports a stable -SH form of cysteine because the
molecule is higlily reactive in alkaline conditions and is rapidly oxidized to cystine in the pres-
ence of iron. At neutral or alkaline pHs, cysteine spontaneously forms addition compounds with
aldehydes, ketones, and unsaturated molecules (5, 7, <*?).

38

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

Ag^w^ :mm

0

• c^

%

V

^ I .

\. /

A/

-r"L '■-

*. «r- - 1 /

'^ * 'J ^ / |/ -'

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*

b

Figure 7. Morphological aspects (1350 X) of the Philadelphia 1 strain of the Legionnaires' disease bacterium
grown for:

A. 3 days at 37°C in 0.8% casein hydrolysate semisynthetic broth with shaking in air.

B. 4 days at 37°C in 0.8% casein hydrolysate semisynthetic broth with shaking in air + 5% COj .

C. 4 days at 37°C in 0.4% casein hydrolysate semisynthetic broth with shaking in air + 5% CO2 .

D. 7 days at 25°C on the semisynthetic agar slants (0.8% casein hydrolysate); incubated in air.

39

Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media

Many of tliese complexes are reversible, e.g., the reactions with glucose and pyruvate (S),
but others may cause new stable compounds to form (7). Since aldehydes and keto compounds
are less reactive under acid conditions, the marked stimulation by pyruvate and alpha-ketoglu-
taric acid at pH 6.5 compared to 7.2 may be due to lower reactivity of the acids with cysteine at
pH 6.5. But the lower pH may also permit these acids to enter the cell more readily by shifting
the dissociation equilibrium to the side of the non-ionized acids.

Although the LDB has shown sensitivity to citrate, malate, and acetate -recognized chelat-
ing agents of calcium, magnesium, and iron— pyruvate and alpha-ketoglutaric acid did not inhibit
growth even though their chelating effects were observed when certain experimental media were
being neutralized.

In addition, the organism must attempt to utilize amino acids as a source of energy and
carbon, with pHs subsequently rising as higli as 8.2. These amino acid substrates appear to be
primarily serine and threonine. With the media we developed, the buffering effect of carbon
dioxide is significant and can in part explain the results observed in liquid shake cultures where
the CO; helped maintain an acid pH. However, carbon dioxide may have other functions and an
acid pH would be contrary to its solution as bicarbonate.

Finally, the LDB appears to be quite sensitive to oleic acid; presumably this is the toxic
factor in agar which is reversed by adding either 1.0% starch or 0.2% charcoal (Norit A). How-
ever, our experience with purified Tween 80 and with certain inocula suggest that the LDB itself
may be a source of inhibiting fatty acids. Thus, with the excretion of a soluble lipase described
by W. B. Baine et al. (Personal communication), one can visualize the release of toxic fatty acids
from purified Tween 80 or from lysed cells of the inoculum.

Nevertheless, in the simple, chemically defined medium (Table 2) without added vitamins,
oleic acid, and starch, 10 LDB strains tested have grown extremely well during several sequential
transfers.

REFERENCES

1. Feeley, J.C, G.W. Gorman, R.E. Weaver, D.C. Mackel, and W.H. Smith. 1978. Primary isolation media tor
the Legionnaires' disease bacterium. J. Clin. Micro. 8:320-325.

2. Feeley, J.C, R.J. Gibson, G.W. Gorman. N.C. Langford. J.K. Rasheed. D.C. Mackel. and W.B. Baine. 1979.
CYE Agar: A primary isolation medium for the Legionnaires" disease bacterium. J. Clin. Micro. (In press).

3. Fraser, D.W., T.F. Tsai, W. Orenstein, W.E. Parkin, H.J. Beecham, R.G. Sharrar, J. Harris, G.F. Mallison, S.M.
Martin, J.E. McDade, C.C. Shepard. and P.S. Brachman. 1977. Legionnaires' disease: Description of an
epidemic of pneumonia. N. Engl. J. Med. 297: 1 18*^)-! 1^7.

4. Pine, L. 1955. Studies on the growth of liistoplasnia capsulatum. II. Growth of the yeast phase on agar
media. J. Bacteriol. 70:375-381.

5. Pine, L. and C.L. Peacock. 1955. Reactions of fumaric acid with cysteine. J. Am. Chem. Soc. 77:31-53.

6. Pine, L., J.R. George. M.W. Reeves, and W. Knox Harrell. 1979. Development of a chemically defined liquid
inedium for the growth of the Legionnaires' disease bacterium. J. Clin. Micro. (In press).

7. Schubert, M.P. 1936. Compounds of thiol acids with aldehydes. J. Biol. Chem. 114:341-350.

8. Schubert, M.P. 1939. The combination of cysteine with sugars. J. Biol. Chem. 130:601-603.

9. Slack, J.M. and M.A. Gerencser. 1975. Actinomyces, filamentous bacteria. //; Biology and Pathogenicity, pp.
127-128. Burgess Publishing Company, Minneapolis.

10. Weaver, R.E. 1978. Cultural and staining characteristics. //; "Legionnaires'" - the Disease, the Bacterium
and Methodology. G.L. Jones and G.A. Hebert (eds.), October Ed. U.S. Dept. Health. Education, and
Welfare, Public Health Service, Center for Disease Control, Atlanta, GA. pp. 39-43.

40

Electron Microscopy of the
Legionnaires' Disease Bacterium

Francis W. Chandler

John A. Blackmon

Martin D. Hickhn

Roger M. Cole*

Carey S. Callaway

Pathology Division

Bureau of Laboratories

Center for Disease Control

National Institute of Allergies and Infectious Diseases*
National Institutes of Health

41

Electron Microscopy of the
Legionnaires' Disease Bacterium

F. W. Chandler. J. A. Blackmon, M.D. Hicklin,
R.M. Cole, and CS. Callaway

We studied the Legionnaires' disease bacterium (LDB) by examining cultures, preparations
of iiuman lung tissue, and chicken egg yolk sacs with transmission electron microscopy (TEM).
Samples of consolidated iiuman lung were obtained at autopsy from twelve patients with con-
firmed Legionnaires" disease. Formalin-fixed pieces of these lung tissues were embedded in
paraffin, sectioned<5 /im, stained by the Dieterle silver impregnation method (2), and examined
by light microscopy. If numerous bacteria characteristic of the LDB were seen, the piece of tissue
from which the section was taken was diced into 1 mm cubes for TEM studies. Normal yolk sacs
and those infected with the LDB were fixed either in cold 4% (v/v) buffered paraformaldehyde
or cold 2% (v/v) glutaraldehyde in 0.2 M S-collidine buffer, pH 7.0, for 1 h. Both lung specimens
and yolk sacs were postfixed in 1% osmium tetroxide (v/v), buffered in 0.2 M S-collidine, pH 7.2
to 7.4 (7), for 1 h at 4°C. The tissue specimens were then dehydrated through graded alcohols,
embedded in Maraglas 732, and cut on a Reichert 0MU2 ultramicrotome fitted with a diamond
knife. Sections were picked up on uncoated copper grids and stained with saturated uranyl
acetate and lead citrate {13). Selected 4-day cultures of the LDB in Mueller-Hinton broth supple-
mented with IsoVitaleX were prepared by the method of Ryter and Kellenberger (6). For whole
cell preparations, broth cultures of the LDB were dried on Formvar-coated grids and stained with
2% aqueous uranyl acetate. All sections were examined with a Philips 200 transmission electron
microscope at 40 KV.

The LDB is ultrastructurally identical in human lung tissue, experimental lesions of guinea
pigs, bacteriologic media, or yolk sac membranes of embryonated hens' eggs. In sections, the
LDB is a blunt or tapering rod which usually measures 0.3 to 0.9 ^tm in diameter and >2.0 /im
in length (Figs. 1 and 2). Its sides may not be parallel, and it varies considerably in length depend-
ing on its environment. After 4 to 7 days of growth, greatly elongated forms are commonly
found in cultures and less commonly found in yolk sac membranes; they are rarely found in
human lung or in experimentally induced lesions in guinea pigs. The LDB is clearly prokaryotic
(!S,9) in that it lacks eukaryotic features such as mitochondria, nuclear membranes, endoplasmic
reticulum, and mitotic division. Prominent features include electron-lucent, filamentous nucleoids
interspersed among areas of well-defined ribosomes; enclosure by a double envelope, each por-
tion of which consists of a triple-layered "unit" membrane (4.10): and division by a pinching,
nonseptate process (Figs. 2 and 3). This pinching type of division and double envelope enclosure
are characteristic of gram-negative bacteria (4. 5. 10. II).

With the techniques used, we have not been able to see any definite structure that could
represent a peptidoglycan layer (11) in the periplasmic space. Cleanly circumscribed, round
electon-lucent vacuoles are frequently seen within the LDB (Fig. 1). These vacuoles stain readily
with Sudan black B in smears made from cultures, and their lipid content is probably removed by
the solvents used in preparing samples for electron microscopy. Their ultrastructural appearance
is suggestive of poly-jS-hydroxybutyrate granules in sections (-5,72). Rarely, membranous profiles

42

:i^

"Jf;,

%M

V

IK'Ctron Microscopy of the Legionnaires' Disease Bacterium

*#

Figure 1. Electron micrograph of Legionnaires' disease bacteria grown on bacteriologic medium. Note enclosure
of organisms by a double envelope consisting of inner (IM) and outer (OM) triple-layered "unit" membranes.
Vacuoles (v) are present in some organisms, (osmium fixation. X90.000).

43

Elcctrdn Microscopy of the LcL'ionnaires" Disease Bacterium

Figure 2. Whole preparation of Legionnaires' disease bacteria grown on bacteriologic medium. Note dividing
organism (arrows indicate region of central pinching) and delineation of outer envelope (blunt arrows) (uranyl
acetate. X43. 700).

which appear to be extensions from the inner envelope are seen in the LDB cytoplasm. However,
membranous inclusions or other organelles, such as those seen in some gram-positive bacteria
(9), are not found in the LDB.

Autopsy specimens from patients with LD included lung tissues obtained diu-ing the 1976
Philadelphia outbreak as well as those from patients associated with sporadic or epidemic cases
of LD since then. A confirmed diagnosis of LD was based either on results of indirect immuno-
fluorescent serodiagnosis, direct immunofluorescence of lung sections or smears, isolation on
artificial media, guinea pig inoculation, or some combination of the four as described elsewhere
in this manual. The electron microscopic features of the LDB in areas of typical pulmonary
consolidation, determined by light microscopy of hematoxylin-eosin and Dieterle silver impregna-
tion stained sections ( J .2), are consistent. However, the concentration of organisms varies from
specimen to specimen and case to case. The LDB is predominately found in phagosomes of
degenerating or necrotic alveolar macrophages (Fig. 3). Enormous phagosomes, which occupy
most of the host cell cytoplasm, may contain as many as 40 organisms in a single plane of section.
Extracellular LD bacteria may also be present and are often intimately associated with cellular

44

<

f

Electron Microscopy of the Legionnaires' Disease Bacterium

Figure 3. Electron micrograph of Legionnaires' disease bacteria witliin phagocytic vacuole (blunt arrow) of
degenerating alveolar inflammatory cell. Extracellular organisms are also seen (arrows) (XI 2,750). Inset: Detail
of dividing organism within alveolar macrophage. Note central pinching (arrows) and enclosure by a double
envelope (uranyl acetate and lead citrate, X61,712).

debris and fibrin (Fig. 3). Multiplying as well as degenerating organisms are observed intracellu-
larly and extracellularly. We have seen fewer LD bacteria within neutrophils and, when present,
only one to six organisms usually occupy a phagocytic vacuole in one plane of section.

Because the ultrastructural features of the LDB are not unique, it may be impossible to
differentiate this organism from other small gram-negative bacilli by fine structure alone. How-
ever, our studies confirm the characteristic gram-negative ultrastructure of the LDB and earlier
reports of its gram-negative staining when isolated from cultures.

45

Electron Microscopy of the Legionnaires' Disease Bacterium

REFERENCES

1. Blackmon. J. A., M.D. Hicklin. and F.W. Chandler. 1978. Legionnaires' disease. Pathological and historical
aspects of a "new" disease. Arch. Path. Lab. Med. 102:337-343.

2. Chandler, F.W.. M.D. Hicklin, and J. A. Blackmon. 1977. Demonstration of the agent of Legionnaires'
disease in tissue. N. Engl. J. Med. 297:1218-1220.

3. Forsyth, W.G.C., A.C. Hayward, and J.B. Roberts. 1958. Occurrence of poly-(3-hydroxybutyrate in aerobic
gram-negative bacteria. Nature 182:800-801.

4. Glavert, A.M. 1962. The fine structure of bacteria. Brit. Med. Bull. 18: 245-250.

5. Kellenberger, E., and A. Ryter. 1958. Cell wall and cytoplasmic membrane o\' Escherichia coli. J. Biophys.
Cytol. 4:323-326.

6. Kellenberger. E., A. Ryter, and J. Sechaud. 1958. Electron microscope study of DNA-containing plasma II.
Vegetative and nature phage DNA as compared with normal bacterial nucleoids in different physiological
states. J. Biophysic. Cytol. 4:671-678.

7. Lynn, J. A., J.H. Martin, G.J. Race. 1966. Recent improvements of histologic techniques for the combined
liglit and electron microscopic examination of surgical speciinens. Am. J. Clin. Pathol. 45:704-713.

8. Murray, R.G.E. 1974. A place for bacteria in the living world, pp. 4-19. In Bergey's Manual of Determina-
tive Bacteriology, Eighth edition. R.E. Buchanan and N.E. Gibbons (Eds.). The Williams and Wilkens. Co.,
Baltimore.

9. Murray, R.G.E. 1962. Fine structure and taxonomy of bacteria. Symp. Cos. Gen. Microbiol. 12:1 19-144.
10. Sulton. M.R.J. 1964. The Bacterial Cell Wall. Elsevier. New York.

1 1 . Steed, P., R.G.E. Murray. 1966. The cell wall and cell division of gram-negative bacteria. Can. J. Microbiol.
12:263-270.

12. Stockdale. H.. D.W. Ribbons, and E.A. Dawes. 1968. Occurrence of poly-j3-hydroxybutyrate in \hi Azoto-
bacteriaceae. J. Bacteriol. 95:1798-1803.

13. Venable, J.H.. and R. Coggeshall. 1965. A simplified lead citrate stain for use in electron microscopy.
J. Cell Biol. 25:407413.

46

Cellular Fatty Acid Composition of the
Legionnaires' Disease Bacterium

C. Wayne Moss
R.E. Weaver*

S.B. Dees
W.B. Cherrv

Analytical Bacteriology Branch

and

Clinical Bacteriology Branch*

Bacteriology Division

Bnreaii of Laboratories

47

Cellular Fatty Acid Composition of the
Legionnaires' Disease Bacterium

C.Wayne Moss. R.E. Wearer. S.B. Dees, and W.B. Cherry

Identification and classification of microorganisms has been a central theme in the develop-
ment of microbiology as a science. Since the early work of Pasteur, classification has been based
largely on morphological, physiological, serological, and biochemical data. In recent years, a new
dimension-the use of chemical data-has been added to bacterial classification, and the term
"chemotaxonomy" is now well-established in the literature. To the microbiologist, tiie term
usually denotes techniques such as cell wall analyses, DNA composition, and DNA homologies. In
a broader sense, the tenn includes studies of cell composition with respect to sugars, proteins,
amino acids, and lipids, and studies of metabolic products of microorganisms.

For the last several years our laboratory has done chemotaxonomic studies using gas liquid
chromatography, mass spectrometry, and associated analytical techniques to develop new meth-
odology for rapid and sensitive detection, identification, and classification of organisms. We have
done extensive studies with cellular fatty acids and have found that a number of closely related
bacterial species can be distinguished on the basis of qualitative differences in their fatty acids
((S"). Moreover, we have obsei^ed that the cellular fatty acid compositions of strains of a species
are essentially identical. Our initial interest in the Legionnaires" disease bacterium (LDB) was to
examine the cellular fatty acid composition of all isolates to assess their degree of chemical
relatedness. At the time the study was begun, few data were available from conventional cultural
and biochemical tests. Thus, the data could provide valuable information for establishing the
relationship of isolates obtained in the future and those from the 1*^)76 Legionnaires' disease
outbreak.

Four isolates from Philadelphia and an isolate each from Flint and Pontiac, Michigan, were
included in the initial study. Each of the six strains was inoculated onto a plate of Mueller-Hinton
agar supplemented with 1% hemoglobin and 1% (v/v) IsoVitaleX (BBL) and incubated in a candle
jar at 35°C for 72 h. After about 3 ml of sterile distilled water was added, the heavy cell growth
on the plates was removed with glass rod spreaders. Smears were prepared for Gram staining to
check the purity of the cultures. The cells were saponified, and the fatty acids were methylated
by the procedure described previously {10) and outlined in detail elsewhere in this manual.
Pseiidomonas cepacia, an organism of known cellular fatty acid composition (lV), was used as a
control; it was grown and processed under the same conditions as the six test cultures.

Methyl esters were analyzed on a Perkin-Elmer Model 900 gas chromatograph (Perkin-
Elmer, Norwalk, CT) equipped with a tlame ionization detector and a Disc integrator recorder.
The instrument contained a 0.16-in (4.06 mm, l.D.) x 1 2-ft (3.Ci6 m) coiled glass column packed
with 3'j^ OV-101 methyl silicone which was coated on 100-120 mesh Gas-Chrom Q (Applied
Science Lab., State College, PA). The carrier gas was helium at a flow rate of 60 ml/min. The
initial column temperature was 160°C; after the sample was injected, it was increased to 265°C at
a rate of 5°C/min. Fatty acid methyl ester peaks were tentatively identified by comparing their
retention times to those of methyl ester standards (Applied Science Lab.; Analabs, North Haven,
CT; Supelco. Bellefonte, PA). Final identification was established by hydrogenation (10) and

48

Cellular Fatty Acid Composition of the Legionnaires' Disease Bacterium

mass spectrometry iJO. 1(\ IS). Combined gas chromatography-mass spectrometry of metliyl
esters was done with a DuPont instrument type 2 1^9 IB equipped witli a combination electron
impact(EI)-chemical ionization (CI) source. Isobutane was used as the reagent gas for CI. Details
of the gas chromatographic procedure and identification of fatty acids are presented elsewhere in
the manual.

The cellular fatty acid profile (as methyl esters) of the Flint 1 strain of the LDB is shown in
the chromatogram in Fig. 1. The single most abundant acid in the chromatogram is a saturated
branched-chain 16 carbon acid (i-!6:0) with the methyl branch at the iso-( penultimate )carbon
atom. The next most aboundant are a mono-unsaturated 16 carbon straight chain acid ( 16: 1 ), a
saturated 15 carbon branched-chain acid (a-15:0) with the methyl branch at the anteiso-(anti-
penultimate)carbon atom, a saturated 14 carbon branched-chain acid (i-14:0), a saturated 17
carbon branched-chain acid (a- 17:0), and a mono-unsaturated 16 carbon iso-branched-chain acid
(i-l6:l). With the exception of 16:1, the nonnal straight-chain saturated (15:0, 16:0, etc.) and
unsaturated (14:1) acids were present in only small to trace amounts. The identities of the
labeled fatty acid methyl esters in the chromatognun were confimied by both EI and CI mass
spectrometry. The EI spectra of anteiso-branched methyl esters were clearly distinguished from
iso-branched and nomial straight-chain esters by comparing the ratio of the m/e = M-29 and m/e
= M-3 1 peaks in the spectra. With anteiso esters the M-29 is equal to or greater than the M-31,
whereas the M-31 peak is approximately twice the size of the M-29 peak in iso-branched and
normal straight-chain esters (76, IS). Unsaturation was confinned by hydrogenating the methyl
ester sample (10), which resulted in the disappearance of the i-16: 1 and 16:1 peaks with concom-
itant increases in the size of the i-16:0 and 16:0 peaks, respectively. The absence of hydroxy
acids was confirmed by the fact that there was no change in retention time of any peak in the
chromatogram when the methyl ester sample was treated with trinuoroacetic anhydride (10).
The profiles of the other five strains were strikingly similar, as shown in Fig. 2. The fatty acid
profile of the control culture, P. cepacia was essentially the same as that reported previously {10).

The cellular fatty acid composition of each of the six strains is shown in Table 1 . Peak areas
from gas-liquid chromatography (GLC) were determined with the Disc integrator, and the per-
centage content of each acid was calculated from the ratio of the area of its peak to the total area
of all peaks. Relative response factors detemiined for each acid were used in the calculation. The
data clearly show the similarity of the fatty acid compositions of the six strains. In each strain
the iso-l6:0 acid was the major component; concentrations ranged from 32% to 43% of the total
acid content. With the exception of 16:1, the other four major acids in each strain were also
branched-chain acids. These acids (i-14:0, a-15:0, i-16:l, a-17:0) were present in each strain but
in different proportions. The total proportion of branched-chain acids in each of the six strains
ranged from 81% to 90%. The only qualitative difference in the cellular fatty acid profiles of the
six strains was the presence of relatively small amounts (2%-4%') of a 1 7-carbon cyclopropane
acid (17A) in the Pontiac and the Philadelphia 2, 3, and 4 strains, which was not detected in the
other two strains. Results of retesting the cellular fatty acid composition of each strain through
the entire procedure (growth, saponification, extraction, GLC) were virtuahy identical (see Table
1).

Since the initial study, 62 additional strains of the LDB have been isolated from clinical
materials and from environmental sources at diverse geographical locations. We examined each of
these for cellular fatty acids after growing them on enriched Mueller-Hinton (M-H) agar or on
other recently developed growth media [Feeley-Gomian (F-G) agar (7), charcoal yeast extract
(CYE) agar (7), semisynthetic medium (14)]. The cellular fatty acid composition of each strain
was similar to that observed in the initial study (12, Figs. 1 and 2, and Table 1). No major
differences in cellular fatty acids were observed among strains of the four recognized serogroups
( 7).

49

Cellular Fatty Acid Composition of the Legionnaires' Disease Bacterium

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Cellular Fatty Acid Composition of the Legionnaires' Disease Bacterium

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51

Cellular I'atty Acid Composition of the Legionnaires' Disease Bacterium

Each new growth medium used for the LDB has been evaluated for its effect on cellular
fatty acids. The fatty acid compositions of two LDB strains grown on M-H and CYE agars are
described in Table 2. The data show no qualitative differences in fatty acids of cells hai-vested
from the two media. However, the total proportion of branched-chain fatty acids was higher for
cells grown on the M-H medium than for those grown on CYE agar (7% higher for strain Berkeley
and 6% higher for strain Flint 2). The same relationship was observed for several other strains: the
average total cellular branched-chain fatty acid content for 36 strains was 79% on M-H medium
and 68% on CYE agar (Table 2). The cellular fatty acid compositions of strains grown on F-G
agar and on Pine's semisynthetic medium were essentially identical to those of strains grown on

Table 1 .

Cellular fatty acid composition of six strains of the

Legionnaires" disease bacterium.

Philadelphia

Flint

Pontiac

Fatty Acid^

i

,

3

4

=1

_2b

=1

^">

-1

—2

=1

—2

=1

-2

= 1 -2

i-14:0

1 r'

12

13

1 1

14

14

lU

13

1 1

1 1

1 1 12

14:1

T

T

T

T

T

T

T

T

T

T

T T

14:0

T

T

T

T

T

T

T

T

T

T

1 T

a-I5:0

20

20

13

14

14

1 1

12

13

20

20

1 1 12

15:0

T

T

T

T

T

T

T

T

T

T

T T

1-16:1

11

1 1

11

1 1

13

9

12

10

12

12

1 1 10

i-16:0

34

32

3*^)

42

35

43

40

40

36

32

40 40

16:1

13

14

14

15

16

14

15

14

10

14

14 14

16:0

T

T

T

T

T

T

T

T

T

T

T T

a-17:0

1 1

11

8

5

5

5

7

6

1 1

1 1

9 8

17A

—

-

■^

")

^T

4

4

4

-

-

4 4

17:0

T

T

T

T

T

T

T

T

T

T

T T

Br 18:0

T

T

T

T

T

T

T

T

T

T

1 T

18:0

T

T

T

T

T

T

T

T

T

T

r T

Br 19:0

T

T

T

T

T

r

T

T

T

T

T T

19:0

T

T

T

T

T

r

T

T

T

T

T T

Br 20:0

T

T

T

T

T

T

T

T

T

T

r T

20:0

T

T

T

T

T

1

T

T

T

T

T T

Total of branclied-

chain acids

87

86

84

83

81

82

81

82

00

86

82 82

a Number to the left of tlie colon refers to tlie number of carbon atoms; number to the right refers to the number of double
bonds: i- indicates a methyl branch at the iso-carbon atom; a- indicates a methyl branch at the anteiso-carbon; Br indicates a
branched-chain acid.

b Data obtained from a second experiment of cells processed through the entire procedure of growth, saponification, extraction.
and GLC analysis.

c Numbers refer to percentages of total acids; T = less than 2%.

52

Cellular I'atty Acid Composition of the Legionnaires' Disease Bacterium

M-H agar, with some slight increase (5%-7%) in tiie relative amount of a-15:0 acid on the
semisynthetic medium. Even though the relative proportions of total branched-chain fatty acids
were lower for all strains grown on CYE agar than for those grown on M-H medium, LDB strains
grown on any medium contained relatively large amounts of i-16:0, a-15;0, a-17:0, and i-14:0
acids.

In general, branched-chain fatty acids are characteristic of gram-positive bacteria (5, 6, 17),
but they are present in major amounts (> 50% of total) in relatively few genera such as Bacillus
L\ -/), Listeria {15), Propiouibacterium (1 1), Micrococcus (2), and Staphylococcus (2). Of the
gram-negative organisms, only Then)ius aquaticus and Flavobacterium are known to contain
major amounts of these acids (9, 13). A striking feature of the cellular fatty acids of LDB is their
high content of branched-chain acids (> 77%). Although branched-chain acids have been found in
other bacteria, their presence and relative concentrations and the absence of other acids which
are generally present in other bacteria produce a unique cellular fatty acid profile for the LDB.
Data show that this unique cellular fatty acid composition is a relatively consistent characteristic
of the numerous LDB strains tested. The cellular fatty acid profiles are typical for cells from each
growth medium developed to date. Limited data on some other factors known to influence
cellular fatty acid composition (3. 6) indicate that they do not significantly affect the general
fatty acid profile. Thus, detemiination of cellular fatty acid content is valuable for rapid identi-
fication and classification of suspected isolates of the LDB.

Table 2.

Comparison of the cellular fatty acid composition of strains of

the Legionnaires' disease bacterium grown on two different media^

Fatty

Acid''

Berkeley

1

Flint

-)

Av. of 36 Strains

M-H

CYE

M-H

CYE

M-H

CYE

i-14;0

7^'

7

7

6

10

8

14:0

T

T

T

T

T

T

a-l5:0

17

21

14

18

15

14

15:0

T

T

T

3

T

T

i-16:l

7

7

3

4

6

3

i-16:0

36

26

49

35

39

32

16:1

7

14

10

12

15

13

16:0

7

5

7

8

2

10

a-17:0

17

16

10

12

9

11

17A

T

T

T

T

T

3

17:0

T

T

T

T

T

2

18:0

2

2

T

2

2

2

19:0

T

T

T

T

T

T

20:0

T

2

T

T

2

2

Total of branc
acids

lied-ciiain

84

77

83

77

79

68

a M-H; enriched Mueller-Hinton agar

CYE; charcoal yeast extract agar
bThe number to the left of the colon refers to the number of carbon atoms; the number to the right refers to the number of

double bonds; (i-) indicates a methyl branch at the iso-carbon atom; (a-) indicates a methyl branch at the anteiso-carbon; and

(A) indicates a cyclopropane ring,
c Number refers to percentage (%) of total fatty acids; T = less than 2%.

53

Cellular Fatty Acid Composition ol' the Legionnaires' Disease Bacterium

REFERENCES

1. Feeley. J.C. G.W. Goniian, and R.J, Gibson. 1978. Priniaiy isolation media and metliods. //; "Legion-
naires"" — The disease, the bacterium and methodology. G.L. Jones and G.A. Ilebert (eds.). U. S. Depart-
ment of Health, Education, and Welfare, Public Health Service, Center for Disease Control, Atlanta, Ga., pp.
107-117.

2. Ishizuka. I., N. Ueta, and T. Yamakawa. 1966. Chromatographic studies of microbial components. II.
Carbohydrate and fatty acid constitution of the family Micrococcaceae. Jpn. J. L,\p. Med. 36:73-<S.v

3. Kaneda, T. 1971. Factors affecting the relative ratio of fatty acids in BuciHus cercus. Can. J. Microbiol.
17:269-275.

4. Kaneda, T. 1967. Fatty acids in the genus Bacillus. 1. iso- and anteiso-fatty acids as characteristic constitu-
ents of lipids in 10 species. J. Bacteriol. 93:894-903.

5. Kates, M. l064. Bacterial lipids. Adv. Lipid Res. 2:1 7-90.

6. Leclievalier, M.P. 1977. Lipids in bacterial taxonomy - A ta.xonomist's view, pp. 109-210. //; A.I. Laskin
and H. Lechavalier (eds.), Critical Review of Microbiology, vol. 5. CRC Press, Cleveland.

7. McKinney, R.M.. L. Thacker, P.P. Harris. K.R. Lewallen, G.A. Hebert, P.H. Edelstein, and B.M. Thom-
ason. 1979. Four serogroups of Legionnaires" disease bacteria defined by direct immunotluorescence. Ann.
Intern. Med. 90:621-624.

8. Moss, C.W., and S.B. Dees. 1976. Cellular fatly acids and metabolic products of Pscuclnmonas species
obtained from clinical specimens. J. Clin. Microbiol. 4:492-502.

9. Moss, C.W., and S.B. Dees. 1978. Cellular fatty acids o( Flavohacterhiin mcningosepticiini and Flawhacte-
riiim species group lib. J. Clin. Microbiol. 8:772-774.

10. Moss, C.W., and S.B. Dees. 1975. Identification of microorganisms by gas chromatographic-mass spectro-
metric analyses of cellular fatty acids. J. Chromatogr. 112:595-604.

11. Moss, C.W., V.R. Dowell, Jr., D. Farshtchi, L.J. Raines, and W.B. Cherry. 1969. Cultural characteristics and
fatty acid composition of propionibacteria. J. Bacteriol. 97:561-570.

12. Moss. C.W., R.E. Weaver. S.B. Dees, and W.B. Cherry. 1977. Cellular fatty acid composition of isolates from
Legionnaires" disease. J. Clin. Microbiol. 6:140-143.

13. Oshima, M., and A. Miyagawa. 1974. Comparative studies on the fatty acid composition of moderately and
extremely themiophilic bacteria. Lipids 9:476-480.

14. Pine. L., J.R. George, M.W. Reeves, and W.K. Harrell. 1979. Development of a chemically defined liquid
medium for the growth of the Legionnaires" disease bacterium. J. Clin. Microbiol. (In press).

15. Raines, L.J., C.W. Moss. D. Farshtchi, and B. Pittnian. 19(i,S. Fatty acids of Listeria nionocytitgcncs. J.
Bacteriol. 96:2175-2177.

16. Ryhage. R.. and E. Stenhagen. I960. Mass spectrometry in lipid research. J. Lipid Res. 1 :361-390.

17. Shaw, N. 1974. Lipid composition as a guide to the classification of bacteria. Adv. Appl. Microbiol. 17:63-
108.

18. Tyerrell. D. 1958. The fatty acid composition of some Lntoniophtlioraceae. II. The occurrence ofbranched-
chain t'atty acids in Cdiiidinhotus dciiaesporus Drechsl. Lipids. 3:368-372.

54

Antimicrobial Susceptibility of the
Legionnaires' Disease Bacterium

Clyde Thornsberry
Linda A. Kirven

Clinical Bacteriology Branch

Bacteriology Division

Bureau of Laboratories

55

Antimicrobial Susceptibility of the
Legionnaires' Disease Bacterium

Clyde Thornsberry and Linda A. Kirreii

In 1976 an outbreak of acute respiratory disease, later called "Legionnaires' disease,"
occurred among people who had attended a convention in Pliiladelpiiia, Pennsylvania (5). The
causative agent, later identified as a bacterium (9), is referred to as the Legionnaires" disease
bacterium (LDB). Since 1976, more than 500 cases with 79 fatalities have been identified from
outbreaks (2). As of October 31, 1978, 453 sporadic cases with 86 fatalities had been confirmed
in 43 states and the District of Columbia (4).

A review of the antimicrobial therapy used for patients with Legionnaires' disease showed
that case-fatahty rates were highest for those treated with cephalosporins and were intermediate
for those treated with aminoglycosides, chloramphenicol, ampicillin. or penicillin. Lowest rates
were for patients who received tetracycline or erythromycin (5). Some patients have been success-
fully treated with a combination of intravenous oxytetracycline and rifamycin ilO). Doxy-
cycline was effective in another patient, but gentamicin was not (7).

In vivo studies have been performed with a guinea pig animal model {.\ 6. 11 ) and with
an embryonated hens' egg model (S). In the first in vivo study of therapy for an infection in an
animal model, erythromycin was demonstrated to be effective in treating infected guinea pigs iS).
All of the guinea pigs treated witli erythromycin survived, whereas all the infected controls died.
Later the same investigators used five other antimicrobial agents to treat guinea pigs infected
with lethal doses of the LDB (77). The most effective drug was minocycline hydrochloride,
which was associated with a 50^''f survival rate. Rifampin was less effective than minocycline, and
the aminoglycosides (gentamicin, tobramycin, and amikacin) were essentially ineffective, even
though they are very active in vitro.

In another in vivo study, guinea pigs were infected with the LDB and then treated with
antibiotics (6). Erythromycin and rifampin were the most effective drugs in preventing mortahty.
Penicillin, chloramphenicol, tetracycline, and gentamicin had no significant effect (6).

In the embryonated hens' egg model studies (6'), rifampin (followed by gentamicin, strepto-
mycin, erythromycin, sulfadiazine, and chloramphenicol, in that order) most effectively pre-
vented death of the embryo. The least effective drugs were cephalothin. which protected only if
large amount were used, and oxytetracycline, whicii partially protected embryos. Ampicillin at
the highest levels tested did not prevent or delay death of the embryo (A).

In vitro studies, with agar dilution and broth microdilution techniques, showed that rifam-
pin was the most active agent, but that the macrolides (erythromycin and rosamicin), the amino-
glycosides (gentamicin, tobramycin, and amikacin), cotrimoxazole (sulfamethoxozole, 19 parts
plus trimethoprim, 1 part), and chloramphenicol were very active (J J. 13). Minimum inhibitory
concentrations (MICs) obtained in these studies indicate that the LDB would be only slightly
susceptible to commonly used (3-lactam antibiotics (cephalosporins and penicillins). The most
active |3-lactam antibiotic tested was cefoxitin (a cefamycin, which has only recently been ap-
proved by the Food and Drug Administration for general use) {IJ).

56

Antimicrobial Susccptibilit\ of the Legionnaires' Disease Bacterium

All clinical and environmental strains of the LDB tested produce a /3-lactamase which is
more active on cephalosporins than on penicillins (13). The greater activity of cefoxitin on these
organisms probably reflects its resistance to the j3-lactamase produced by the LDB.

On the other hand, cefamandole, a cephalosporin that is resistant to some (3-lactamases of
gram-negative bacilli, is not resistant to the LDB j3-lactamase. As new |3-lactum antibiotics are
synthesized, they should be tested for susceptibility to the enzyme of the LDB. If they are
resistant, they should be candidates for clinical trials of treatment for Legionnaires' disease.

The in vitro results show that minocycline and doxycycline are more active than tetra-
cycline. All the drugs (except for cotrimoxazole) that were active on the LDB on the basis of
MICs were bactericidal at levels comparable to the MIC. The minimum bactericidal concentra-
tions (MBCs) for cotrimoxazole were 33-fold greater than the MIC, but were still within achiev-
able blood levels.

In vitro susceptibility results must be interpreted cautiously because of the difficulty in
growing the organisms. None of the artificial media supports the rapid growth that is needed for
susceptibility tests.

One reason for the lack of correlation between the in vitro and in vivo results may be the
relative differences in the intracellular bactericidal action of the drugs. The LDB are frequently
found within macrophages in lung specimens from patients who died with Legionnaires' disease
(/) and within peritoneal macrophages in experimentally infected guinea pigs (Chandler. F..
cited in 6). This may explain why rifampin and erythromycin are effective both in vitro and in
vivo, whereas an aminoglycoside is effective only in vitro.

All strains of LDB that have been tested have shown similar susceptibility patterns. Because
of this, and because the methods for performing susceptibility tests are far from optimal, we
recommend that susceptibility tests not be performed routinely. Reference laboratories, such as
ours, can monitor future isolates for changes in susceptibility.

All the LDB have produced a jS-lactamase when tested by a chromogenic cephalosporin test
(see Appendix) but not by the starch-iodine or acidometric techniques {13). The chromogenic
cephalosporin test is very simple to perform. Although jS-lactamase data may not be useful in
formulating therapeutic strategy, tliey may be useful in characterizing the LDB.

REFERENCES

1. Blackmon. John A.. M.D. Hicklin, F.W. Chandler, and the Special Expert Pathology Panel. 1978. Legion-
naires" disease, pathological and historical aspects of a 'new' disease. Arch. Patiiol. Lab. Med. 102:337-343.

2. Brenner, D.J., A.G. Steigerwalt, R.E. Weaver, J.E. McDade, J.C. Feeley. and M. Mandel. 1978. Classifi-
cation of the Legionnaires' disease bacterium: An mterim report. Current Microbiology 1:71-75.

3. Center for Disease Control. 1977. Follow-up on Legionnaires' disease-Pennsylvania. Morbidity and Mortal-
ity Weekly Report 26:152.

4. Center for Disease Control. 1978. Legionnaires" disease - United States. Morbidity and Mortality Weekly
Report 27:439-441.

5. Eraser, D.W., I.E. Tsai, W. Orenstem. W.E. Parkin, H.J. Beecham, R.G. Sharrar, J. Harris. G.E. Mallison,
S.M. Martin, J.E. McDade, C.C. Shepard. P.S. Brachman, and the field investigation team. 1977. Legion-
naires" disease: Description of an epidemic of pneumonia. N. Engl. J. Med. 297:1 189-1197.

6. Eraser, D.W., l.K. Wachsmuth, C. Bopp. J.C. Feeley. and I.E. Tsai. 1978. Antibiotic treatment of guinea
pigs infected with agent of Legionnaires" disease. Lancet i: 175-178.

7. Keys, T.F. 1977. A sporadic case of pneumonia due to Legionnaires' disease. Mayo Clin. Proc. 52:657-660.

8. Lewis, V.J., W.L. Thacker, C.C. Shepard. and J.E. McDade. 1978. In vivo susceptibility of the Legionnaires'
disease bacterium to ten antimicrobial agents. Antimicrob. Agents Chemother. 13:419-422.

57

Antimicrobial Siisceptibilit\' of the Legionnaires" Disease Bacterium

9. McDade. J.E., C.C. Shepaid. D.W. Fraser. T.F. Tsai. M.A. Rediis, W.R. Dowdle, and tlie laboratory investi-
gation team. 1977. Legionnaires' disease: Isolation of a bacterium and demonstration ot" its role in other
respiratory disease. N. Eng. J. Med. 297: 1 197-1203.

10. Meenhorst. P.L.. J.W.M. van der Meer, A. Blusse. and H, Mattie. 1978. Treatment of suspected sporadic
cases of Legionnaires" disease (letter). Lancet i:71 1.

1 1. Nash. P., L. Sideman. V. Pidcoe, and B. Kleger. 1978. Minocycline in Legionnaires' disease (letter). Lancet
i:45.

12. Thornsberry, C, C.N. Baker, and L.A. Kirven. 1978. In vitro activity of antimicrobial agents on Legion-
naires' disease bacterium. Antimicrob. Agents Chemother. 13:78-80.

13. Thornsberry. C. and L.A. Kirven. 1978. |3-lactamase of the Legionnaires' bacterium. Current Microbiology.
1:51-54.

58

Legionella pneumophila sp. nov.:
The Legionnaires' Disease Bacterium

Don J. Brenner
Arnold G. Steigerwalt

G. Ann Hebert*
Joseph E. McDade**

Enterobacteriology Branch

and

Analytical Bacteriology Branch*

Bacteriology Division

and

Leprosy and Rickettsia Branch**

Virology Division

Bureau of Laboratories

59

Legionella pneumophila sp. nov.:
The Legionnaires' Disease Bacterium

Don J. Brenner. Arnold G. Steigenvalt.
G. Ann Hebert, and Joseph E. McDade

In the 2 years since the outbreak in Philadelphia (7) that brought Legionnaires" disease
worldwide attention and led to the isolation of the Legionnaires" disease bacterium (LDB) at the
Center for Disease Control (7/). intense effort has been focused on understanding the "new"
disease and the "new"' bacterium. We have learned that the LDB is a gram-negative, weakly
oxidase-positive. catalase-positive, fastidious organism that has narrow optimal pH and tempera-
ture ranges and will not grow anaerobically. It does not reduce nitrates, utilize carbohydrates,
degrade urea, or appear to possess decarboxylases for lysine and ornithine or an arginine dihydro-
lase (79). It has a unique fatty acid profile and no major differences in cellular fatty acids among
strains (75). Four different serogroups have been identified (6, 12).

Deoxyribonucleic acid (DNA) hybridization has proved extremely useful in the classifi-
cation of bacteria (7. 8. 76. 7 7). We have characterized the DNA of LDB by genome size, guanine
plus cytosine (GC) content, and DNA hybridization in order to determine the extent of related-
ness between strains of LDB and possible relatedness of the LDB to other microorganisms.

DNA relatedness studies were first done before biochemical data were available for LDB and
before its GC composition had been detemiined. Thus the initial experiments were designed to
rule on the possibility that the LDB was a fastidious Klebsiella or another fastidious member of
Enterobacteriaceae. Since DNA relatedness studies have previously been done on all described
species within Enterobacteriaceae (7, 2. 3). we were able to test possible relatedness between the
LDB and Enterobacteriaceae. DNAs from Philadelphia 1 and Flint 1 were reacted with labeled
DNA from Escherichia coli K-12, Proteus mirabilis 1. Yersinia enterocolitica 498-70, and Ed-
wardsiella tarda 3889-64 (Table 1). Relatedness was between 0% and 3% (5). All members of
Enterobacteriaceae are at least 15% related to one or more of these four species (7. 2, 3).
Therefore, the LDB is not a member of Enterobacteriaceae. Relatedness values below 5% are not
considered to be significant. Similar experiments proved that LDB had no relatedness to Staphy-
lococcus epidermidis, Bordetella pertussis, Aeromonas hydrophila, and Vibrio cholerae (Table I).
Although Vibrio parahaemolyticus and Vibrio alginolyticus were not tested directly (Table 1),
these and some other vibrios were ruled out on the basis of their known DNA relatedness to V.
cholerae.

Preliminary approximations, using optical thermal denaturation, indicated that the GC con-
tent of LDB DNA was slightly lower than that o( Proteus mirabilis. GC content in strains of P.
mirabilis is 39% to 42%. Quantitative determinations of GC content by optical thermal de-
naturation (9) and by CsCI buoyant density ultracentrifugation (70) showed that both Phila-
delphia 1 and Flint 1 DNA contained 39% GC. With this knowledge we were able to restrict DNA
relatedness tests mainly to those organisms with known GC values between 35% and 43%.

Highest priority was given to detemiining relatedness between LDB and the organisms
shown in Table 2. Rochalimaea (formerly Rickettsia) qui)itana, which has a GC content of 37%,
was given high priority because its pattern of growth on artificial media is comparable to that of

60

Legionella pneumophila sp. nov.: The Legionnaires' Disease Bacterium

the LDB. Since the LDB was mitially isolated by methods designed for rickettsiae (77), com-
parisons with R quintana were especially indicated. Flavobacterium meningosepticum and Flavo-
bacterium IIB were tested because these organisms have similarities to the LDB. The only known
group of gram-negative organisms that have predominantly branched-chain fatty acids are in the
genus Flavobacterium (15). They are pigmented, their antibiotic resistance profile is similar to
that of the LDB US), and the GC content in the DNA of some flavobacteria is similar to that of
the LDB. FrancisL'Ua tularensis, yersiniae, and Pasteurella multocida were tested because some
patients from whom F. tularensis and Yersinia pestis had been isolated had elevated serum titers
to the LDB. Additional species in the genus Pasteurella and several species found in the environ-
ment were also tested.

Table 1 .

DNA relatedness of LDB strains to several species

selected at random

Reaction^ % DNA relatedness at 60°C''

E. coli K-\2*IE. coll K.-12 100 (85)

£. CO/; K-I2*/Philadelphia 1 ' 3

£■. c-o//K-i:*/Flint 1 (D8711) 1

P. mirabilis 1 *IP. mirabilis 1 100 (74)

P. mirabilis 1 */Philadelphia 1 2

P. mirabilis 1*/Flint 1 (087 11) 3

}'. enterocolitica 498-70*/}', enterocoUtica 498-70 100 (74)

Y. e/!rero<:o//r;t'a498-70*/Philadelphia 1 0

Y. enterocolitica 498-70*/Flint 1 (D87 11) 0

E. tarda 3889-64*/£', tarda 3889-64 100 (64)

E. tarda 3889-64*/Philadelphia 1 -
a: ?fl/-(7ff3889-64*/Flint 1 (08711) . 0

V. cholerae 1845-73*/F, cholerae 1845-73 100 (81)

V. cholerae 1845-73*/F. cholerae 1320-72 100

V. cholerae 1845-73*/K cholerae 5540-70 95

V. cholerae 1845-73*/F, parahaemolyticus A8633 32

V. cholerae 1845-73*/K, alginolyticus C68 14 22

F, c/zo/erae 1845-73*/Philadelphia 1 0
V. cholerae 1845-73*/Flint 1 (0871 1) 0

A. hydrophila*IA. hydrophila 100 (67)

A. /7vt/ro/;/H7a*/Philadelphia 1 ' 0

A. hydrophila* l¥\m\ 1 (0871 1) 0

S. epidermidis */S. epidermidis 1 00 ( 6 1 )

S. ep/<ie/TO/d/s*/Philadelphia 1 1

B. pernissis*IB. pertussis 100 (65)
B. pem/is/s*/Philadelphia 1 0

B. pertussis* If \\M \ {D87 \ V) 0

a Labeled DNA is indicated by an asterisk.

bThc figures in parentheses are the actual values obtained in homologous reactions. These are arbitrarily designated 100%, and
heterologous values are nomialized to them. The B pertussis reactions were done at 65°C due to the high GC of B. pertussis
DNA.

61

Legionella pnciiinoplula sp. nov.: Tlie Legionnaires' Disease Bacterium

Table:.

DNA relatedness of LDB strain Philadelphia 1 to organisms

of similar guanine plus cytosine content and organisms isolated

from patients with high serum titers to LDB

7r relatedness to LDB strain Philadelphia L'
Source of unlabeled DNA 60°C 75°C

Rochalimaea quintana ATCC VR35ii 5 U

Flavobactehum meningosepticum 2

FlavobacteriumW^^'S.X^O 3

Flavnbacteriitm IIB B6829 3

Francisella tularensis 4 1

Yersinia entewcolitica 498-70'' 0

Y. enterocolirica 27^ 0

Y. entewcolitica 1474'' 0

Y. entewcolitica 481^ 0

Y. entewcolitica 867'' 4

Y. pseudotuberculosis P262 0

Y. pseudotuberculosis P27 0

Pasteurella haemolytica KC228 3

P.multocida ATCC 12947 1 0

P. multocida ATCC \0544 1 0

P. multocida ATCC 6535 0 0

P.ureaeKC5\8 1 4

Cytophaga johnsonae AJCC 29586 0 2

Cylophagajohnsonae ATCC 29588 0 0

Fle.xibacter canadensis V ASM 9D 0 0

Microcyclus major B859 3 0

a DNA reassociation in homologous Pluladelplua 1 was approxnnately 70'; in 60°C reactions and 60';f in 75"C" reactions.

b There are four different DNA relatedness groups among strains presently called Yersinia cnterocolitica {3). These groups are
characterized by their reactions for sucrose (sue), rhamnose (rha), raffinose (raf), and melibiose (mel). Y. enterocolitica sU-d'uK
498-70 and 27 give the typical fermentation pattern: suc+, rha-, raf-, mel-. Strain 1474 is atypically sue-. Strain 48 is
atypically rha-i-, raf-t-. meH. Strain 867 is atypically ^ha-^.

Pasteurella ureae and LDB were 7% related in 60°C reactions and 4% related in 75°C
reactions. DNA relatedness of Philadelphia 1 to all these organisms at 60°C was 5% or less (Table
2). None of these values below 5% was considered significant. Therefore, the LDB is not related
to any of these organisms at the species or genus level. While )'. pestis was not directly ruled out,
it is more than 90% related to Y. pseudotuberculosis (13) and can be ruled out indirectly.

The time required for reassociation was similar for LDB DNA and DNA from E. co//K-12.
DNA from this E. coli strain has a molecular weight of 2.5 X 10' daltons. Therefore, LDB DNA
is also approximately 2.5 X 10' daltons; enough genetic information to specify some 3,000
genes. Isolates of LDB are extremely similar in growth, serology, biochemical tests, fatty acid
composition, and antibiotic susceptibility profiles (77. 75. 7iS'). DNA relatedness was detennined
for 15 of the LDB isolates available at the Center for Disease Control (CDC) to see if these
relatively few phenotypic markers reflected similarity over the entire genome of LDB. Most

62

Legionella pneumophila sp. nov.; The Legionnaires' Disease Bacterium

workers find that members of a given species are at least 70% related (1. S. 76, J7). It lias been
our experience that relatedness among strains of a species remains above 60% when reactions are
done at the stringent 75°C incubation temperature. Furthemiore, divergence in DNA relatedness
between strains of a species is almost always less than 6% (1, J). Strains of LDB were 70% to 99%
related to Philadelphia 1 (Table 3). Divergence in related sequences was 3% or less, and re-
latedness remained almost unchanged in 75°C reactions. Thus on the basis of these tests all of
these LDB isolates belong to a single species.

Table 3.
DNA relatedness among 15 strains of LDB

Reaction w

th labeled Philadelphia 1 DNA^

Source of unlabeled DNA

% relatedness

7p divergence

60°C

75°C

Philadelphia 1

100(74)

100(60)

0(87.8°C)

Philadelphia 2

96

Philadelphia 3

94

90

Philadelphia 4

93

100

2.0

Pont lac 1

79

74

2.5

Flint 1 (08711)

90

74

2.5

Flint2(E1035)

98

Knoxville 1

84

80

3.0

Buriington 1

86

93

0.5

Bellinghani 1

81

82

2.5

Albuquerque 1

94

77

3.0

Berkeley 1

. 97

Bloomington 1

80

71

1.8

Blooniington 2

85

85

2 2

Togus 1

85

a Figures in parentheses are the actual % of DNA reassocialion in homologous Philadelphia 1 reactions, and the Tp, (temperature
at which 50% of labeled DNA is detected as single-stranded) for homologous, reassociated DNA. % divergence is the decrease in
Tni (in °C) of heterologous DNA duplexes compared to that of the homologous DNA duplexes. It can be expressed as %
because each degree decrease in thermal stability is caused by approximately 1% unpaired bases in double-stranded DNA {3).

The first four strains of^ LDB isolated were called Philadelphia 1, 2, 3, and 4. Strains
subsequently isolated were also identified by city to distinguish them from the Philadelphia
strains. This convention has now been adopted at CDC, and all strains are designated by city of
origin and the number of the isolate from that city. For instance, Flint 1 and Flint 2 were both
isolated in Flint, Michigan. If a strain has a city designation without a number, it can be assumed
that this is the first isolate from that city: Albuquerque = Albuquerque 1. Specific culture
collection numbers are put in parentheses after the city name and number; Flint 1 (D871 1), Flint
2 (El 03 5), etc.

LDB strains Philadelphia 2, 3, and 4 are very closely related to Philadelphia 1. This is not
surprising since these strains were all isolated from the same outbreak. The strain from the
Burlington outbreak and strains from sporadic cases were also very closely related to Philadelphia
1.

63

Legionella pneumophila sp. nov.: The Legionnaires' Disease Bacterium

LDB strains Togas 1, Bloomington 1, and Bloomington 2 were at least 80% related to
Philadelpliia 1 in reactions done at 60°C. These results are significant in tiiat botli the Togus and
Bloomington isolates were serologically atypical and/or isolated from the environment. Togus 1 is
the first strain whose O antigen does not react with the direct fluorescent antibody conjugate to
LDB Knoxville (12). Bloomington 1 reacts with the Knoxville conjugate, but was isolated from
the environment {14). Although Bloomington 2 is also an environmental isolate, it is serologically
different from both Knoxville and Togus 1 {12. 14).

It is an impossible task to prove directly that LDB is different from all described species at
the level of species, genus, and family. Our approach to this problem was to assume that LDB
could not be related to organisms that had GC content in their DNA outside the range of 35% to
45%, or were gram-positive, strict anaerobes, spore fomiers, psychrophiles, thennophiles, acido-
philes, or alkalinophiles. One can argue with any of these criteria, but the alternative is an
irrational screening approach.

Genera containing one or more species still under consideration for some level of relatedness
to LDB are listed in Table 4. Direct or indirect DNA relatedness results can be used to rule out at
least most species in these genera except for those of Aciiietobacter. Bdelloribrio. B rani nun el la.
Fle.xithri.x. Kingella. Microscilla. Moraxella, and Simousiella. There are persuasive reasons to
exclude these remaining genera. For example, Biwihemella are cocci; Fle.xithri.x are catalase
negative, gelatin negative, and grow well at alkaline pH. One cannot, however, rule out the
possibility that LDB is related to these genera at the family level. Therefore, species in all of these
genera should be tested for DNA relatedness to LDB.

Table 4.

Genera in which relatedness to Legionella pneumophila sp. nov.

has not been totallv ruled out

Acinetobacter

ActinobacHlus

Bdellcribrio

BrauliamcUa

Cytophaga

Flavobacterium

Francisella

Flexihacter

Fle.xithri.x

Haemophilus

Kingella
Microscilla
Mora.xella
Pasteurella
Simon siella

It is unlikely that the LDB is related to any well-described pathogen. We believe that the
LDB is a previously undescribed species. Its biochemical reactions, growth pattern, and DNA
relatedness are sufficiently unique to warrant placing it in a new genus. The fact that to date LDB
is apparently totally unrelated to other organisms is consistent with the creation of a new family.

We have proposed the name Legionella pneumophila McDade et al. (//) sp. nov. for the
LDB. Le-gi-on-ella. M.L. Icgio M.L.n. legion or army;M.L. dim. ending ella. pneu-moph"ila. Gr.
n. pneinno lung; Gr. adj. philos loving (4). The type strain of Legionella pneumophila is Phila-
delphia 1. Legionella pneumophila was proposed as the type species of the genus Legionella.
Legionella was proposed as the type genus for a new family Legioneilaceae (4).

SUMMARY

DNA from strains of the LDB was characterized in order to aid in the proper classification
of this organism. The genome size of LDB DNA was estimated at 2.5 X 10'' daltons by reasso-
ciation kinetics; a GC content of 39% for LDB DNA was established by optical thennal denatura-
tion and buoyant density ultracentrifugation measurements.

64

Legionella pneumophila sp. nov.: The Legionnaires' Disease Bacterium

DNA relatedness was used to classify strains of tlie LDB. Tliese DNA comparisons showed
that all strains of LDB were members of the same species. Included were strains isolated from the
environment and strains with three different O antigens. DNA from LDB was not significantly
related to DNA from any other group of bacteria tested. Biochemical data, growth character-
istics, and GC ratios were used to rule out the possibility that LDB was significantly related to
members of genera whose DNA was not tested.

On the basis of these data, we propose that LDB be named Legionella pneuiiiophila sp. nov.,
the type species of the genus Legionella.

ACKNOWLEDGEMENTS

We are greatly indebted to Drs. W. B. Cherry, J. C. Feeley, C. W. Moss, C. C. Shepard, and
R. E. Weaver for providing strains, allowing access to their unpublished data, and for their critical
comments and encouragement.

We are grateful to Dr. M. Mandel for purified DNA from Microcycliis major. Dr. E. Shotts
for the Aeromonas hydwphila DNA, and Dr. W. Kloos for the Bonletella pertussis DNA and the
Staphylococcus epiderniidis DNA.

Cytophaga johnsonae UASM E-1-25 (ATCC 29586), C. johnsonae UASM 4539 (ATCC
29588), and Flexibacter canadensis UASM 9D were sent to CDC by P. Christensen of the
University of Alberta Soil Microbiology Laboratory. They were cultivated and given to us by J.
C. Feeley. Pasteurella lutemolytica KC228 and P. ureae KC518 were cultivated and given to us by
R. E. Weaver.

REFERENCES

1. Brenner. D.J. 1973. DNA reassociation in the taxonomy of enteric bacteria. Int. J. Syst. Bacteriol. 23:
298-307.

2. Brenner. D.J., G.R. Fanning, K.E. Jolinson, R.V. Citarella, and S. Falkow. 1969. Polynucleotide sequence
relationships among members of Enterobacteriaceae. J. Bacteriol. 98:637-650.

3. Brenner, D.J., A.G. Steigerwalt, D.P. Falcao, R.E. Weaver, and G.R. Fanning. 1976. Characterization of
Yersinia enterocolitica and Yersinia pseudotuberculosis by deoxyribonucleic acid hybridization and biochem-
ical reactions, hit. J. Syst. Bacteriol. 26:180-194.

4. Brenner, D.J., A.G. Steigerwalt, and J.E. McDade. 1979. Classitlcation of the Legionnaires' disease bacte-
rium: Legionella pneumophila , genus novum, species nova, of the family Legionellaceae, familia nova. Ann.
Intern. Med. 90:656-658.

5. Brenner, D.J., A.G. Steigewalt. R.E. Weaver, J.E. McDade, J.C. Feeley, and M. Mande!. 1978. Classification
of the Legionnaires' disease bacterium: An interim report. Current Microbiology L71-75.

6. Cherry, W.B., B. Pittman, P.P. Harris. G.A. Hebert, B. Thomason, L. Thacker. and R.E. Weaver. 1978.
Detection of Legionnaires' disease bacteria by direct immunofluorescent staining. J. Clin. Microbiol. 8:329-
338.

7. Eraser, D.W., I.E. Tsai. W. Orenstein. W.E. Parkin. H.J. Beecham, R.G. Sharrar. J. Harris, G.F. Mallison. S.M.
Martin, J.E. McDade, C.C. Shepard, P.S. Braclniian, and the field investigation team. 1977. Legionnaires'
disease: Description of an epidemic. N. Eng. J. Med. 297:1 189-1 197.

8. Johnson, J.L. 1973. Use of nucleic acid homologies in the taxonomy of anaerobic bacteria. Int. J. Syst.
Bacteriol. 23:308-315.

9. Mandel, M.. and J. Mamiur. 1968. Use of ultraviolet absorbance-temperature profile for determining the
guanine plus cytosine content of DNA. Methods in Enzymology 128:195-206.

10. Mandel, M., C.L. Schildkraut. and J. Mamuir. 1968. Use of CsCl density gradient analysis for determinitig
the guanine plus cytosine content of DNA. Methods in Enzymology 12B: 184-195.

11. McDade, J.E., Q £ . Shepard. D.W. Eraser. T.F. Tsai, M. A. Redus,W.R. Dowdle.and the laboratory investiga-

65

Legionctla pncuinoplula sp. nov.: The Lesijonnaires' Disease Bacterium

tion team. l')77. Legionnaires' disease; Isolation of a bacterium and demonstration of its role in other
respiratory disease. N. Eng. J. Med. 297:1 197-1203.

12. McKinney, R.M., L. Thacker. P.P. Harris, K.R. Lewallen, G.A. Hebert. P.H. Edelstein, and B.M. Thoniason.
1979. Four serogroups of Legionnaires' disease bacteria defined by direct immunofluorescence. Ann. Intern.
Med. 90:621-624.

13. Moore. R.L.. and R.R. Brubaker. 1975. Hybridisation of deo.xyribonucleic sequences of Yersinia cntcrocDli-
tica and other selected members of Enterobacteriaceae. Int. J. Syst. Bacteriol. 25:336-339.

14. Morris. G.K., CM. Patton, J.C. Feeley.S.E. Johnson, G. Gorman. W.T. Martin. P. Skaliy.G.F. Mallison. B.D.
Politi. and D.C. Mackel. 1979. Isolation of Legionnaires" disease bacterium from environmental samples.
Ann. Intern. Med. 90:664-666.

15. Moss. C.W.. R.E. Weaver, S.B. Dees, and W.B. Cherry. 1977. Cellular fatty acid composition of isolates from
Legionnaires" disease. J. Clin. Microbiol. 6:140-143.

16. Palleroni. N.J., R. Kunisawa, R. Contopoulou, and M. Duodoroff. 1973. Nucleic acid homologies in the
genus Pseudomonas. Int. J. Syst. Bacteriol. 23:333-339.

17. Staley, T.E., and R.R. Colwell. 1973. Deo.xyribonucleic acid reassociation among members of the genus
Vibrio. Int. J. Syst. Bacteriol. 23:3 16-332.

18. Thoinsberry, C, CM. Baker, and L.A. Kirven. 1978. In vitro activity of antimicrobial agents on the Legion-
naires" disease bacterium. Antimicrob. Agents and Chemother. 13:78-80.

19. Weaver, R.H. 1978. Cultural and staining characteristics, pp. 18-21. /;; ""Legionnaires""": The Disease, the
Bacterium and Methodology. G.L. Jones and G.A. Hebert (eds.). Center for Disease Control. Public Health
Service, U. S. Department of Health, Education, and Welfare, Atlanta, GA.

66

PART THREE

Methodology

Primary Isolation Using Guinea Pigs
and Embryonated Eggs

Joseph E. McDade

Leprosy and Rickettsia Branch

Virology Division

Bureau of Laboratories

69

Primary Isolation Using Guinea Pigs
and Embryonated Eggs

Joseph E. McDade

Tlie properties of the Legionnaires" disease bacterium (LDB) have already been described.
Briefly, the LDB is a gram-negative, non-acid-fast bacterium tliat does not grow on most bacterio-
logic media (7) but can be cultivated on enriched Mueller-Hinton (M-H) agar (9), F-G agar (J), a
semi-synthetic medium derived from the supplemented M-H formula, and charcoal yeast extract
(CYE) agar, recently developed for cultivating the bacterium {2).

Tile LDB was originally isolated in guinea pigs from autopsy specimens and propagated in
embryonated hens' eggs (7). Later, after the organism was successfully cultivated /// vitro, com-
parison studies were done to evaluate the relative sensitivities of the various isolation procedures
(6). Although guinea pigs were found to be somewhat less sensitive than bacteriologic media for
isolating the LDB. the fact that these animals are relatively resistant to most other bacteria
enhances the likelihood of isolating the LDB from specimens that contain contaminants. Ad-
mittedly, the contamination problem with the //; vitro systems could be solved by the addition of
appropriate antibiotics (5. 12), but the fact remains that guinea pigs and embryonated eggs are
good systems for isolating the LDB, having been used successfully in the first isolations of the
LDB from clinical (7) and environmental samples (4. J J).

The following sections outline the procedures involved in isolating the LDB, with particular
emphasis on using guinea pigs and embryonated eggs. Although the discussion is aimed specifi-
cally at isolating the LDB from human lung tissues, the procedures can be modified slightly and
used for other types of specimens.

COLLECTION AND PROCESSING OF SPECIMENS (see Figure 1)

Several tliumbnail-sized pieces of lung tissue should be obtained from the affected areas at
autopsy. At least one tissue sample should be placed in 10% neutral Formalin and used in
subsequent pathology studies and in direct fluorescent antibody (FA) tests (7) for the presence
of the LDB. Another piece of tissue should be stored at -70°C as reference material, and the
remaining tissue should then be taken or sent to the appropriate diagnostic laboratory for /// vitro
isolation attempts.

If these initial isolation efforts are unsuccessful, //; vivo isolation can be attempted with
guinea pigs and embryonated eggs if appropriate facilities are available. If not, tissue suspensions
should be prepared and shipped to the appropriate reference laboratory in accordance with the
instructions in Part Four of this manual. Specimens received by the reference laboratory can be
safely held at -70°C until processed.

A. Initial Processing (see Figure I )

(NOTE: All personnel who work with specimens suspected of containing the LDB should
wear surgical masks and gloves, and specimens should be processed in a biological safety cabinet.)
Grind a piece of lung tissue with a sterile mortar and pestle using alundum as an abrasive.

70

Primary Isolation Using Guinea Pigs and Embryonated Eggs

Tissue Specimen Obtained

4-
I

Laboratory Service
Immediately Available

If Yes

If No

I

Store tissue at
-70° C until . .

4-

4-

ROUTINE

DIAGNOSTIC

LABORATORY

Place tissue specimens in
10% Formalin for
subsequent use in:

Prepare 10% tissue suspension
in PBS for subsequent use in:

Pathology
studies

Direct FA
tests

Direct FA
tests

Culture
attempts

±.

in vivo/in vitro
isolation attempts

4-

CAN BE REFERRED TO
APPROPRIATE REFERENCE
LABORATORY, AS NECESSARY

Guinea pigs

i

i

Agar

Eggs

i

Agar

Figure 1. Treatment of Tissue Specimens for Isolation of the Legionnaires' Disease Bacterium

Primary Isolation Using Guinea Pigs and Embryonated Eggs

Suspend the minced tissue in enougli phosphate buffered sahne (PBS), pH 7.2, to form a 10%
suspension. Inoculate aliquots of the suspension onto bacteriologic media which have been suc-
cessfully used in growing the LDB and onto other bacteriologic media such as blood agar or
Trypticase soy (TS) agar to serve as controls. Place other aliquots of the suspension on glass
slides, air dry them, fix them in acetone or 10% neutral Formalin, and evaluate them with the
direct FA test. Dispense 1-ml aliquots of the rest of the suspension into labelled 13- X 100-mm
screw-capped test tubes; Hash-freeze the samples in a dry ice-alcohol bath, and store them at
-70° C as reference materials.

B. Interpretating FA Results and Direct Culture Attempts

The four possible combinations of results can be described as follows:

1. Both the FA results and attempts to culture the organism //; vitro are negative. Such
results indicate that the LDB is not present in the specimen, and attempts to isolate other
pathogens should be considered.

2. Both the direct FA results and the in vitro isolation attempts are positive. Such results
strongly indicate that the LDB is present in the specimen and that it may have been isolated.
However, confirmational testing must be done to verify that the organism in question is the
LDB; i.e., the bacterium isolated on agar should have cultural and staining characteristics
and direct FA results comparable to those of prototype strains of the LDB.

3. Direct FA test results for tissue suspensions are negative, but cultural results are positive
on media known to support the growth of the LDB. This combination of results must be
interpreted cautiously. In some cases the LDB miglit be isolated directly on agar when it
cannot be demonstrated with direct FA tests of the tissues. The resolution of such a
situation led to the discovery of the second serogroup of LDB (<S'. 10). However, a more
likely explanation for this conflicting pattern of direct FA and culture results is that a
bacterium other than the LDB has been isolated. In any case, the reason for the discrepant
results must be determined and the data reconciled by appropriate staining, FA, and cultural
techniques before isolates are reported to be LDB.

4. Direct FA test results are positive for the LDB, but the organism is not isolated in direct
cultures. If this combination of results occurs, attempts should be made to isolate the
organism with guinea pigs and embryonated eggs as previously discussed and as described
below.

C. Inoculating Guinea Pigs

1. Prebleed each guinea pig and store the serum for future reference. Thaw an aliquot of
the tissue suspension which was stored at -70° C, and dilute it to approximately 5 ml in
PBS. Inoculate (25-gauge needle) each of four adult male guinea pigs (approximately 600 g)
iutraperitoneally with a 1-ml aliquot of the tissue suspension.

2. Observe the guinea pigs daily for signs of illness. Symptoms sometimes include ruffled
fur, watery eyes, and prostration. Determine their rectal temperatures at a specified time
each day for 10 days. Temperatures >39.5° C are considered to be frank fevers.

3. Sacrifice febrile guinea pigs on the second day they have frank fevers by exposing them
to excessive levels of CO2 vapor. Aseptically remove a portion of each guinea pig's spleen
with sterile forceps and scissors. (Use one set of forceps and scissors to open the animal and
a second set to remove the spleen.) Grind a piece of the spleen with alundum and dilute in
PBS to form a 10% suspension. Dispense aliquots of the guinea pig spleen into labelled test

72

Primary Isolation Using Guinea Pigs and Embryonated Eggs

tubes and store as described above. Save one aliquot to be inoculated into embryonated
eggs.* Any remaining guinea pigs should be allowed to convalesce and then should be bled
by cardiac puncture approximately 4 weeks after they were inoculated with the tissue
suspension in order to obtain sera for serologic tests.

D. Inoculating Embryonated Eggs

1. Select twelve, 6- to 7-day-old embryonated hens' eggs from antibiotic-free tlocks. Candle
the eggs just before inoculating them to confinn that they are healthy. Label the eggs from
1 through 12 with a soft-lead pencil.

2. Working in a biological safety cabinet, dilute a 1-ml aliquot of the lO'r guinea pig spleen
suspension with 9 ml of PBS to form a 1% suspension. Draw up 6 ml of the 1% suspension
into a 6-ml syringe fitted with a 20-gauge, ll/z" needle. Cover the needle with the shield.

3. Disinfect the top of each egg with 70% ethanol (or tincture of Merthiolate). Allow the
disinfectant to air dry. Disinfect an egg punch by dipping it in 70% alcohol and passing it
through a tlame. Use it to puncture the top of each egg. Inoculate each egg with 0.5 ml of
the 1% guinea pig spleen suspension. (Be sure to insert the needle completely into the egg.)
Seal the eggs with Duco Cement or its equivalent, and then incubate them at 35°-37° C in a
humid incubator for 10 days.

4. Candle the eggs daily and discard all that die within the first 3 days after inoculation.
Harvest eggs that die between the 4th and 10th days postinoculation on the day they die to
obtain infected yolk sacs.

Harvesting Infected Yolk Sacs

1. Disinfect the top of an egg with 70%' ethanol.

2. Disinfect a pair of blunt forceps by dipping them in 70% ethanol and passing them
through a tlame. Crack the shell of the egg with one prong of the forceps, and remove the
uppermost quarter of the shell with the forceps.

3. Disinfect a second pair of blunt forceps as above. Use them to separate the yolk sac
membrane from the other membranes; remove the yolk sac membrane, and place it in a
sterile petri dish. Aseptically tear off a small piece of the membrane and drop it onto a 2" x
2" sterile gauze pad. Place the remainder of the yolk sac into a sterile 13- X 100-mm
screw-capped test tube labelled with the appropriate egg number and date.

4. Blot the excess yolk from the small piece of yolk sac membrane on the gauze pad with
another pad. Hold the tissue with sterile forceps, and use it to make smears on glass slides.
Label each shde with the appropriate egg number and date. (Be sure that a particular egg,
the smears of its yolk sac tissue, and the test tube used to store that yolk sac are assigned
the same numbers.)

5. Wipe the excess tissue from the forceps with a clean 2" x 2" gauze pad, disinfect the
forceps as above, and continue to harvest any remaining infected yolk sacs as instructed.

6. Flash-freeze the yolk sacs in a dry ice-alcohol bath, and store at -70° C.

*lf embryonated eggs of the proper age are not available at the time the guinea pigs are sacrificed, guinea pig spleen suspensions
can be quick-frozen and stored at -70° C until such eggs are available.

73

Primary Isolation Using Guinea Pigs and Embryonated Eggs

F. Staining Smears

1. Allow tlie yolk sac smears to air dry. Heat fix.

2. Stain the smears by the Gimenez method as follows;

a. Filter working carbol fuchsin onto slide (tiirough Whatman No. 2 filter paper) and
let stand 1 to 2 m in.

b. Wash slide thoroughly with tap water over sink.

c. Cover smear with malachite green for 6 to '^ sec.

d. Wash slide thoroughly with tap water.

e. Cover smear a second time with malachite green for 6 to 9 sec.

f. Wash thoroughly with tap water.

g. Blot slide dry and examine stained yolk sac smears uilder an oil immersion objective
with light microscopy.

3. In properly prepared slides, the LDB stains red. and the yolk sac tissue appears as
blue-green background cells.

G. Confirming that Isolates are Legionnaires' Disease Bacterium

1. After examining the Gimenez-stained yolk sac smears, select infected yolk sacs for
further study that contain 50-100 organisms/field { lOOOX).

2. Thaw the test tube containing the selected yolk sac by placing it under running tap
water.

3. Remove the yolk sac from the tube, aseptically grind it with alundum. and mix it with
sterile PBS to form a 5% suspension.

4. Inoculate aliquots of the suspension onto TS agar, blood agar, F-G agar, and CYE agar
(see Feeley et al., this manual). Incubate F-G agar cultures at 35°-37° C under 2.5% CO2 .
Incubate the other types of cultures at 3 5° -3 7° C (not in a candle jar). Incubate all cultures
for 10 days.

5. Test an aliquot of the yolk sac suspension by direct FA to determine whether the isolate
is an LDB. Draw the rest of the yolk sac suspension up into a Pasteur pipette, dispense it
into sterile, 1-dram vials, and store them at -70° C as reference material.

6. The LDB should grow on F-G or CYE agar, but not on the other media listed above.
Growth observed on the other media represents isolates other than LDB. In order to give a
definitive report of the presence of the LDB, the isolate's growth and morphological prop-
erties must be confirmed as characteristic for the LDB, and its serologic reactivity in direct
FA tests with standard conjugates to the LDB must be appropriate.

7. Bleed all surviving guinea pigs by cardiac puncture (with a I 2-nil syringe and an 18-gauge
IVi" needle") approximately 1 month postinoculation, and process the blood to obtain sera.
Test these sera, and the sera obtained prior to inoculation, for reactivity with standard LDB
antigens in indirect FA tests. Even if the LDB has not been successfully isolated with the
procedures already described, the seroconversion of guinea pigs to a standard LDB antigen
indicates that the LDB was present in the original patient sample that was submitted for
analysis.

74

Primary Isolation Using Guinea Pigs and Embryonated Eggs

ADDENDUM

REAGENTS FOR GIMENEZ STAIN

1. Carbol basic fuchsin stock solution:

a. 10% basic fuchsin in 95% ethanol 100 ml

b. 4%. aqueous phenol 250 ml

(10 ml phenol in :50 ml distilled H, O)

c. distilled H.O 650 ml

Mix well; incubate at 37° C for 48 h before use.

2. Stock buffers:

a. 0.2MNaH2PO4 2.84 gm in 100 ml distilled water

b. 0.2MNa2HPO4 2.76 gm in 100 ml distilled water

3. Buffer solution:

a. 0.2MNaH2PO4 3.5 ml

b. 0.2MNa2HPO4 15.5 ml

c. distilled water 19.0 ml

4. Carbol basic fuchsin working solution:

a. Carbol basic fuchsin stock ^ 4 ml

b. 0.1 M sodium phosphate buffer solution, pH 7.45 1 0 ml

c. Filter immediately and again before each use. Remains suitable for use for about 48 h.

5. Malachite green oxalite:

0.8% solution in distilled water

REFERENCES

1. Cherry, W.B., B. Pittnian. P.P. Hams, G.A. Hebert, B.M. Thomason, L. Thacker, and R.E. Weaver. 1978.
Detection of Legionnaires" disease bacteria by direct immunofluorescent staining. J. Clin. Microbiol. 8:329-
338.

2. Feeley, J.C. R.J. Gibson, G.W. Gorman, N.C. Langford, J.K. Rasheed, D.C. Mackel. and W.B. Baine. CYE
agar: A primary isolation medium tor the Legionnaires' disease bacterium, (in press).

3. Feeley, J.C, G.W. Gorman, R.E. Weaver, D.C. Mackel, and W.H. Smith. 1978. Priinary isolation media for
the Legionnaires' disease bacterium. J. Clin. Microbiol. 8:320-325.

4. Kaufmann, A., et al. Pontiac fever: Isolation of the etiologic agent, its mode of transmission, and its
relationship to the Legionnaires' disease bacterium, (in preparation).

5. Lewis, v., L. Thacker, C.C. Shepard. and J.E. McDade. 1978. In vivo susceptibihty of the Legionnaires'
disease bacterium in ten antimicrobial agents. Antimicrob. Agents Chemotherap. 13:78-80.

6. McDade, J.E., and C.C. Shepard. 1979. Virulent to avirulent conversion in the Legionnaires' disease bacte-
rium and its effect on isolation techniques. J. Infect. Disease. (In press).

7. McDade, J.E., C.C. Shepard, D.W. Eraser. T.R. Tsai, M.A. Redus, W.R. Dowdle, and the laboratory investiga-
tion team. 1977. Legionnaires' disease: Isolation of a bacterium and demonstration of its role in other
respiratory disease. N. Engl. J. Med. 297: 1 197-1203,

8. McKinney, R.M., B.M. Thomason. P.P. Harris, L. Thacker, K.R. Lewallen. H.W. Wilkinson, G.A. Hebert, and
C.W. Moss. 1978. Recognition of a new serogroup of the Legionnaires' disease bacterium. J. Clin. Microbiol.
9:103-107.

75

Primary Isolation Using Guinea Pigs and Embryonated Eggs

9. Center for Disease Control. 1977. Followup on respiratory disease: Pennsylvania. Morbid. Mortal. Weekly
Rep. 26:93.

10. Center for Disease Control. 1978. Identification of a new serogroup of Legionnaires" disease bacterium.
Morbid. Mortal. Weekly Rep. 27:293.

11. Center for Disease Control. 1978. Isolates of organisms resembling Legionnaires' disease bacterium from
environmental sources: Bloomington, Indiana. Morbid. Mortal. Weekly Rep. 27:283.

12. Thornsberry, C. C.N. Baker, and L.A. Kirven. 1978. /« vitro activity of antimicrobial agents and Legion-
naires' disease bacterium. Antimicrob. Agents Chemotherap. 13:78-80.

76

Primary Isolation Media and Methods

James C. Feeley

George W. Gorman

Robert J. Gibson

Epidemiologic Investigations Laboratory Branch

Bacterial Diseases Division

Bureau of Epidemiology

77

Primary Isolation Media and Methods

James C. Fcclcy. George H'. Gurnnin. and Robert J. Gibson

Rickettsial tecliniques were used by McDade et al. in tiie initial isolation of the Legion-
naires' disease bacterium (LDB) (4). In attempts to grow the LDB on artificial media, Dr. Robert
Weaver inoculated portions of suspensions of LDB-infected embryonated hens' egg yolk sacs
obtained from Drs. McDade and Shepard onto 17 different bacteriological agars. Of the media
tested, the only one that supported growth of the LDB was Mueller-Hinton agar, supplemented
with I'A hemoglobin and 1% IsoVitaleX (MH-IH) (/).

After studying MH-IH agar, we developed two solid media that better support the growth of
the LDB from tissue. We also developed a diphasic medium for culturing blood. All these
media-Feeley-Gorman (F-G) agar (/), charcoal yeast extract (CYE) agar {2). and CYE diphasic
blood culture medium (.?)— contain chemicals necessary for growing the LDB. Soluble ferric
pyrophosphate or ferric nitrate replaces hemoglobin, and L-cysteine HCl replaces IsoVitaleX in
these new media.

The CYE agar is the most sensitive artificial medium developed to date for isolating the
LDB, but it does not offer the differential clues for LDB colony identification that the F-G agar
allows. Consequently, we recommend that both CYE and F-G agars be prepared as described
below and used in combination. Although we no longer use MH-IH agar for isolating the LDB, we
include the instructions for preparing it as a reference. For blood cultures, we recommend a CYE
diphasic medium.

Preparation of Media

L MH-IH Agar

Component A Mueller-Hinton agar 38 gm

Distilled water 490 ml

Component B Hemoglobin powder 10 gm

Distilled water 490 ml

Component C IsoVitaleX (#1 1876) 20 ml

Prepare components A and B separately, and autoclave at 12rC for 15 min; cool to 50° C,
combine A and B, and hold at 50°C in a water bath. Prepare Component C by adding 10 ml of
sterile distilled water to each of two vials containing lyophilized IsoVitaleX. Add the contents of
both vials to the A-B mixture. Before pouring 20-mI quantities of medium into each of a series of
plastic dishes ( 1 5- X 100-mm), adjust the pH so that the final pH of the cooled medium will be
6.9. Allow agar plates to cool to room temperature. Measure the final pH of a plate of cool agar
(procedure in the Quality Control section of this chapter). The final pH must be between 6.85
and 6.95 in order to consistently support growth of the LDB.

78

Primary Isolation Media and Methods

II. F-G Agar

Casein (acid liydrolysis) 17.5 gm

Beef extractives 3.0 gm

Starcii 1.5 gm

Agar 1 7.0 gm

L-cysteine HCl • H. 0 0.4 gm

Ferric pyrophosphate, soluble* 0.25 gm

Distilled water 1.0 liter

* Available on request from Dr. Morris Suggs, Director. Biological Products Division, Center tor Disease Control, Atlanta, Georgia
30333.

Baltimore Biological Laboratories (BBL) Mueller-Hinton (M-H) agar, lot #105621 , contains
all the ingredients of F-G agar except for L-cysteine HCl and ferric pyrophosphate, soluble, and
provides a source of casein, beef extractives, starch, and agar. (NOTE: Other lots of M-H agar
may vary too widely in formulation and stability to serve as the base for satisfactory F-G agar.)

Add the casein (acid hydrolysate), beef extractives, starch, and agar (or their equivalent-38
gm of M-H agar) to 980 ml of distilled water, autoclave at 121°C for 15 niin, cool to 50°C in a
water bath, and hold until L-cysteine HCl and soluble ferric pyrophosphate are added.

Prepare separate fresh solutions of L-cysteine HCl (0.4 gm in 10 ml distilled water) and
soluble ferric pyrophosphate (0.25 gm in 10 ml distilled water). Note the precuation for ferric
pyrophosphate stated below. Filter sterilize each solution separately. Add the L-cysteine HCl to
the agar mixture first, and then add the ferric pyrophosphate.

Before pouring the warm medium into petri plates, adjust its pH so that the final pH of the
cooled medium will be 6.9. Pour 20 ml into each of a series of plastic petri dishes (15- X
100-mm). Allow agar plates to cool to room temperature. Measure the pH of a plate of cool agar
(procedure in the Quality Control section of this chapter). The final pH must be 6.85 to 6.95 to
consistently support growth of the LDB.

Precaution: Soluble ferric pyrophosphate must be kept dry and stored in the dark; it is no
longer usable if its color changes from green to yellow or brown. Prepare fresh solution of the
compound each time it is needed for media. Do not heat over 60°C to dissolve. The mixture can
be readily dissolved by placing in a 50° C water bath.

III. CYEAgar

Yeast extract (DIFCO) 10.0 gm

Activated charcoal (Norit SG)* 2.0 gm

L-cysteine HCl ■ Hj O 0.4 gm

Ferric pyrophosphate, soluble 0.25 gm

Agar (DIFCO) . ~. 1 7.0 gm

Distilled water 1 .0 hter

Add all other ingredients of CYE agar except L-cysteine HCl and soluble ferric pyrophos-
phate to 980 ml of distilled water; dissolve by boiling; autoclave at I21°C for 15 min, and cool to
50°C in a water bath.

Prepare separate fresh solutions of L-cysteine HCl (0.40 gm in 10 ml distilled water) and
soluble ferric pyrophosphate (0.25 gm in 10 ml distilled water). Filter sterilize each solution
separately. Add the L-cysteine HCl to the basal medium first, and then add the ferric pyrophos-
phate. Note the above precaution for ferric pyrophosphate.

* Available through Sigma Chemical Co., St. Louis, MO., Catalogue # C5510. Activated charcoal, washed witli phosphoric and
sulfuric acids.

79

Primary Isolation Media and Methods

Adjust the complete medium to exactly pH 6.9 at 50°C by adding 4.0 to 4.5 ml of 1.0 N
KOH. The pH of this medium is critical.

Pour 20-ml quantities of medium into sterile petri dishes ( 1 5- X 100-mm). Swirl the medium
between pouring plates to keep charcoal particles suspended.

IV. CYE Diphasic Blood Culture Medium

Agar Phase:

Activated charcoal (Norit SG) 2.0 gm

Agar(DIFCO) 17.0 gm

'Distilled water 500.0 ml

Broth Phase:

Yeast extract (DIFCO) 20.0 gm

L-cysteine HCI • H2 O 0.40 gm

Fe(N03)3 ■ 9H,0 0.10 gm

Distilled water 500.0 ml

Prepare agar first. Combine ingredients, boil, and dispense as 20-ml aliquots into each of a
series of 125-ml Wheaton serum bottles. Stopper each bottle loosely with both a rubber stopper
and a metal cap. Autoclave the bottles at HTC for 20 min, cool to 50°C. remix the warm
charcoal and agar thoroughly, and place the bottles at an angle so that an agar slant with a
vertical height of 6.0 cm is formed. This procedure should leave a portion of the agar protruding
above the 25 ml of liquid-broth plus specimen-that are added later.

Prepare the broth by first autoclaving the yeast extract and water at 12rC for 15 min.
Allow to cool, and then add fresh sterile solutions of L-cysteine HCI and ferric nitrate in that
order. Adjust the pH to 6.9 by adding 6.0 ml of 1 N KOH. Dispense the broth in 20-ml aliquots
into the Wheaton bottles of charcoal agar slants. Seal the bottles by crimping the metal caps over
the rubber stoppers. Check for sterility by preincubation at 35°C for 2 days.

Quality Control

Each batch of media must be tested for pH and ability to support the growth of the LDB.
Ideally, all media should be tested with a standardized tissue inoculum. However, since this
material is not generally available, stock culture inoculum is recommended with the understand-
ing that it is a minimal quality control test.

1. The pH of the media must be 6.9 ± 0.05. Measure with a pH meter. To check solid media,
use a surface electrode (if available), or emulsify the agar from one plate in distilled water, and
measure the pH of the emulsion. The pH is critical. If given lots of ingredients do not conform to
the acceptable pH range, adjust the pH of the complete liquid medium by adding 1 N HCI or 1 N
KOH.

2. Check for support of growth in the following manner:

a. Prepare a standardized inoculum by emulsifying some actively growing LUB in sterile
distilled water to a turbidity of a McFarland ^4 standard.

b. Seed the media with 0.05 ml of the standard moculum. Streak the plates for isolated
colonies, and incubate at 35°C aerobically (except for F-G agar, which is incubated in air
plus 2.5?; CO: ).

c. Examine the media daily. On agar plates, growth should be present in the heavily
inoculated area after 2 days. Isolated colonies should be macroscopically visible in 4 to 5

80

Primary Isolation Media and Methods

days. Hold blood bottles for 2 wk, and tilt once each day so that the agar slant is inoculated
with any growth in the broth. Look for turbidity of the broth and growth on the slant
protruding from the broth.

3. Store media in the dark, in sealed plastic containers, in a refrigerator. (Media have been used
successfully for growing the LDB after being stored for as long as 2 mon under these conditions.)

Maintenance of Stock Cultures

Stock cultures of the LDB have remained viable for several months in our laboratory when
stored as follows:

1. Prepare CYE agar and pour (5 ml/tube) into 16- X 125-mm screw-capped tubes. Allow to
cool in a slanted position.

2. Inoculate the surface of the CYE agar slants with strains of the LDB.

3. Incubate the slants at 35°C for 2 days.

4. Remove, tigliten the screw caps, and store in the dark at room temperature.

Clinical Specimens

Clinical specimens can be either liquid or solid. The initial preparation, media inoculation,
and subculturing of colonies thouglit to be LDB must all be done in a biological safety cabinet by
personnel protected with masks and gloves.

I. Initial Processing and Media Inoculation

A. Liquid specimens (pleural fluid, transtracheal aspirate, sputum, blood, etc.) re-
quire no special preparation before being inoculated directly on media. Inoculate each
container of F-G and CYE agar in two spots. Leave one spot undisturbed, and streak
the other with a bacteriological loop for isolated colonies. Inoculate blood specimens
directly into botdes of CYE diphasic medium. Use a sterile needle and syringe to put 5
ml of fresh blood into each bottle; mix well.

B. Solid specimens (lung, spleen, liver) should be processed (sliced or ground) as
follows:

1. To prepare sliced specimens, cut the tissue with a sterile scalpel, and slide the
freshly cut surface of the tissue over the surfaces of media to be inoculated. This
procedure must be followed for tissues obtained from patients who received
extensive antibiotic therapy.

2. Prepare 10% suspensions of ground tissue by emulsifying approximately 1.0
gm of tissue in 9.0 ml of sterile phosphate buffered saline (PBS), pH 7.2. Use a
sterile mortar and pestle with sterile 60-mesh alundum as an abrasive. Mix well,
and dispense into several sterile screw-capped test tubes. Use one aliquot for steps
3 and 4. Quick freeze the remainder for future use.

3. Inoculate CYE and F-G agars with one aliquot of the unfrozen suspension as
described for liquid specimens.

4. Also, dilute the above 10% tissue suspension 1:50. Inoculate CYE and F-G
agars with 0.1 ml of this 0.2% suspension. Spread the inoculum over the entire
surface of each medium with a sterile glass spreading rod.

C. Incubate aU inoculated CYE media aerobically in a humidified atmosphere at
35°C. Incubate the inoculated F-G agar at 35°C in air plus 2.5% CO2 •

Primary Isolation Media and Methods

II. Examination of Cultures

Examine all ciiltiu-es macroscopically and microscopically each day.

A. MH-IH Agai :

Examine plates for macroscopic growth. Colonies which are visible before 3 days
of incubation probably are not LDB, because colonies of LDB do not usually appear
on this medium before 5 days of incubation. Examine colonies suspected of being LDB
with a dissecting microscope. They should have a cut-glass appearance, and there
should be a distinctive brownish darkening of the surroundmg agar.

B. F-G Agar:

Examine this medium macroscopically with a desk lamp, allowing the light to pass
through the agar. In areas of heavy inoculum, confluent growth will be visible in 2 to 3
days, and there will be a distinctive brownish darkening of the colonies and the sur-
rounding agar. In liglitly inoculated areas, colonies of LDB should appear in 4 to 5 days
as white pin-point dots. These colonies grow larger after longer incubation. Examine
heavy growth in the dark with a long-wave (366 nm) ultraviolet light; a butter-yellow
color should be detectable in the 4- to 5-day growth. Examine suspected LDB colonies
microscopically by placing plates on the stage of a dissecting microscope and illuminat-
ing from one side with a light source held at an angle slightly > 10° to the horizontal.
LDB colonics will have a cut-glass texture.

C. CYE Agar:

Examine CYE plates as described for F-G plates. LDB colonies will appear in 3 to
5 days. Young colonies have a slight cut-glass texture which is rapidly lost after addi-
tional incubation. The browning of the agar is not visible on this black mediiun.

D. CYE Diphasic Blood Culture Medium:

Examine bottles daily for growth on the portion of agar slant extending above the
broth. Reinoculate this part of the slant daily by tilting the bottle so that broth flows
over it. Growth should be observed in about 6 to 8 days. Subculture colonies to CYE
agar. Keep bottles for 14 days before discarding as negative.

in. Presumptive Identification of LDB Colonies

Subculture colonies with a characteristic cut-glass appearance to both LDB-growth-
supporting media (F-G and CYE agar) and non-LDB growth-supporting medium (a blood
agar plate that does not contain L-cysteine). Incubate media at 35°C in air plus 2.59? CO,
for 5 days. Examine plates daily for growth. Test all cultures that grow on F-G and/or CYE
agar but not on blood agar. Cultures which grow on plain blood agar are not the LDB. A
culture that has typical morphological and biochemical characteristics, a compatable cellular
fatty acid profile, and a higli DNA-relatedness value to LDB strain Philadelphia 1 is the
LDB.

Figure 1: Growth of the Legionnaires' disease bacterium (LDB). Suspensions of guinea pig spleen tissue infected

with LDB strain Philadelphia 1 were inoculated onto CYE, F-G. and MH-IH agars. MH-IH and F-G agars received

a 1/5,000 dilution: CYE agars received a 1/500,000 dilution. Plates were incubated at 35°C in air + 2.5% COj-

Pictures show growth at 4, 6 and 10 days on CYE (black plates), F-G (clear plates), and MH-IH (red plates)

media.

Figure 2: Heavy growth of the LDB on F-G agar showing brown pigment production.

Figure 3: The LDB on F-G agar showing cut-glass appearance of colonies resulting from oblique lighting.

Figure 4: CYE diphasic blood culture medium; Wheaton bottle with a charcoal agar slant and brotli inoculated

with blood.

82

Primary Isolation Media and Methods

4 Days

6 Days

10 Days

Fimire 2.

Figure 1 .

Figure 4.

Figure 3.

k

^

l-^^M

li^B

83

Primary Isolation Media and Methods

REFERENCES

1. Feeley, J.C., G.W. Gorman, R.E. Weaver, D.C. Mackel. and W.H. Siiiitli. 1078. Priinary isolation media for the
Legionnaires' disease bacterium. J. Clin. Micro. 8:320-325.

2. Feeley, J.C, R.J. Gibson, G.W. Gorman, N.C. Langford. J.K. Rasiieed, D.C. Mackel, and W.B. Baine. CYE
Agar: A primary isolation medium for the Legionnaires' disease bacteriuin. (Manuscript submitted for publica-
tion).

3. Feeley, J.C, G.W. Gorman, P.H. Edelstein, and R.J. Gibson. CYE diphasic blood culture medium. (Manu-
script in preparation).

4. McDade, J.E., C.C. Shepard, D.W. Fraser, et al. 1977. Legionnaires' disease. Isolation of a bacterium and
demonstration of its role in other respiratory diseases. N. Engl. J. Med. 297:1 197-1203.

84

Method for Isolating

Legionnaires' Disease Bacterium

from Soil and Water Samples

George K. Morris

Peter Skaliy

Charlotte M. Patton

James C. Feeley

Epidemiologic Investigations Laboratory Branch

Bacterial Diseases Division

Bureau of Epidemiology

85

Method for Isolating

Legionnaires' Disease Bacterium

from Soil and Water Samples

George K. Morris, Peter Skaliy.
Charlotte M. Patton and James C. Feelev

INTRODUCTION

Thirteen documented outbreaks of Legionnaires" disease (LD) iiave occurred in the United
States since 1965 (2). Epidemiologic investigations in several outbreaks indicated tiiat the etio-
logic agent was transmitted in air. Among the environmental sources implicated were water from
air-conditioning cooling towers or evaporative condensers (3) and soils disrupted by constmction
work and presumably transported as dust by air currents (7.6). In order to document environ-
mental sources of the Legionnaires" disease bacterium (LDB) microbiologically, we modified the
primaiy isolation system that utilizes guinea pigs and embryonated eggs (described by McDade in
this manual). We used these procedures to isolate the LDB from 38 of 176 environmental samples
of water and soil collected during 1 1 epidemic investigatons in 10 states (5, unpublished data).
All samples were subjected to two types of analyses: (a) primary evaluation using the direct
fluorescent antibody (FA) test and culture media and (b) guinea pig inoculation and evaluation.

COLLECTION AND PRIMARY ANALYSIS OF SAMPLES

Water or soil and other solid samples should be collected asceptically in chemically-inert
sterile containers and refrigerated until analyzed. Personnel analyzing these samples should wear
proper safety attire and manipulate the materials only in a biological safety cabinet.

Primary analysis involves the direct FA test and primary culture on Feeley-Gomian (F-G)
and charcoal yeast extract (CYE) agars. The media are described by Feeley et al. in this manual.

Direct FA. Screen samples with direct FA to detemiine which contain bacteria which stain
with the LDB conjugate(s) and are tluis most likely to yield isolates of the LDB when cultured.
Prepare smears of water samples and aqueous suspensions of soil and other solid material by
completely filling the circles on a microscope slide with the sample. Let the slides air dry, fix
with gentle heat and then with 10% neutral Formalin. The procedure is described fully by Cherry
and McKinney in this manual.

Four serogroups of the LDB have been defined to date (4). Although ideally all specimens
should be screened for the presence of all known serogroups, appropriate FA conjugates must be
available in order to implement such a policy.

Primary Culture of Water Samples. Prepare a water sample for direct culture on media by
making serial 10-fold dilutions to lO""* in sterile distilled water. Inoculate portions (0.1 ml) of the
undiluted sample and of each dilution to individual F-G and CYE agar plates, in triplicate, and
spread with a smooth glass rod. Incubate the plates at 35°C in air plus 2.5% COj . Examine plates
daily for growth. After 2 to 3 days of incubation, calculate colony fonning unit (CFU) value for

86

Method tor Isolating Legionnaires" Disease Bacterium I'roiii Soil and Water Samples

growth on each medium, and record as follows:

sparse growth <10'' CFU/ml

' moderate growth lO"* to 10* CFU/ml

heavy growth >10*' CFU/ml

Use the calculated microbial density or CFU for each water sample as a guide to determine
amount of sample to inject into guinea pigs in the procedure described in a later section of this
chapter.

Examine plates at 3 days and daily thereafter for the development of additional colonies
resembling the LDB. Colonies of the LDB are usually visible within 4-7 days; however, incubate
plates with no growth or well-dispersed colonies of nomial tlora for 14 days before discarding as
negative. Confirm isolates suspected of being LDB with tests described elsewhere in this manual.

Primary Culture of Soil Samples. Culture soil samples on F-G and CYE agar media. To
prepare a soil sample for culture, first add 10 gm of soil to 100 ml of sterile distilled water
containing 0.5% Tween 60. Then shake the suspension vigorously for 30 min on a mechanical
shaker. Let the heavy particles settle for 5-10 min, and then plate the aqueous layer as described
for liquid samples using serial 10-fold dilutions through 10"". Examine all plates for LDB as
described above for water samples.

PREPARATION OF SAMPLE AND INOCULATION OF GUINEA PIGS

Although the guinea pig procedure is a laborious and tedious means of isolating the LDB,
researchers have not yet found a satisfactory alternative for working with soil and water samples.
Only once in our laboratories has the LDB been isolated directly from a water sample without
inoculating guinea pigs. Results of direct FA tests (performed with polyvalent conjugates against
all known serogroups) show which samples are most likely to contain the LDB when tested
further. Environmental samples are frequently much more contaminated with other microflora
than are clinical specimens. If a guinea pig is inoculated with a large number of microbial tlora,
the animal is likely to die of causes other than an LDB infection. Therefore, it is important to be
aware of the nonnal tlora concentration in the samples when choosing the inoculum for the
guinea pigs. We currently do this by culture. Other potentially suitable methods for estimating
microbial density of non-LDB which we have not yet evaluated are direct microscopic observa-
tion of Gram-stained slides and growth of non-LDB on total plate count media. Before injecting
guinea pigs with samples, establish each animal's average baseline temperature from measure-
ments taken on each of the 5 days before inoculation.

Water Samples (See Figure 1). If the CFU/ml described above from cultures of a water
sample is less than 10", concentrate the sample. Centrifuge 100 ml at high speed (ca. 2900 X g)
for 30 min; discard the supernatant. Resuspend the sediment in 10 ml of sterile water. Mix well,
and inoculate two guinea pigs intraperitoneally (IP) with 3 ml each of the sample. When the
CFU/ml for a sample is moderate (10'* to 10*), inject 3 ml of the untreated sample into each of
two guinea pigs. If the CFU/ml for a sample is high (> 10* ), dilute it to 10* CFU/ml and inject 3
ml of the diluted sample.

Soil Samples (See Figure 2). Suspensions of soil samples to be used as inoculum are prepared
as follows: Add 10 gm of soil sample to 100 ml of sterile distilled water containing 0.5% Tween
60. Place the mixture on a shaker at moderate speed for 30 min. Allow 5-10 min for the heaviest
particles to settle, decant and save the top portion, and discard the heavy sediment. Centrifuge
the top portion (approximately 90 ml) of the suspension at 400 X g for 5 min. Carefully remove
the supernatant and discard the sediment. Centrifuge the supernatant at 2900 X g for 30 min.
Discard this tlnal supernatant, and resuspend the sediment in 10 ml of sterile distilled water.

87

Method for Isolating Legionnaires' Disease Bacterium from Soil and Water Samples

Determine the total CFU/ml in tiie final suspension, adjust to 10*^ CFU/ml, and inject 3 ml into
each of two guinea pigs. Refrigerate the final suspension for 2 to 3 days until bacterial quantita-
tion is completed before inoculating the guinea pigs.

Water Sample

Growth (CFU/ml) on Agars

1

1

If low
KIO^)

Centrifuge

100 ml sample

2900 X g for 30 min.

Discard
supernatant.

Resuspend

sediment in

10 ml sterile HjO.

Inject
3 ml/animal.

If moderate
(104 to 106)

Inject
3 ml/animal.

If high
(>106)

Dilute with

sterile HjO

to 106 CFU/ml.

Inject
3 ml/animal.

Figure 1 . Preparation of Water Samples for Guinea Pig hioculation

Obsei-ving the Inoculated Guinea Pigs and Collecting Spleens. Measure the temperature of
the inoculated guinea pigs at a predetennined time each day for 7 days, and observe the animals
for signs of illness, i.e., ruffled fur, water eyes, prostration, and hypothermia. If there is a rise in
temperature of 0.6°C for 2 consecutive days or any rise in temperature with 1 of the above
symptoms, sacrifice and examine the affected animal. Sacrifice and examine all other guinea pigs
in the study at 7 days. Aseptically remove splenic tissue from the animals for culturing. Culture
peritoneal exudates and material swabbed from peritoneal walls by direct plating on CYE and
F-G agars.

88

Method for Isolating Legionnaires' Disease Bacterium from Soil and Water Samples

CULTURING GUINEA PIG SPLENIC TISSUE

The procedures for preparing tissue homogenates, inoculating emhryonated eggs, and col-
lecting yolk sacs are described by McDade in this manual. Splenic tissue and yolk sacs are
inoculated to F-G and CYE agar according to methods described by Feeley et al. in this manual.
Isolates suspected of being LDB must be confirmed as such with tests described elsewhere in this
manual.

Add 10 g soil

to

100 ml sterile distilled

HjO containing 0.5%

Tween 60.

Agitate on shaker for 30
min.— let settle for 5-10 min.

Discard
sediment.

I

Centrifuge I i

supernatant also
400 X g for 5 min. i 1

Discard
sediment.

Use supernatant

for

primary analysis:

screen by

direct FA,

plate to

F-G and CYE agars

Centrifuge supernatant
2900 X g for 30 min.

Sediment

+

10 ml sterile water

Discard
supernatant.

Determine CFU/ml

Adjust to <106 CFU/ml

Inoculate guinea pigs
3 ml/animal.

Figure 2. Preparation of Soil Samples for Guinea Pig hioculation

89

Method for Isolating Legionnaires' Disease Bacterium from Soil and Water Samples

REFERENCES

1. Center for Disease Control. 1*^)65. Institutional outbreak of pneumonia, Washington, D.C. Morbid. Mort:il.
Wkly. Rep. 14: 265-266.

2. Center for Disease Control. 1978. Legionnaires" disease -United States. Morbid. Mortal. Wkly. Rep. 27(45):
439.

3. Click, T.H., M.B. Gregg, B. Berman. G. Mallison, W.W. Rhodes, and I. Kassanoff. 1978. Pontiac fever: An
epidemic of unknown etiology in a health department. I. Clinical and epidemiologic aspects. Am. J. Epidemiol.
107:149-160.

4. McKinney, R.M., L. Thacker, P.P. Harris, K.R. Lewallen. G.A. Hebert, P.H. Edelstein, and B.M. Thomason.
1979. Four serogroups of Legionnaires' disease bacteria defined by direct immunofluorescence. Ann. Intern.
Med. 90:621-624.

5. Morris, G.K., CM. Patton, J.C. Feeley, S.E. Johnson, G. Gorman, W.T. Martin, P. Skaliy, G.F. MaUison, B.D.
Politi. and D.C. Mackel. 1979. Isolation of Legionnaires' disease bacterium from environmental samples. Ann.
Intern. Med. 90:664-666.

6. Thacker, S.B., J.V. Bennett, T.F. Tsai, D.W. Eraser, J.E. McDade, C.C. Shepard, K.II. Williams, Jr., W.H.
Stuart, H.B. Dull, and T.C. Eickhoff. 1978. An outbreak in 1965 of severe respiratory illness caused by the
Legionnaires' disease bacterium. J. Infect. Dis. 138(4):512-519.

90

Detection of Legionnaires' Disease
Bacteria in Clinical Specimens

by
Direct Immunofluorescence

William B. Cherry
Roger M. McKinney

Analytical Bacteriology Branch

Bacteriology Division

Bureau of Laboratories

91

Detection of Legionnaires' Disease

Bacteria in Clinical Specimens

by

Direct Immunofluorescence

William B. Cherry and Roger M. McKinney*

INTRODUCTION

Direct fiuorescent antibody (FA) staining is useful for detecting Legionnaires' disease bac-
teria (LDB) in clinical specimens and in environmental samples. In early studies of the LDB, a
fluorescein isothiocyanate (FITC) conjugate of rabbit antibodies to the Knoxville 1 strain stained
all of 13 available isolates (i, 4). LDB isolates have since been obtained that do not stain with the
Knoxville 1 conjugate. Four serogroups of LDB have been recognized to date by direct FA
staining. Knoxville 1 , isolated by McDade from a postmortem lung specimen from a patient in
Knoxville, Tennessee, is representative of serogroup 1 ; Togus 1 , isolated by McDade from a
postmortem lung specimen from a patient in Togus, Maine, is representative of serogroup 2 (9);
Bloomington 2, an environmental isolate obtained from a creek in Bloomington, Indiana (2, 11),
is representative of serogroup 3; Los Angeles 1. obtained from a liunian lung specimen (5) is
representative of serogroup 4. Serogroup-specific FA conjugates for each of the four recognized
serogroups (70), and a polyvalent conjugate containing all four group-specific conjugates have
been prepared. The current practice in diagnostic direct FA staining at the Center for Disease
Control (CDC) is to first test clinical specimens by staining with the polyvalent reagent. If the
tissue is FA positive, the serogroup is determined by staining with each of the four group-specific
conjugates. Although the great majority of FA positive clinical specimens tested at the CDC have
reacted only to serogroup 1 conjugate, four clinical specimens have been found positive only with
serogroup 2 conjugate, three only with serogroup 3 conjugate, and two only with serogroup 4
conjugate. It is likely that additional serogroups of LDB will be recognized in the future. There-
fore, when isolates are obtained that have the growth characteristics and colonial appearance of
LDB but do not stain with polyvalent conjugate, further definitive tests for LDB such as bio-
chemical tests, gas4iquid chromatography of cellular fatty acids, and measurements of DNA
relatedness must be performed.

Legionnaires' disease (LD) is naturally acquired by the respiratory route, and organs other
than the lung rarely appear to be involved. Therefore, this chapter deals almost exclusively with
detecting LDB in lung tissue or lung exudates. If all available clinical, epidemiological, micro-
biological, and serological data are considered in interpreting direct FA test results, they are
extremely valuable for indicating the scope of an outbreak and for establishing retrospective and
current diagnoses.

*The contributions of Bertie Pittman, Patricia P. Harris, G. Ann Hebert, Berenice M. Thomason. LeRoy Tliacker, and Karen
Lewallen of the Immunofluorescence Section. Analytical Bacteriology Brancli, who participated in tlie development of the
methods described, are gratefully acknowledged.

92

Detection ol' Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunonuorescence

PREPARATION OF SPECIMEN MATERIAL
Tissue scrapings of Formalin-fixed tissue (■/)

1. Select one or more areas of the lung or other tissue for testing. With hing tissue, choose dense
areas of gray or reddish consolidation.

2. Transfer each tissue block to a sterile petri dish.

3. With a sharp scalpel, cut through these areas to produce new tissue faces for scraping.

4. Grasp the tissue with forceps, and holding the scalpel at a right angle to the tissue face, scrape
it to produce a fine puree of tissue particles. The lung tissue of victims of Legionnaires' disease is
usually quite friable. If the tissue is rubbery or spongy, a positive test is unlikely.

5. Using the scalpel blade, smear the particles of tissue and tissue tluids onto two 1.5-cm circles
on each of three microscope slides. Stain one smear on the first slide with preimmune or normal
conjugate and the other with the polyvalent conjugate. Reserve the other four smears for staining
with the four monovalent serogroup conjugates if the polyvalent conjugate gives positive results.

6. Let the smears air dry. Gently heat fix. It is not necessary to remove the Formalin before
staining.

Fresh or fresh-frozen tissue (process in a safety cabinet)

When fresh or fresh-frozen tissue is obtained through autopsy or biopsy, the first laboratory
procedure should be to inoculate culture media for attempts to isolate the LDB. This will
minimize contamination of the tissue. Then select tissue for other tests, including direct FA.

1. Using sterile instruments, cut a fresh face of tissue; with the forceps, press and squeeze the
tissue against three clean microscope slides, making impression smears within the two 1.5-cm
circles on each slide. If the tissue is so moist that smears may be too thick, blot on sterile gauze
or culture media before imprints are made.

Alternatively, grind the tissue with sterile phosphate buffered saline (PBS) and alundum in a
sterile mortar, or homogenize the tissue in a Ten Broeck or comparable tissue grinder. Use
sufficient PBS to give an approximate 10% tissue homogenate. Prepare smears in the same
manner as described for Formalin-fixed tissue. With larger pieces of tissue this method produces a
more representative sample and reduces sampling error.

2. Air dry and heat fix.

3. Fix smears for 10 min by covering them with 10% neutral Formalin. Do not let the Formalin
dry on the smears.

4. Drain off the Formalin and gently and briefly dip each slide into distilled water to remove the
remainder of the Formalin. (If slide is not rinsed, Fomialin will not interfere with staining.)

5. Air dry.

Tissue sections

Legionnaires' disease bacteria maintain their serologic integrity through histopathological
processing and can easily be demonstrated in tissue sections if they are present in reasonable
numbers. However, they are not as easily demonstrated in such sections as they are in scrapings
of Fomialin-fixed lung or imprints of fresh lung tissue because many of the bacteria are intra-
cellular, lie at many different levels in the section, and are shrunken by the histological process-
ing. Counterstains, such as rhodamine-labeled rabbit serum or rhodamine-labeled bovine serum
albumin, make the organisms much more visible in tissue sections. (See Addendum at the end of

93

Detection of Legionnaires' Disease Bacteria in Clinical Specimens b\ Direcl Imniuiuilliiorescence

this chapter for counterstain preparation.) Hvans" blue or otiier counterstains will also probably
be helpful, but their relative value has not been sufficiently documented.

1 . Cut tissue sections as thin as possible (4 fjni or less) from paraffin blocks.

2. Affi.x the sections to slides by heatinu them for appro.ximately 15 min at 58° to (iO°C.

3. Remove the paraffin and hydrate the tissue sections by sequentially dipping the slides in two
changes each of xylene, absolute ethancil, 95'^? ethanol, and distilleti water.

4. Air dry.

Lung exudates and pleural fluids (process in a safety cabinet) ( /, 6. iV )

Expectorated sputa, transtracheal aspirates (TTA), bronchial washings, pleural fluids, and
other specimens from the lower respiratory tract (LRT) are all satisfactory materials for study.
Patients with Legionnaires' disease (LD) frequently do not produce much sputum, and what they
produce may not be purulent. The secretions are usually extremely viscid and tenacious. Smears
usually contain tissue cells which will autofluoresce or stain nonspecifically, so counterstains are
needed. Until selective media are developed, it is probably not worthwhile to culture expec-
torated sputum; however, clean specimens such as TTA, bronchial washings, and pleural tluids
that are taken by invasive procedures should be cultured. Several isolates have been obtained in
this way.

1. Select a viscous portion of the sputum, aspirate, washing, or other LRT specimen, and
prepare two smears of moderate thickness on each of three microscope slides. Pleural tluids tend
to form a fibrin clot on the slide, and unless processed carefully, the entire film can be dislodged.

2. Air dry and heat fix.

3. Fix smears in \07o neutral Formalin for 10 min. Either place each slide in a separate coplin jar
or lay each one flat and flood it, but do not let the Fonnalin dry on the slide.

4. Drain off the Formalin.

5. Airdi-y.

Blood cultures

Inoculate 5 ml of patient's blood into a bottle of charcoal yeast extract (CYE) diphasic
blood culture medium (see chapter by Feeley et al., this manual). Incubate at 35°C aerobically.
Each day flood the agar slope of the diphasic medium with- the bnUh portion. Growth may not
be apparent for four or more days. When growth is apparent on the agar medium, subculture to
slants of CYE agar and Feeley-Gonnan (F-G) agar. Prepare smears for direct FA, Gram's and
other stains by standard and previously described methods.

Culture smears (process in a safety cabinet)

1. Make suspensions of known or suspected LDB cultures in 1',^ Fomialm m 0.85/f NaCl
solution to give a light turbidity (McFarland No. 2).

2. Prepare smears on double-ring slides.

3. Air di7 and heat fix.

FA STAINING PROCEDURE

1. Methods for preparing the specific LDB conjugate and a counterstain reagent are detailed in
the Addendum to this chapter.

94

Detection ol Lcpionnnircs; Disease Baeteiia in Clinical Specimens by Direct Ininiunotltiorescence

2. Stain all preparations by covering the smear nearest the labeled end of the slide with 1-2
drops (0.05 ml) of the preimnuine or other control conjugate.

3. Cover the second smear with 1-2 drops of the use dilution of the polyvalent LDB conjugate.

4. Place the slides in a covered chamber to prevent evaporation during staining.

5. Stain for 20 min.

6. Remove excess conjugate by holding the slide level, but perpendicular, and tapping it against
a towel. Quickly and gently rinse smears with a stream of PBS from a wash bottle while keeping
the slide rougjily horizontal with the long edge tipped slightly downward. Prevent the specific
conjugate from coming into contact, even momentarily, with the control smears.

7. Immerse slide in PBS for 5 min. Use a separate PBS container for each slide to prevent
cross-contamination with organisms that may wash off of LDB positive slides.

8. Rinse each slide in distilled water from a wash bottle.

9. Air dry.

10. Add a small drop of buffered glycerol (pH 9.0) and a 1 or 1'/: (Coming) cover sHp to the
smears.

EXAMINATION OF STAINED SLIDES

Examine first under the lOX objective of the tluorescence microscope. In strongly positive
preparations the bacteria may be visible as unifonnly sized dots. Select areas of the smear where
organisms may be present, and switch to a 40X or 63X oil immersion objective for screening. Use
the lOOX oil objective for close study and confinnation. The bacteria will be visible as single
short rods or small intracellular or extracellular clumps of organisms showing strong peripheral
staining with darker centers. No specimen should be reported as negative until after at least a
5 -min search.

If organisms of appropriate morphology are stained by the polyvalent LDB conjugate and
not be the control conjugate, stain the additional smears with the LDB serogroup-specific re-
agents.

CONTROLS

The most important controls consist either of preimmune conjugate from the same animals
which furnished the specific reagent or conjugates prepared from the sera of unimnumized
animals of the same species as those immunized. These control reagents should have approxi-
mately the same protein content and F/P ratio as the specific conjugate.

As controls, the conjugates should be used at either the same dilution as that of the specific
conjugate, or preferably, at twice that concentration, as a margin of safety.

Each specimen preparation showing specific staining must be tested with the control reagent
to insure that the observed reaction is serologically specific. Other essential controls are culture
suspensions, and positive and negative preparations of tissue scrapings, tissue imprints, sections,
and smears of lower respiratory tract secretions, depending upon the types of diagnostic material
being submitted for examination.

Positive and negative smears should be carried through the specimen staining procedure each
time a group of specimens is processed. Negative smears may be made from suspensions of any
heterologous bacteria which are not related serologically to the LDB.

95

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immiinonuorescence

INTERPRETATION OF RESULTS AND TEST LIMITATIONS

In all clinical specimens, except for lung exudates (LE), the following criteria are used to
evaluate the test results:

Result Report

>25 strongly fluorescing bacteria/smear* FA +

<25 strongly fluorescing bacteria/smear #'s only

0 strongly fluorescing bacteria/smear FA -

*In many lA-positive specimens, an average of 50 or more LDB per field ( 1 250X) may be observed.

Positive FA staining should be reported as FA+ (group 1, 2, 3, or 4) depending on which
diagnostic conjugate gave positive FA staining. In LE, organisms are seldom numerous. Thus, the
observation of more than five brightly stained small rods morphologically typical of the LDB
constitutes a positive result in this type of specimen.

Inteipreting the results of stained tissue scrapings, sections of Formalin-fixed lung, fresh
lung imprints, cultures, and pleural fluids is rather straightforward {3, 4). Organisms in culture are
usually longer rods than those seen in tissues. In older cultures, long filaments, swollen rods, and
other bizarre fomismay be seen.

Interpreting the results of stained LE specimens such as expectorated sputa, transtracheal
aspirates, and those obtained by bronchial lavage or by bronchoscopy is more difficult. Tissue
cells and white blood cells may be highly autotluorescent. Bacteria such as staphylococci, dip-
lococci, and streptococci may fluoresce because of natural antibodies which are in the senmi of
the immunized rabbit. Frequently, many of the LDB are disintegrating from the effects of
cellular defense mechanisms. Ragged, swollen, atypical fomis and fluorescent debris are com-
monly observed. A smear stained with the negative control conjugate must be studied as carefully
as one which is presumed to be positive. Familiarity with the moiphology and staining charac-
teristics of the LDB is imperative if false-positive reports are to be avoided. For example, one of
23 strains oi Pseiidomuiias fliiorescens tested was brightly and specifically stained by the working
dilution of the serogroup 1 LDB conjugate. The fluorescent Pseiidomo}uis rods may, however,
appear wider than the LDB rods. The use of counterstains for sputum and tissue examination has
proved very helpful.

Because of the size of the particles of tissue obtained by scraping Formalin-fixed lung, many
particles are lost from the slide during processing. The free bacteria and tissue cells will, however,
remain on the slide to give a good test substrate.

Although isolation and characterization of the LDB is discussed elsewhere in this Manual,
we emphasize the necessity of attempting to culture these organisms from lung tissue, pleural
Ouid and LE of patients suspected of having the disease. Culture should be attempted even
though no LDB-like bacteria are seen by direct FA staining. The organisms may be present in
numbers too small to be detected by FA staining or new serogroups of the LDB may be
producing infection. All LDB isolates should be fully characterized culturally, serologically, and
physiologically to deteimine if they fit the accepted criteria for classification as the LDB.

DISCUSSION

Recently, Lattimer, Rhodes, and Cepil {8) commented as follows: "the degree of fluores-
cence in the I.E. A. and D.F.A. tests used for diagnosing Legionnaires" disease is dependent not
only on the presence of the bacterium but also on the materials from which the organism is
isolated (lung, egg yolk sac, or artificial media) and by the method used to prepare the antigen.

Until a standard antigen with defined sensitivity and specificity is developed, serodiagnosis should

96

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Immunonuorescence

be considered as only suggestive for the diagnosis of Legionnaires' disease and definitive diagnosis
should be established by culture." In respect to the direct fluorescent antibody (D.F.A.) test, this
view places unjustified constraints on the application of the test and limits its proven value. Our
experience with large numbers of human and animal diagnostic specimens over the past 2 years
supports the reliability of the test if the methodology presented in this chapter is used. In the
hands of experienced workers, the specificity of the direct test is high. Failure to isolate LDB
from clinical materials that are positive by direct FA staining does not necessarily indicate
cross-reactivity. The best available media are probably still suboptimal for growth and the LDB
are quite susceptible to inhibition by contaminants. The organisms may be drug inhibited or they
may have lost viability during storage or handling of the specimens. If non-LDB are present and
responsible for positive staining, they must be more difficult to isolate than the LDB because we
have not encountered them during the examination of thousands of colonies from human and
guinea pig tissues and from sputum and environmental specimens. We isolated only one organism,
a single strain of Ps. fluorescens, that is antigenically related to the serogroup 1 LDB. It was
present as a contaminant in commercial Evans' blue counterstain. Twenty-two laboratory-
maintained strains of Ps. fluorescens were nonreactive with the group specific and polyvalent
LDB conjugates. Over 400 pure cultures representing 25 genera and 60 species and 54 unidenti-
fied cultures were negative. Lung tissues from a number of patients with pneumonia known to be
caused by other bacteria or fungi also were negative when tested with LDB conjugates.

Undoubtedly the future will reveal some additional antigenic relationships between LDB
and other organisms. Certainly, every effort should be made to isolate and identify organisms
which are stained by the LDB conjugates. However, sufficient experience has been accumulated
attesting to the specificity of the direct FA test for the LDB to place the burden of proof of
nonspecificity on those who suggest otherwise.

The fact remains that direct FA staining of LDB is still the only rapid diagnostic test for
these organisms and also the only specific laboratory test that can be used by clinicians for
guidance in instituting therapy. Autopsy and biopsy tissue can be screened for LDB more quickly
by the direct FA test than by pathological examination. In addition the direct FA test has a
dimension of serological specificity not possessed by the Dieterle silver impregnation stain and
other histological staining techniques used to demonstrate the organisms in tissue.

PERFORMANCE CHARACTERISTICS

1. The diagnostic dilution of an LDB polyvalent or monovalent conjugate should stain, at least
to a 3+ intensity, all known strains of LDB belonging to the serogroups (polyvalent) or to a
serogroup (monovalent).

2. The corresponding control conjugate at its use dilution should not stain any of the stock
strains to more than a 1 + intensity.

SUMMARY

In this chapter we discuss the basic steps required for detection of the LDB by direct
immunofluorescence. The question of controls and the inteipretation of results and limitations of
the direct FA tests are also discussed. Questions related to specificity and sensitivity are treated
in some detail. An Addendum includes the preparation of vaccine for antiserum production, the
preparation of reagents, and a listing of LDB isolates by serogroup.

Finally, the color plate (Figures 1-8) shows the appearance of the LDB in pure culture at
different ages, in Formalin-fixed tissue sections, in scrapings of wet Formalin-fixed tissue, and in
sputum from LD patients.

97

Detection of Legionnaires' Disease Bacteria mi Clinical Specimens liy Direct Immunonuorescence

Legends for Figures

Figure 1. Smear of 48-h culture of LDB stained witli a 1:80 dilution of hotnologous cotijugate. Culture was
grown on modified Mueller-Hinton medium and suspended in phosphate buffered saline containing l^c Formalin.
Note that these cells resemble those seen in tissue. Photographed with a lOOX oil objective on 135-mm Ekta-
chrome 200 film. Magnification .X400 as photographed.

Figure 2. Smear of 5-day-old culture of LDB grown on a different modification of Mueller-Hinton medium.
Stained and photographed as in Figure 1.

Figure 3. Deparaffinized section of LDB-infected human lung tissue showing positive FA staining of serogroup 2
LDB with serogroup 2 conjugate. The Atlanta 1 isolate (serogroup 2) was obtained from a fresh-frozen specimen
of the same lung tissue. Photographed with 25X objective.

Figure 4. Deparaffinized section of lung tissue showing negative FA staining with serogroup 1 conjugate on tissue
infected with serogroup 2 LDB. The lung tissue was from the same paraffin-embedded block as that used in
Figure 3. Photographed with 40X objective.

Figure 5. Deparaffinized section of heavily infected human lung tissue showing positive FA staining of LDB with
serogroup 3 conjugate. The tissue was FA negative when stained with conjugates for serogroups 1,2, and 4.
Photographed with 40X objective.

Figure 6. LDB as they appear in a smear of scrapings of wet Formalin-fi.xed human lung tissue. Note large number
of extracellular bacteria, many of which have been released from broken tissue cells. Note absence of chains or
filamentous forms. Stained with a 1:80 dilution of homologous conjugate. Photographed as in Figure 1.

Figure 7. LDB in sputum from a patient with LD. Stained with serogroup 1 conjugate. Photographed with lOOX
oil objective on 135-mm Ektachrome 400 film. Magnification X400 as photographed.

Figure 8. LDB in sputum from a patient with LD. Stained and photographed as in Figure 7. Note filamentous

forms.

98

•

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunofluorescence

ligurc }■.

1 «•

t *

^5- rr-

^^

Mte^lN^^

^^BE^^

-c

-.4

Kiaure 5.

Figure 7.

Figure 8.

99

Detection of Legionnaires' Disease Bacteria in Clinical Speciments by Direct Immunotluorescence

ADDENDUM
I. PREPARATION OF FLUORESCENT ANTIBODY REAGENTS

A. Production of Antisera

1 . Preparation of vaccine for antisenmi production

The bacterial cell vaccine is prepared as a suspension of cells harvested from solid
media, killed by suspending overnight in 1% Fomialin-0.85% saline, centrifuged, and
resuspended and adjusted with 0.5% Formalin-0.85% saline to give a turbidity reading
of 40 International Units (Maaloe, 0. 1955. The International Reference Preparation
for Opacity - Notes and Description. Bull. World Health Organ. 12: 769-775). This is
roughly comparable to 4 x lO"* bacterial cells/ml. For injection, a portion of the
suspension is mixed with an equal volume of Freund's complete adjuvant.

2. Immunization schedule

Day 1 - Pre-bleed the rabbits for control sera. Distribute 2 ml of the cell suspension-
Freund's adjuvant mixture by intracutaneous injection into 20-40 sites along the
shaved back of each rabbit.

Day 31 - Inject 1 ml of the cell suspension-Freund's adjuvant mixture deep into the
thigh muscle of each hind leg of the rabbit ( 2 ml total).

Day 38 - Take 50 or 60 ml of blood from the ear for antiserum. Inject 2 ml of the
Formalinized cell suspension intravenously into each rabbit (no adjuvant).

Day 45 - Take 50 or 60 ml of blood from the ear. Inject 2 ml of FomTalinized cell
suspension intravenously into each rabbit.

Day 52 - Exsanguinate the rabbits.

B. Preparation of Conjugates

1. Specific LDB conjugates

Specific antisera for the four known serogroups of LDB were raised in rabbits by
immunizing with cells of the representative strains according to the above schedule.
Sera were fractionated and labeled with fluorescein isothiocyanate according to the
methods of Hebert et al. (7). Serogroup 1 comprises strains of LDB, such as Knoxville
1, that are stained brightly (3+ to 4+ intensity) with relatively high dilutions of the
Knoxville 1 conjugate, and others, such as the Bellingliam 1 strain, that are only
stained to a 3-l- to 4+ intensity when relatively low dilutions of the Knoxville 1
conjugate are used. Thus, serogroup 1 conjugates must be titered against both types of
the group 1 strains to insure that a sufficiently low working dilution is used to detect
all serogroup 1 LDB organisms. A polyvalent conjugate was prepared by combining
appropriate volumes of the four serogroup-specific conjugates so as to give 3-1- to 4-1-
staining of all of the available isolates of the four known serogroups of LDB. The
number of serogroup-specific conjugates that can be practically included in a poly-
valent preparation is limited because the total fluorescein concentration is higher in a
polyvalent reagent. Eventually, as the number of group specific conjugates is increased,
nonspecific or background fluorescence of the stained tissue specimen becomes a prob-
lem. Thus, if additional serogroups of LDB are found it will probably be necessary to
use two polyvalent conjugates for diagnostic staining.

100

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunofluorescence

2. Counterstain (rhodamiiie-labeled rabbit serum)

Deparaffinized tissue sections and sputum smears are difficult to examine for LDB by
tiie direct FA test because of the brigiit background formed by tlie nonspecific uptake
of fluorescein-labeled globulin. Whole nomial rabbit serum labeled with tetramethyl-
rhodamine isothiocyanate has been found to be useful as a counterstain with these
types of specimens.

a. Materials

1. Whole serum from nonimmunized rabbits.

2. Tetramethylrhodamine isothiocyanate (TMRD. This reagent can be purchased
in 100-mg lots from Baltimore Biological Laboratories, Baltimore, Maryland, or
from Research Organics, Inc., Cleveland, Ohio.

3. Phosphate buffered saline (PBS) - 0.5 M sodium phosphate, pH 7.6, contain-
ing 0.85% NaCl and 0.1% NaNj .

4. Dim ethyl form amide (DMF).

5. 0.5MNa3PO4.

6. Sephade.x G-25 fine (Phamiacia Fine Chemicals, Piscataway, New Jersey).

b. Procedures

A 40-ml aliquot of whole serum is adjusted to pH "5.5 by adding 0.5 M Na,, PO4 . A
100-mg portion of TMRI is dissolved in 2 ml of DMF before being mixed with
serum. The reaction mixture is allowed to stand for at least 1 h at room tempera-
ture and then centrifuged to remove particulate matter. Unreacted rhodamine
products are removed by gel filtration on a Sephadex column. A column 5 cm in
diameter by 30 cm in length packed with 100 g of Sephadex G-25 fine that has
been presoaked in PBS is adequate for this purpose. The first colored fraction to
emerge from the column is the rhodamine-labeled serum. This fraction is followed
by an orange fraction and a red fraction which are discarded. The column can be
washed fairly clean of rhodamine products and used repeatedly. The whole
serum -rhodamine conjugate should be dialyzed against PBS to remove traces of
DMF, adjusted to a 160-ml volume by adding PBS, and filtered through a 0.45 ^m
fOter.

c. Application

The anti-LDB conjugate, at four times the usual working concentration, is com-
bined with three parts of the counterstain. This mixture is used in the usual
manner for FA staining of LDB in tissue sections or in sputum specimens. If the
anti-LDB conjugate is in the lyophilized state, it is rehydrated by adding the
appropriate volume of counterstain to produce the desired working concen-
tration. When a counterstain is used with the diagnostic conjugate, a similarly
prepared control conjugate must also be used.

101

Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunofluorescence

II. LIST OF LDB ISOLATES

A. Serogroup 1 isolates of the Knoxville 1 type include:
Knoxville 1 Pontiac 1 (E)*
Philadelpiiia 1-4 Miami Beach 1
Flint 1 and 2 Orlando 1

Detroit 1-4 Indianapolis 1 and 2

Beri<eley 1 Oak Lawn 1

Bimiinghani 1 Baltimore 1

Burlington 1 and 2 Houston 1

Buffalo 1 Chicago 1

Allentown 1 Long Beach 1 and 2

Albuquerque 1 Cambridge 1 (England)

Los Angeles 2-14 Atlanta 3

New York 1 and 2 Bellingliam 1

Olda Gastonia 1

Rochester 1 TOTAL = 50

B. Serogroup 2 isolates obtained to date include:
Togus 1

Atlanta 1 and 2

C. Serogroup 3 isolates obtained to date include the environmental representative Bloom-
ington 2 (E). The human isolates Chicago 2 and 3 and Houston 2 also stain with
serogroup 3 conjugate.

D. Serogroup 4 is represented by the Los Angeles 1 strain obtained from human tissue.

All strains listed above were obtained from human patients with LD except Blooming-
ton 2, an environmental isolate which is the type strain of serogroup 3, and Pontiac 1, which
was isolated from a guinea pig naturally infected by the aerosol route. Thus 49 or 87. 59^ of
the 56 liuman isolates listed above belong to serogroup 1 . A number of other environmental
isolates not included in the above list have been obtained that belong to serogroups 1-4. Still
other environmental isolates have been found that apparently represent new serogroups but
these cultures have not been completely characterized.

REFERENCES

1. Broome, C.V., W.B. Cheny, W.C. Winn, and B.R. MacPherson, Jr. IQV). Rapid diagnosis of Legionnaires'
disease by direct immunofluorescent staining. Ann. Intern. Med. 90:1-4.

2. Center for Disease Control. 1978. Isolates of organisms resembling Legionnaires' disease bacterium from
environmental sources - Bloomington, Indiana. Morbid. Mortal. Weekly Rept. 27:283-285.

3. Cherry, W.B., P.P. Harris, G.A. Hebert, B.M. Thomason, and R.M. McKinney. 1978. Legionnaires' disease -
the disease and the bacterium with special reference to the laboratory diagnosis. In Immunofluorescence and
Related Staining Techniques, W. Knapp, K. Ilolubar, and G. Wick (eds.). Elsevier/North-Holland Biomedical
Press, Amsterdam.

4. Cherry, W.B., B. Pittman, P.P. Harris, G.A. Hebert. B.M. Thomason, L. Thacker, and R.H. Weaver. 1978.
Detection of Legionnaires' disease bactena by direct immunofluorescent staining. J. Clin. Microbiol. 8:329-
338.

EnvironmenUil isolate

102

Detection of Legionnaires' Disease Bacteria ni Clinical Specimens by Direct InimunoniiDresccnce

5. Edelstein, P.H., R.D. Meyer, and S.M. Finegold. I'^VS. Isolation of a new serotype of Legionnaires' disease
bacterium. Lancet ii: 1 172-1 174.

6. Gump. D.W., R.O. Frank, W.C. Winn, Jr.. R.S. Foster, Jr., C.V. Broome, and W.B. Cherry. 1979. Legion-
naires" disease in patients with associated serious disease. Ann. Intern. Med. 90:538-542.

7. Hebert. G.A.. B. Pittman, R.M. McKinney, and W.B. Cherry. 1972. The Preparation and Physicochemical
Characterization of Fluorescent Antibody Reagents. U. S. Department of Health, Education, and Welfare,
CDC, Atlanta, GA.

8. Lattimer, G.L., L.V. Rliodes III, and Beth Cepil. 1979. Legionnaires' serotypes. Lancet i: 109.

9. McKinney, R.M.. B.M. Thomason. P.P. Harris, L. Thacker, K.R. Lewallen, H.W. Wilkinson, G.A. Hebert, and
C.W. Moss. 1979. Recognition of a new serogroup of the Legionnaires' disease bacterium. J. Clin. Microbiol.
9:103-107.

10. McKinney, R.M., L. Thacker, P.P. Harris, K.R. Lewallen. G.A. Hebert. P.H. Edelstein. and B.M, Tliomason.
1979. Four serogroups of Legionnaires' disease bacteria defined by direct immunonuorescence. Ann. Intern.
Med. 90:621-624.

1 1 . Morris. G.K., CM. Patton. J.C. Feeley , S.E. Johnson, G. Gorman, W.T. Martin, P. Skaliy , G.F. Mallison, B.D.
Politi, and D.C. Mackel. 1979. Isolation of Legionnaires' disease bacterium from environmental samples.
Ann. Intern. Med. 90:664-666.

103

Demonstration of the Bacterium
in Tissue by a Modification of the
Dieterle Silver Impregnation Stain

A.E. Van Orden
P.W. Greer

Pathology Division
Bureau of Laboratories

105

Demonstration of the Bacterium in Tissue by a Modification
of the Dieterle Silver Impregnation Stain

A.E. Van Ordeii and P.W. Greer

The modification of the Dieterle silver impregnation stain described below is our adaptation
of the staining procedure which Dr. Robert Dieterle developed for demonstrating Spiruchaeta
pallida in single microscopic sections. We modified his procedure to meet our need for a batch
staining method and have used it successfully for a number of years. Recently, interest in this
modified method increased after we used it successfully to demonstrate the Legionnaires' disease
bacterium (LDB) in paraffin-embedded tissue sections. The routine staining procedures for bac-
teria and fungi were not satisfactory; therefore, the modified Dieterle staining procedure is a
valuable diagnostic tool. Instructions for the procedure are detailed below.

TISSUE AND SLIDE PREPARATION

Fix lung tissue for at least 24 hours in 20 times its volume of \Q7r buffered Formalin.
Trim the tissue with a sharp blade to not more than 3 x 1.5 \ 0.5 cm. Process according to tlie
following schedule:

1. Place in 70% alcohol (two changes of 1 hour each).

2. Transfer to 957c alcohol (two changes of 1 hour each).

3. Transfer to absolute alcohol (two changes of 1 iiour each).

4. Place in equal parts of absolute alcoiiol and chloroform for 1 hour.

5. Transfer to chloroform (two changes of 1 liour each).

6. Place in melted paraffin (two changes of 2 hours each).

7. Place tissues in a third change of melted paraffin until tiiey are embedded.

8. Transfer tissue to an embedding mold and fill the mold with fresh melted paraffin.
Allow the paraffin to harden, remove the paraffin block from the mold, and trim its face of
excess paraffin.

9. Cut thin sections (4-6 micrometers) from the paraffin-embedded tissue blocks and
float the sections on warm water in a floatation water bath. If a pinch of gelatin was dis-
solved in the warm water, pick up a floating tissue section with a plain glass slide. When
gelatin was not used, pre-coat the glass slides with egg albumin before picking up sections.
(NOTE: Albumin on the slide surface may be a source of nonspecifically staining artifacts.)

STAINING PROCEDURE

Glass or plastic staining racks must be used or the staining can be done in coplin jars. Metal
containers must not be used because the staining reagents react witii metals to form a black pre-
cipitate which adheres to the tissue sections. All glassware must be thoroughly cleaned. Include
a known positive control in each staining rack or coplin jar.

106

Demonstration of the Bacterium in Tissue by a Modification of the Dieterle Silver Impregnation Stain

1 . Remove the paraffin and hydrate the tissue sections by sequentially dipping the slides
in two changes each of xylene, absolute alcohol, 95% alcohol, and distilled water.

2. Place slides in preheated 5% alcohohc uranyl nitrate at 55°- 58°C for 1 hour.

3. Dip slides in distilled water.

4. Dip slides in 95% alcohol.

5. Place slides in 10% alcoholic gum mastic for 3 minutes.

6. Dip slides quickly in 95% alcohol.

7. Place slides in distilled water for 1 minute.

8. Allow slides to drain for 15-20 minutes. (Slides can be left to dry overnight and the
procedure completed the following day.)

9. Place slides in preheated 1% silver nitrate solution in 55°- 58°C oven in the dark for
at least 4 hours. (The silver will precipitate if the oven temperature exceeds 60°C.)

10. Dip slides twice quickly in distilled water.

1 1 . Dip sHdes in developing solution until sections are yellow to tan-usually in 1-2 minutes
(check control slide microscopically for proper development of organisms).

12. Dip slides twice in distilled water.

13. Dip slides twice in 95% alcohol.

14. Dip slides twice in acetone.

15. Dip slides twice in xylene.

16. Place shdes in a second dish of xylene.

1 7. Coverslip as soon as possible with Permount or Protex mounting medium.

INTERPRETATION

Scan the sections on a microscope with the low power objective. Locate areas of consolida-
tion in which the alveoli are filled with fibrin and leukocytes. In most cases, we have found more
organisms in the field containing tlie most degenerated leukocytes.

Under the high dry objective, the LDB appears as a 0.4-0.8 micrometer by 2-4 micrometer
dark-brown to black rod against a yellow to tan background. Although these organisms may be
intracellular or extracellular, most are found within macrophages. Spirochetes and some other
bacteria also stain dark-brown to black. In paraffin-embedded tissue sections, bacteria which
stain dark-brown to black with this procedure and have the morpiiological characteristics of the
LDB but do not stain with tissue Gram stains can be presumed to be the LDB (Figure 1 ).

Other structures including melanin granules, chromatin, Formalin pigment, and some
foreign material in macrophages may also stain by this method, but they can be readily distin-
guished from tiie LDB.

DISCUSSION

The modified Dieterie silver impregnation stain has also been successfully applied to de-
stained sections previously stained by hematoxylin and eosin or by tissue Gram stains. After
removing the coverslips (by soaking slides in xylol) and destaining the sections in acid alcohol
(by dipping until colorless), we have laed the modified Dieterle procedure to demonstrate LDB
in sections tiiat have been filed for as long as one year.

107

Demonstration of tlic Bacterium in Tissue by a Modification of the Dieterie Silver Impregnation Stain

We should mention tliat stained organisms may fade unpredictably. Although some of our
slides have retained the stain for over a year, others have faded within hours— especially if they
were not coverslipped immediately after staining.

We have had some success in preventing this fading by modifying the staining procedure as
follows;

(1) After the distilled water rinse in step 111. dip the slides in a lO't solution of 88%
formic acid.

(2) Rinse the slides once in distilled water and then proceed with step 13.

We have not been able to stain Zenkers'-fixed material successfully: otlier metal-containing
fixatives may also adversely affect the staining of organisms.

To determine whether tissues subjected to decalcification solutions could be successfully
stained, we used some tissue that had been treated with our routine decalcification solution
(equal parts of 5'/i formic acid and 5% Fomialin) and other tissue treated with a commercial
decalcification solution (RDO from Dupage Kinetic Laboratories). Staining was not affected by
either decalcification procedure.

As previously stated, a known Dieterie positive control sHde should be stained in each rack
of tissue sections. Control tissue for this procedure is available from the Center for Disease
Control. Attention: Mrs. Pat Greer. MT (ASCP). Control Tissue Repository, Bureau of Labora-
tories, Atlanta. Georgia 30333.

ji

.mm^ 9 ^ •*

• •

#

li

/ . .' *^

^ If. ^ 'SI ^'

• \

f.

V

-< :"-'^

'%

Figure 1. Legionnaires' disease bacteria in an area of consolidation. (Dieterie silver iinpregnation stain, X1500j.

108

Demonstration of tlio Bactorium in Tissue by a Modification of the Dieterle Silver Itiiprcgnation Stain

REAGENTS

1 . 5% alcoholic uranyl nitrate. Dissolve 50 gm uranyl nitrate in 1,000 ml of 70% alcohol.
Stable in refrigerator for months.

2. 10% alcoholic gum mastic. Mix 100 gm gum mastic in 1,000 ml absolute alcohol,
and allow 2-3 days to dissolve. Filter solution and store in refrigerator in a well-stoppered
bottle. Gum mastic is available from O.G. Innes Corp., 10 East 40th Street, New York,
NY 10016.

3. 1% silver nitrate. Dissolve 10 gm silver nitrate in 1,000 ml of distilled water. Store in
refrigerator. Discard solution if it turns dark.

4. Developing solution - Mi.x in order:

Hydroquinone 15.0 gm

Sodium sulfite 2.5 gm

Distilled water 600.0 ml

Acetone 100.0 ml

Formalin (3 7%-40%) 1 00.0 ml

Pyridine 100.0 ml

1 0% alcoholic gum mastic 1 00.0 ml

Swirl flask gently as each solution is added. Solution turns milky yellow as the gum mastic
is added and light brown after standing in a well-lighted area. As brown streaks appear,
swirl the flask gently. Developing solution is ready to use in about 6 hours and can be used

until it turns dark brown (usually in 2-3 days).

REFERENCES

1. Dieterle, R.R. 1927. Method for demonstration of Spirochaeta pallida in single microscopic sections. Archives
of Neurology and Psychiatry. 18:73-80.

2. Van Orden, A.E., and P.W. Greer. 1977. Modification of the Dieterle spirochete stain. J. of Histotechnology.
1:51-53,

109

Indirect Immunofluorescence Test
for Legionnaires' Disease

Hazel W. Wilkinson

Donna D. Cruce

Bonnie J. Fikes

Lynn P. Yealy

Carol E. Farshy

Bacterial Immunology Branch

Bacteriology Division

Bureau of Laboratories

Indirect Immunofluorescence Test
for Legionnaires' Disease

Hazel W. Wilkuisoii, Donna D. Cruce, Bonnie J. Fikes.
Lynn P. Yealy, and Carol E. Farshy

INTRODUCTION

The indirect immunofluorescence (IF) test for Legionnaires' disease (LD) has been modified
several times since it was first used by McDade et al. to provide serological evidence that an or-
ganism which had been isolated from patients with Legionnaires' disease was actually the causa-
tive agent of that disease. We now know that at least four different serogroups exist, with indi-
viduals responding immunologically in different ways to serogroup-specific antigenic determi-
nants. Some patients appear to respond to the Legionnaires' disease bacterium (LDB) with a
group-specific antibody response; others respond to antigenic determinants presumably common
to serogroups 1-4. Preliminary data obtained at the Center for Disease Control (CDC) suggest
that a polyvalent antigen (not yet in general use) can be used to screen sera for LDB antibodies.
Positive sera are then titrated against monovalent antigens. We now use heat instead of diethyl
ether to kill the organisms because ether extracts or destroys the serogroup 2 (Togus 1) antigen.
Further modifications of the test may be necessary as we gain more knowledge about the organ-
ism and about the immune response to the LDB.

Perhaps because specific diagnostic criteria for LD have not been completely formulated,
expectations of sensitivity, specificity, and reproducibility have been unusually liigh for the LD
indirect IF test. Nevertheless, it is limited by the same immunologic parameters that affect
other serological tests. For example, repeat tests in which titers are obtained that vary by no
more than one 2-foid dilution are generally considered acceptable within limits of experimental
error. Therefore, paired sera should be tested simultaneously. A 4-fold rise in titer from the
acute to the convalescent phase of illness is usually considered the only solid serologic evidence
of infection. Some sera from patients with LD do not contain detectable antibodies against the
identified "infecting" strain of the LDB, whereas sera from some other patients infected with
dissimilar pathogens may contain antibodies that react with LDB, presumable because of sim-
ilarities of the heterologous organisms" antigenic structure or perhaps because of nonspecific
stimulation of the reticuloendothelial lymphocytic system. It is interesting that most of the
reported "cross-reactions" between LDB and other pathogens have not been confirmed. It
would be unusual, however, for serologic cross-reactions not to occur with antigenic mimicry
being so common in nature. No serologic test is lOO'^r sensitive or specific. For these reasons,
the indirect IF test should be used in conjunction with other diagnostic criteria.

The indirect IF test for LD has the following components: (a) an antigen composed of
heat-killed, whole LDB cells, (b) appropriate dilutions of the human serum to be tested or of
a control serum, (c) rabbit antihuman conjugate [fluorescein isothiocyanate(FITC)-labeled rabbit
antibody with specificity for human immunoglobulins G and M|, and (d) an appropriately
equipped fluorescence microscope.

Indirect Immunofluorescence Test lor Legionnaires' Disease

PREPARATION OF BACTERIAL ANTIGENS

1 . Inoculate tlie organism lieavily on an agar slant of charcoal-yeast extract medium
(20- X 150-nim screw-capped test tube containing 10 ml of medium). Incubate the loosely
capped agar tube in a candle extinction jar at 35°C until good growth occurs (approxi-
mately 2 days).

2. Add 2 ml of sterile distilled water to the tube. Gently rub the growth off the slant
with a Pasteur pipette, and transfer the suspension to a screw-capped test tube.

3. Place the tube contaming the cell suspension in a boiling water bath for 15 min to kill
the cells.

4. Streak an agar plate to confirm cell death. Use 0.1 ml of the cell suspension and the same
medium and incubation conditions as in Step 1. Observe for growth for at least 10 days.

5. Pack the cells by centrifugation (2000 x g or about 5 min in a tabletop clinical centri-
fuge). Discard the supernatant fluid. Resuspend the bacterial sediment in 2 ml of sterile
distilled water (concentrated antigen suspension).

6. Make working dilutions of the concentrated antigen suspension in 0.5'^^ normal chicken
yolk sacs (NYS) in sterile distilled water. (The procedure for preparation of NYS is de-
tailed in the manual Appendix.) it is generally best to try several antigen dilutions in the
1:20 to 1:80 dilution range in the indirect IF test. High concentrations of bacterial cells
can reduce titers, because proportionately fewer antibody molecules are bound per bac-
terial cell, and low concentrations make slide reading difficult because there are too few
fields of cells and too few cells per field. Most antigens are dispersed optimally at a dilution
of 1:50 (OD of 0.08-0.10 at 660 nm with 1-cm light path or approximately 500-600 organ-
isms per microscopic field at a magnification of 3 15 X).

7. Add the preservative, sodium azide (NaN3 ), to the concentrated antigen and to the
diluted "working" antigen to a final concentration of 0.05%.

8. Store antigens at 4°C. Although it is too early to know the results of long-term stability
studies, antigens are apparently stable at 4°C for at least 2 mon.

INDIRECT IMMUNOFLUORESCENCE TEST PROCEDURE

A. Preparation of antigen slides

1 . Thoroughly mix the working dilution of the heat-killed antigen suspension. Using a
Pasteur pipette, apply enough antigen to each well of a microscope slide to cover the well
with the antigen; remove the excess liquid with the same pipette (e.g., 25- X 75-mm acetone-
resistant glass slide with 12 staggered wells 5 mm in diameter; Cel-Line Associates, Inc.,
Minotola.N.J.).

2. Allow the slide to air dry (may take up to 30 min).

3. Fix the antigen smears to the slide by placing the slide in an acetone bath for 15 min.

4. Allow the slide to air dry.

5. If slide is to be stored for future use, place in freezer at -20°C. Frozen slides are stable
for at least 2 mon.

B. Titration of Sera

1. Prepare a 1:16 dilution of the serum in 2.5%-3.0% normal chicken yolk sac (NYS)

113

Indirect ImimiimnLiorcscencc Test tor Lefioniiaircs" Disease

suspended in 0.01 M phosphate buffered siiUne, pM 7.6 (PBS). Make subsequent doubling
dilutions through 1:2048 in plain PBS. (We use Linbro Seientifie Co. rigid polystyrene
U-bottomed microtitration trays and a Cooke Engineering Co. automatic diluter. We place
0.01 ml of serum in a test tube containing 0.15 ml of NYS suspension and make subsequent
doubling dilutions in wells of the microtitration tray using plain PBS and 0.05-ml volumes.
Alternatively, replace the automatic diluter with hand diluters, or make all dilutions in test
tubes.)

2. Using a Pasteur pipette, place a drop of each serum dilution from 1:64 to 1:2048
(or 1:256 if sera are being screened first) in its well of fixed antigen on a slide. Incubate
the slide in a moist chamber (e.g., petri dish containing moist filter paper) at 37°C for
30 min.

3. Rinse slide quickly with PBS and place in a PBS bath for 10 min. Remove from the
bath and gently blot dry.

4. Place a drop (approximately 20 ^1) of FITC4abeled antihuman immunoglobulin on
each well. Incubate as above for 30 min.

5. Rinse slide quickly with PBS, place in a PBS bath for 10 min, rinse quickly with water,
and gently blot dry.

6. Add several drops of buffered glycerol (1 part 0.5 M carbonate-bicarbonate buffer,
pH 9, in 9 parts glycerol) to the slide and mount with a 24- X 60-mm coverslip (No. I).
(Check the pH of the buffered glycerol mounting fluid frequently; the tluid should not
be used if below pH 8.)

7. Read slides on a tluorescence microscope (see below),

8. The serum titration end point is the highest dilution of serum that gives \+ fluorescence
(see below) of at least 507r of the bacteria estimated per microscopic field. The titer is the
end-point dilution factor.

C. Controls

1 . The primary control is a human serum having a specified titer against tiie serogroup
antigen used. For the test to be considered valid, the titer obtained with the control serum
should not vary more than 2-fold from the titer specified for that serum.

2. The second control is PBS substituted for serum, to test for nonspecific binding of the
conjugate. If bacterial cells are visible, fluorescence intensity must be <\+ (see below).

Legend for color plate:

Indirect IF test of patient's serum for antibodies against Legionnaires' disease bacterium, serogroup 1
(Philadelphia 1 strain). Dilutions of serum and resulting fluorescence intensities are given under each
photograph. Staining endpoint = 1 + . Titer = endpoint dilution factor = 512. Magnification = 315X.
Note that autofluorescence (yellow-brown) of organisms at dilution of 1:1024 must not be interpreted
as positive FITC fluorescence (green) and tliat FITC fluorescence must be read only for nonfilamentous,
isolated cells in an area relatively free of clumps of fluorescent yolk sacs.

114

Indirect liiiiminoriuorescciuc Test for Legionnaires' Disease

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y-

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urS^H

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— 1 <-,

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115

Indirect Inimunonuoresccncc Test tor Legionnaires' Disease

D. Fluorescence Microscopy

1 . Fluorescence intensities are read as follows:

4+ = brilliant yellow-green staining of bacterial cells

3+ = bright yellow-green staining

2+ = definite but dim staining

1+ = barely visible staining

— = no staining, although dim autolluorescence of the cells may occur with

some filter systems (see below)

CAUTION: Disregard staining of filamentous cells and of cells on or adjacent

to fluorescent yolk sac material.

2. Microscope Assemblies

We have found that the following give satisfactory results: Leitz Dialux 20 fluorescence
microscope equipped with an HBO-100 mercury incident light source; the Leitz I-cube
filter system (2 x KP490 and a 1-mm GG455 primary filter. TK5 10 dichroic beam-splitting
mirror and K515 secondary filter) or Leitz K-cube filter system (same as Lcube except
1-mm GG455 filter is replaced by a 2-mm GG475 or a K480 edge filter to reduce back-
ground tluorescence), 40 X dry objective, and 6.3 X eyepiece (magnification- 315 X).
Other microscope assemblies may also be satisfactory.

E. Interjjretation*

1. A 4-fold or greater increase in titer to >128 in sera obtained in the acute and convales-
cent phases of illness provides serological evidence of Legionnaires' disease. Optimal time
intervals from onset of illness to specimen collection appear to be <7 days for the acute-
and >21 days (but <6 weeks) for the convalescent-phase serum.

2. A single or standing titer of >256 provides presumptive evidence of an LDB infection
at an undetermined time. Until more data are accumulated to show (a) how long a detect-
able level of antibody usually persists after infection with the LDB, and (b) tiie average
LDB antibody concentration in sera of apparently nomial individuals, the significance of
a single high titer will remain unknown.

3. All other findings are considered negative.

SELECTED REFERENCES

1. Jones. Gilda L., G. Ann Hebert, and William B. Cherry. 1978. Fluorescent Antibody Techniques and Bac-
terial Applications. Center for Disease Control. Public Health Service, U.S. Department of Health, Education,
and Welfare, Atlanta. GA.

2. McDade. J.E.. C.C. Shepard. D.W. Eraser. I.E. Tsai. M.A. Redus. VAR. Dovvdle. and Laboratory Investigation
Team. 1977. Legionnaires' disease. Isolation of a bacterium and demonstration ofits role in other respiratory
disease. New Engl. J. Med. 297:1197-1203.

3. Tsai, T.E., and D.W. Eraser. 1978. The diagnosis of Legionnaires' disease. Ann. Intern. Med. 89:413-414.

4. Wilkinson, H.W., B.J. Eikes, and D.D. Cruce. 1979. Indirect immunofluorescence test for serodiagnosis of
Legionnaires' disease: Evidence for serogroup diversity of Legionnaires" disease bacterial antigens and for
multiple specificity of human antibodies. J. Clin. Microbiol. 9:379-383.

*Although these interpretations appear to be valid for serogroup 1 infections, they may not be vahd for those caused by members
of other serogroups. The serogroup to which the infecting strain belongs cannot be determined unequivocally by results of the
indirect IF test alone.

116

Analysis of Cellular Fatty Acids

of Bacteria by

Gas-Liquid Chromatography

C. Wayne Moss

Analytical Bacteriology Branch

Bacteriology Division

Bureau of Laboratories

117

Analysis of Cellular Fatty Acids of Bacteria
by Gas-Liquid Chromatography

C. Wayne Moss

INTRODUCTION

Cliemical analyses of cell structure of bacteria and of products from their metabolism
provide useful additional information for identification and classification. Extensive studies with
cellular fatty acids have shown that certain closely related species can be distinguished on the
basis of qualitative differences in their fatty acids {/, 2). A recent example is the Legionnaires'
disease bacterium (LDB), which is different from other gram-negative bacteria because it contains
large amounts (> 779c ) of branched-chain fatty acids (5).

The lipids of bacteria are found in the cell wall/cell membrane fraction, where the unit fatty
acids are chemically bonded to other cellular components. The acids are liberated or cleaved from
these components and subsequently analyzed by gas-liquid chromatography (GLC). GLC is the
method of choice for fatty acids because of its speed, sensitivity, and excellent separating effi-
ciency. The following is a protocol for analysis of bacterial cellular fatty acids by GLC techniques
with special reference to the LDB. A detailed review on GLC and its application to microbiology
appeared recently {2).

PREPARATION OF SAMPLES

A. Saponification - to detemiine "total" cellular fatty acids, bound lipids must be freed by a
saponification or hydrolysis procedure.

1. Add 0.5 ml sterile distilled water to the surface of an agar slant (16 x 150 mm).

2. Gently scrape the cells off the agar, and then transfer the turbid cell suspension to an 18
or 20 X 1 50 mm test tube with a TetTon-lined screw cap.

3. Add 4 ml of 5% NaOH in 50% aqueous methanol, and tighten the Tetlon-lined cap.

4. Heat tiie cell suspension for 30 min in a boiling water batii ( 100°C).

5. Remove the tube from the water bath and cool to room temperature. If necessary, tubes
may be held overniglit in a refrigerator.

B. Methylation - before conversion to esters, the sodium salts of the acids from saponification
must be converted to the free fomi.

1. Add approximately 1.0 ml of 6 N HCl to the cool saponificate to lower the pH to 2.0.

2. Mix well and check with pH indicator paper. If needed, add more 6 N HCl drop by drop
until pH is 2.0.

3. Add 4 ml of 10% boron trichloride-methanol reagent (Applied Science Laboratories,
State College, PA) and mix well. Tighten Tetlon-lined cap.

4. Heat for 5 min in a water bath maintained at 80°C-85°C.

118

Analysis of Cellular I'atty Acids of Bacteria by Gas-Liquid Chromatography

5. Remove tubes from the water bath and cool to room temperature prior to extraction.
For additional information regarding other esterification procedures see reference 4.

Extraction of Methyl Esters

1. Add 10 ml of a 1:1 mixture of ethyl ether:hexane to the tube and tighten cap. Mix well,
i.e., invert the tube 10-20 times and shake firmly for about 10 sec.

2. Allow the phases or layers to separate. Transfer the ether:hexane phase (top layer) to a
50- or 100-ml beaker. CAUTION: Avoid transferring aqueous phase along with the ether:
hexane phase.

3. Add another 10 ml of ether:hexane to the aqueous phase (bottom layer) and repeat the
extraction procedure of Step C-1.

4. Allow the phases to separate, and combine the ether:hexane layer with the first fraction
already in the beaker.

5. Place the beaker under a gentle stream of tlowing nitrogen gas in a chemical hood, and
evaporate to reduce the combined 20 ml of solvent to approximately 0.5 ml. CAUTION:
Never allow the sample to go to complete dryness during evaporation of solvent.

6. Add a small amount (80-100 mg) of Na2S04 to the beaker to remove residual moisture,
and then transfer the sample to a 13- x 100-mm screw-capped tube. Rinse the beaker with a
small volume of hexane, and then add the rin.se to the sample tube.

7. Reduce the final volume (sample plus solvent) in the test tube to approximately 0.1 ml
under a gentle tlow of nitrogen gas in the hood. The methyl ester sample is then analyzed by
GLC or is stored at- 20° C.

GAS-LIQUID CHROMATOGRAPHY

A. Equipment

The gas chromatographs used in this laboratory for cellular fatty acid analysis are equipped
with tlame-ionization detectors (FID) and temperature programs. Glass rather than metal col-
umns is used to avoid possible destruction of hydroxy acids (2). A non-polar stationary-phase
material such as the methyl silicones (SE-30, OV-1, OV-101) is used as the principal analytical
column. These materials have excellent separating efficiency, temperature stability, and low
column bleed, and are readily available from several commercial sources. A 3% concentration of
one of these phases coated on an inert support (i.e., 1 00- 1 20 mesh Gas-Chrom 0) is packed into a
0. 1 6 in (4.06 mm I.D. ) x 12 ft (3.66 m) glass column. The column is placed into the instrument
without connecting it to the detector and "conditioned" overniglit at 290"C with a low carrier
gas flow (20 cc/min) passing through the column. After conditioning, the column is connected to
the detector, checked for leaks, and temperature programmed from IOO°-270°C several times
prior to sample analysis. The carrier gas is adjusted to about 60 cc/min, and the instrument is set
as follows: initial temperature, I60°C; final temperature, 270°C; temperature program rate,
5°C/min. Under these conditions, excellent separation of a fatty acid methyl ester mixture
containing up to 23 components can be accomplished within 30 minutes.

B. Column Control

A measure of the efficiency of column separation on non-polar phases is base line or near
base line resolution of the methyl esters of palmitoleic ( 16: 1) and palmitic (16:0) acids. Appro-
priate adjustments of carrier gas tlow, initial column temperature, and temperature program rates

Analysis of Cellular Fatty Acids of Bacteria bv Gas-Liquid Chromatography

should be made to insure satisfactory separation of tiiese two compounds. If tliese two compo-
nents are resolved on a non-polar phase material, other esters which are present in a bacterial
fatty acid sample will, in general, also be well resolved.

C. Procedure

For analysis, 1-3 /il of the bacterial fatty acid methyl ester sample is injected into the
column, and a tracing (chromatogram) of the separated components of the mixture is recorded
on a strip chart. The retention time (time from injection to the center of the eluted peak) of each
peak is recorded and compared to retention times of those in a reference standard. The presence
of specific fatty acids and their relative concentration compared to those of other acids in the
sample are the bases for ditTerentiation {2).

IDENTIFICATION

A significant amount of information on the identity of the fatty acids in tlie bacterial
sample can be obtained by comparing GLC retention data to highly purified reference standards
on both non-polar (i.e.. OV-101 ) and polar [i.e.. ethylene-glycol-adipate (EGA)] stationary-phase
columns. On non-polar phases, fatty acid methyl esters are separated by boiling points. Thus,
unsaturated acids elute before their saturated homologs, saturated branched-chain acids before
their saturated straight-chain homologs, cyclopropare acids before their straight-chain homologs,
and 2-hydroxy acids before their 3-hydroxy homologs. On polar columns such as EGA, the
elution sequence is reversed for saturated acids and their unsaturated homologs. Iso- and anteiso-
isomers of saturated branched-chain acids also separate to some degree on polar columns (S).
Thus identical retention time matches on both polar and non-polar phase materials give strong
preliminary identification. In addition, the presence of unsaturated acids can be confirmed by
bromination or hydrogenation. Treatment of the methyl ester sample with bromine results in the
addition of this compound across the double bonds to produce a much less volatile species
(bromo-methyl ester), which does not appear in the chromatogram under the initial conditions of
GLC upon subsequent GLC analysis. Similarly, hydrogenation converts unsaturated esters to
their corresponding saturated esters, which causes the GLC peaks representing esters of unsatu-
rated acids to disappear and causes the peaks representing saturated acid esters to increase in size.
The presence of a hydroxy acid can be confinned by treating the methyl ester sample with an
anhydride (e.g., acetic, trifluoroacetic) to form the diester derivative. Upon subsequent analysis
by GLC, the peak representing the methyl ester derivative will disappear from the chromatogram.
and a new peak representing the diester derivative will appear. The diester will elute from the
column more quickly (shorter retention time) as a result of its increased volatility.

A. Hydrogenation of Methyl Esters

1. After analysis by GLC, reduce the methyl ester sample almost to dryness under nitro-
gen.

2. Add approximately 0.5 ml of a 3:1 mixture of chloroform;methanol down the side of
the tube and mix well.

3. Transfer the sample to a 10-ml vacuum hydrolysis tube (Kontes Glass Company).

4. Add a small amount (5 mg) of catalyst (5% platinum on charcoal) and a small, Tetlon-
coated stirring bar.

5. Connect the tube to a ring stand and suspend the tube over a magnetic stirrer. Close the
upper part of the tube and mix the contents by stirring.

120

Analysis of Cellular Fatty Acids of Bacteria by Gas-Liquid Chromatography

6. Turn off the stirrer, open the stopcock in the upper part of the tube, and bubble a small
vokime of hydrogen gas into the tube for approximately 5 sec. CAUTION: Hydrogen is a
highly tlammable gas and must be handled with extreme caution.

7. Close the stopcock and mix the contents for 15 min with the magnetic stirrer. Add a
small volume of hydrogen gas two (more) times, and allow 15-min intervals for stirring.

8. Remove the catalyst by filtration through a small Whatman paper filter (#1) and collect
the hydrogenated sample in a 13- x 100-mm screw-capped tube.

9. Reduce the sample volume to about 0. 1 ml under a gentle fiow of nitrogen gas in the
hood.

10. Analyze 1-3 /.tl of sample by GLC under identical conditions to those described for the
original methyl ester sample.

B. Acetylation of Methyl Ester

1. After analysis by GLC, add 5 drops of trifluoroacetic anhydride to the methyl ester
sample tube with a Pasteur pipette.

2. Mix gently and leave at room temperature for 30 min.

3. Add 0.3 ml distilled water and mix gently to wash the sample.

4. Allow the layers to separate. Carefully transfer the hexane layer (top) to a clean 13- x
100-mm screw-capped tube.

5. Analyze 1-3 /j1 of the acetylated sample by GLC under identical conditions to those
described for the original methyl ester sample.

QUANTITATION

As noted previously, the bases for differentiation among bacteria are the presence of specific
fatty acids and their relative concentration compared to those of other acids in the sample.
Therefore, in addition to observing the overall general fatty acid profile, it is desirable to deter-
mine the relative concentration of peaks in the chromatogram. Peak areas are obtained from the
chromatogram (DISC or electronic integrator), and the percentage of each acid is calculated from
the ratio of the area of its peak to the total area of all peaks. In this laboratory, small peaks with
areas less than 2% of the total are disregarded.

CONTROLS

Because of the extreme sensitivity of the technique, highly purified chemicals, solvents,
reagents, and gases are used in GLC studies. Glassware is a potential source of contamination and
should be checked for cleanness and cracks. Tefion-lined caps should be used, and these and all
glassware should be rinsed with solvents (hexane, acetone) before use. A bacterium of known
fatty acid composition should be included along with the unknown organism. Also, a "reagent
blank" (i.e., distilled water) should be processed occasionally as a check on all reagents, solvents,
and the GLC column.

121

Analysis of Cellular Fatty Acids of Bacteria by Gas-Liquid Chromatography

SUMMARY OF GLC ANALYSIS OF BACTERIAL CELLULAR FATTY ACIDS

A. Saponification - 4 ml of 5% NaOH in 50% methanol is added to cells from a slant culture
and heated for 30 min at 100°C. The sample is cooled.

B. Methylation - The pH is adjusted to 2.0 with 6 N HCl; 4 ml of 10% boron tri-
chloride-methanol reagent is added, and tlie mixture is heated at 80" C for 5 min. Sample is
cooled.

C. Extraction Methyl esters extracted from mixture with two 10-ml aliquots of a 1:1
mixture of diethyl ether;hexane. Solvent containing methyl esters is reduced to about 0.1 ml.

D. GLC 1-3 ij\ of methyl ester sample is injected into non-polar GLC column and analyzed.
Retention times of peaks are recorded and compared to reference standards. Quantitative data
are obtained, and identities of fatty acids are confirmed by additional procedures (hydrogena-
tion, acetylation, analysis on polar column, mass spectrometry).

REFERENCES

1. Lechevalier, M. P. 1977. Lipids in bacterial taxonomy A taxonomist's view. pp. 109-210. //; A. I. Laskin
and H. Lechavalier (eds.). Critical Review of Microbiology, Vol. 5. CRC Press, Cleveland, Ohio.

2. Moss, C.W. 1978. New methodology for identification of nonfermenters: Gas-liquid chromatographic chemo-
taxonomy. pp. 171-201. //; G. L. Gilardi (ed.). Glucose Nonfeimentating Gram-Negative Bacteria in Clinical
Microbiology. CRC Press, West Palm Beach. Florida.

3. Moss, C. W., and W. B. Cherry. 1978. Characterization of the C-15 branched-chain fatty acids of Conv;e-
bactehum acnes by gas chromatography. J. Bacteriol. 95:241-243.

4. Moss, C.W., M. A. Lambert, and W. H. Mervin. 1974. Comparison of rapid methods for analysis of bacterial
fatty acids. Appl. Microbiol. 28:80-86.

5. Moss, C. W., R. E. Weaver, S. B. Dees, and W. B. Cherry. 1977. Cellular fatty acid composition of isolates
from Legionnaires' disease. J. Clin. Microbiol. 6: 140-143.

12:

ELISA for Legionnaires' Disease -

Antibody and Antigen

Detection Systems: An Interim Report

Carol E. Farshy
John C. Feelev

Bacterial Immunology Branch

Bacteriology Division

Bureau of Laboratories

123

ELISA for Legionnaires' Disease -

Antibody and Antigen

Detection Systems: An Interim Report

Carol E. Farshy and John C. Feeley

INTRODUCTION

In many cases, laboratory confimiation of Legionnaires' disease (LD) is accomplished retro-
spectively either by indirect immunotluorescence (IF) tests of acute- and convalescent-phase sera
(7), or by direct IF tests of postmortem lung tissue {2). Previously we reported a micro enzyme-
linked immunosorbent assay (ELISA) for the detection of antibodies against serogroup 1 LDB
soluble antigens (i), using the horseradish peroxidase enzyme. Although the test appears promis-
ing, its sensitivity and specificity need further refinement and evaluation. The primary disadvan-
tage of using antibody detection tests is the delay involved in seroconversion (appro.ximately 3
weeks) and, therefore, in diagnosis.

Earlier diagnosis of LD is needed so that specific therapy can be instituted during the acute
stage of illness. Accordingly, we have investigated the use of ELISA for the detection of sero-
group 1 antigen in urine (/).

In a preliminary evaluation of the ELISA. antigen was detected in the urine of all six guinea
pigs tested within 4 h and for at least 1 wk after they were inoculated with nonviable LDB
serogroup 1 (Philadelphia 1 and 2 and Knoxville 1). Urine specimens from animals inoculated
with the serogroup 2 strain (Togus 1) were negative in the serogroup 1 ELISA, as would be
expected because of the antigenic dissimilarity of these serogroups (5, 7). Urine specimens from
control animals were negative.

Urine specimens from 20 human subjects with no recent history of respiratory disease were
also negative. Antigen was detected, however, in urine which had been collected in 1*^76 from
three of four LD patients during the Philadelphia outbreak (4). Urine from a fifth patient with a
chnically compatible illness but equivocal serological test results was negative in the ELISA. Since
this work was completed, investigators conducting an independent Imiited study have used
ELISA to detect antigen in sputum and urine from two LD patients (ft).

ELISA FOR ANTIBODY

The micro-ELISA for detecting antibodies to the Legionnaires" disease bacterium (LDB)
involves a soluble antigen prepared from heat-killed organisms. Comparison testing of this proce-
dure and the indirect IF test has not been completed. ELISA appears to be a promising labora-
tory tool which continues to be improved with research on other enzyme systems and immuno-
purified conjugates. The instructions for perfomiing the micro-ELISA for antibody titration are
shown below:

A. Antigen Preparation

I. Grow the LDB on Mueller-Hinton agar plates supplemented with 1% IsoVitaleX (BBL)
and \% hemoglobin for 72 h at 37° C in a candle jar.

124

ELISA for Legionnaires' Disease - Antibody and Antigen Detection Systems: An Interim Report

2. Working in a microbiological safety Jiood, harvest growth by adding 3 ml of sterile
phosphate buffered saline (PBS), pH 7.2, to each agar plate and gently scrapping the agar
with a bent capillary pipette.

3. Transfer the cell suspension to a sterile screw-capped flask, and steam in an Arnold
sterilizer for 1 h at lOTC.

4. Centrifuge the killed-cell suspension in sterile 1 5-ml conical centrifuge tubes for 30 min
at 1600 X g. Decant the supernatant, and wash packed cells twice in sterile PBS, pH 7.2.

5. Prepare a stock suspension of the cells by adding 2 ml of sterile PBS, pH 7.2, to each 0.1
ml of packed, centrifuged cells. Store stock antigen suspension at 4°C for 10 days to allow
the release of soluble antigen from the cells.

6. Centrifuge the suspension at 1600 X g. Save the supernatant to be used as the stock
soluble antigen. (By doing block titrations, we found that a 1 :40 dilution was adequate with
our system.)

B. Test Procedure

1. Dispense 0.1-ml volumes of diluted antigen into each well of as many Cooke micro-
ELISA plates as are needed (protein-binding polystyrene no. 1-223-29). Seal plate with
Cooke Microtiter plate sealers, incubate at 37°C for 3 h, and store at 4°C until used.

2. Aspirate excess antigen from wells, and wash each plate at least three times with PBS,
pH 7.2, containing 0.05% Tween 20. Leave PBS in the plate for at least 3-4 min per wash.

3. If numerous sera are to be tested, screen them at a 1;8 or 1:16 dilution. Set up two
wells per specimen.

4. Detemiine the end points of the positive sera by making further 2-fold dilutions. Assign
each specimen to be tested a row of wells on a plate. Also assign one row to a known
negative serum and one row to a known positive serum for each test run. Include PBS
control.

5. With an automatic pipette, dispense 0.1 ml of a 1:8 or 1:16 dilution of the specimen to
be tested into well 1 of the row assigned to it. Prepare serial 2-fold dilutions as follows. To
wells 2 through 8 assigned to a specimen, with a Cooke microdropper add 0.05 ml of PBS,
pH 7.2, containing 0.05% Tween 20. Then, make 2-fold dilutions with Cooke microdiluters
(0.05 ml). (See Figure 1.) Finally, add 0.05 ml of PBS, pH 7.2, with 0.05% Tween 20, to all
wells on each plate, and vibrate or gently mix.

6. Incubate plates at 37°C for 1 h.

7. After they are incubated, wash plates as in Step 2.

8. Predetermine appropriate conjugate dilution by block titration. Dispense 0.1 ml of the
appropriate dilution of enzyme-labeled conjugate [horseradish peroxidase(HRPO)-labeled,
antihuman immunoglobulin] into each well of every plate.

NOTE: Assaying and adding the conjugate are perhaps the most critical steps in the proce-
dure. Impure and nonspecific conjugates adversely affect both specificity and sensitivity.

9. Cover plates, and incubate them at 37°C for 1 h.

10. Wash as described in Step 2 using five washes.

1 1. Add 0.1 ml substrate to each well. The substrate for HRPO-labeled conjugate is ortho-
phenylenediamine (OPD). Stock contains 1% (wt/vol) OPD in methanol (100 mg OPD/10

125

ELISA for LL'gionnaires' Disease - Antibody and Antigen Detection Systems: An Interim Report

0 ^

U

(U

■*-»

Q

c
o

o
I

126

ELISA for Legionnaires' Disease - Antibody and Antigen Detection Systems: An Interim Report

ml ethanol). Stock does not deteriorate if stored in the dari<. for up to 1 wk. Working
substrate is composed of 1 ml of stock plus 99 ml of distilled water containing 0.1 ml of 3%
hydrogen peroxide.

1 2. Cover plates and incubate in the dark at 37° C for 30 min to 1 h, depending upon the
color reaction desired.

13. Remove plates from the incubator; add 1 drop of 8 N Ho SO4 to each well to temiinate
the reaction and deepen the color.

C. Interpretation

Place the plate on a white background or on a reading mirror, and grade the color of each
well against that of the partically colorless negative control. The end point is defined as the
highest dilution of serum which is distinctly different in color from the first dilution of the
negative control. Report titer as the reciprocal of the end point.

D. Limitations

Because the sensitivity and specificity of this developmental procedure are not adequately
defined, sera for which positive or doubtful or equivocal results are obtained should be
referred to the state health department for confimiatory testing with the indirect IF proce-
dure.

ELISA FOR ANTIGEN

This ELISA is a "sandwich" technique with four layers (Figure 2) and uses the alkaline
phosphatase enzyme detection system. The ELISA can detect antigen representing as little as 3
pg of protein in a purified lipopolysaccharide protein complex which is a major antigenic consti-
tuent of serogroup 1 LDB (S). Further antigen-detection studies with respiratory secretions and
with acute-phase sera, as well as efforts to incorporate other serogroups into the test system, are
clearly warranted. In addition, sensitivity and specificity must be determined. Until these parame-
ters are defined a detailed procedure cannot be recommended.

SUMMARY

Although still in the developmental stages, ELISA for both antibody and antigen detection
appear to offer promise as aids in the diagnosis of Legionnaires' disease.

127

ELISA tor Legionnaires' Disease - Antibody and Antigen Detection Systems: An Interim Report

1st layer

2nd layer

3rd layer

4th layer

1. Antibody (Ab) to LDB, adsorbed to plastic well of a tray

2. Wash

Specimen containing LDB antigen (Ag) diluted in normal goat
serum; prepare a duplicate specimen diluted in anti-LDB goat
serum in another well

4. Wash

5 . Rabbit antibody (RAb) to LDB. Will bind to any antigen bound
in previous step

6. Wash

7. Enzyme-labeled antibody (EAb) to rabbit IgG. Goat anti-rabbit
will bind to rabbit anti-LDB being held by LDB antigen from
specimen

8. Wash

develop color

v->

EAb

b

3

%d^

3

9. Substrate(s) to make reaction visible. Any enzyme bound in the
complex will convert substrate to a detectable color.

Figure 2. ELISA: 4-layer antigen detection system
128

ELISA lor Legionnaires" Disease - Antibod>' and Antigen Detection Systems: An Interim Report

REFERENCES

1. Berdal, B.P., C.E. Farshy, and J.C. Feeley. 1979. Detection of antigen of tiie Legionnaires' disease bacterium
in urine by an enzyme-linked immunospecitlc assay (ELISA). J. Clin. Microbiol. (In press).

2. Cherry, W.B., B. Pittman, P.P. Hams. G.A. Hebert. B.M. Thomason, L. Thacker, and R.E. Weaver. 1978.
Detection of Legionnaires' disease bacteria by direct immunofluorescent staining. J. Clin. Microbiol. 8:329-
338.

3. Farshy, C.E., G.C. Klem. and J.C. Feeley. 1978. Detection of antibodies to the Legionnaires" disease organism
by microagglutination and micro-enzyme-linked immunosorbent assay tests. J. Clin. Microbiol. 7:327-331.

4. McDade, J.E., C.C. Shepard. D.W. Eraser, T.R. Tsai, M.A. Redus. W.R. Dowdle. and the laboratory investiga-
tion team. 1977. Legionnaires' disease. Isolation of a bacterium and demonstration of its role in other
respiratory disease. New Engl. J. Med. 297:1 197-1203.

5. McKinney, R.M.. B.M. Thomason. P.P. Harris, L. Thacker, K.R. Lewallen, H.W. Wilkinson, G.A. Hebert, and
C.W, Moss. 1979. Recognition of a new serogroup of the Legionnaires' disease bacterium. J. Clin. Microbiol.
9:103-107.

6. Tilton, R.C. 1979. Legionnaires' disease antigen detected by enzyme-linked immunosorbent assay. Ann.
Intern. Med. 90:697-698.

7. Wilkinson, H.W., B.J. Fikes, and D.D. Cruce. 1979. Indirect immunofluorescence test for serodiagnosis of
Legionnaires' disease: Evidence for serogroup diversity of Legionnaires' disease bacterial antigens and for
multiple specificity of human antibodies. J. Clin. Microbiol. 9:379-383.

8. Wong, K.H.. W.O. Schalla, R.J. Arko, J.C. Bullard, and J.C. Feeley. 1979. Immunochemical, serologic, and
immunologic properties of major antigens isolated from the Legionnaires' disease bacterium. Observations
bearing on the feasibility of a vaccine. Ann. Intern. Med. 90:634-638.

129

PART FOUR

Policies, Resources

and Services

Policies, Resources and Services

This section catalogues most aspects of the Center for Disease Control (CDC) assistance
program pertaining to Legionnaires" disease (LD). Included are procedures to follow in obtaining
( 1 ) technical support and consultation and (2) diagnostic materials and services beyond the scope
of your institution. The expertise of our laboratory staff during the adaptation and development
of diagnostic methods is also available.

Summary Outline: ^^^^

I. Laboratory Safety: Precautions and Practices 134

IL Laboratory Services

A. PoHcy 137

B. Reference Material

1 . Cultures 137

2. Reagents 138

3. Tissues 141

4. Chemicals 141

C. Reference Diagnostic Services

1. Indirect Immunofluorescence (IF) Test 142

2. Isolation from Clinical Specimens 143

3. Direct Immunotluorescence (IF) Test 143

4. Identification of Culture Isolates 144

5. Histopathological Examination 144

D. Submitting Reference Specimens 144

1 . Information Required 144

2. Transporting 144

III. Epidemiologic Services 149

A. PoHcy ^ 149

B. Epidemiologic Consultation and Assistance 149

C. Clinical Consultation 149

133

I. LABORATORY SAFETY: PRECAUTIONS AND PRACTICES

Apparently Legionnaires" disease is acquired by tlie airborne route, presumably after droplet
nuclei containing the Legionnaires' disease bacterium (LDB) are iniialed. Laboratories must also
consider a second route - the puncture of the skin with equipment or broken glass contaminated
with the LDB. The following general precautions and laboratory practices are minimal safety
standards for handling infectious material. In addition, most of the contributors to this manual
included in their procedures or discussions safety measures that are pertinent to the techniques
detailed in their chapters.

A. Aerosols

Many laboratory manipulations generate potentially infectious aerosols; every effort
should be made to minimize aerosol formation and to contain any such aerosols formed.
Appropriate precautions can significantly reduce aerosols, which are generated whenever a
liquid surface is disrupted, e.g., the liquid film between the cap and the wall of a container
stretching and breaking when a screw cap or cover is removed: bubbles of lluid bursting at
the tip of a pipette or on the surface of a fiuid after vigorous shaking or mixing; fluids
spattering from heated wire loops; wire loops vibrating after inadvertently touching the sides
of tubes or containers; tubes breaking or leaking in a centrifuge; streaking agar media in
tubes or plates, especially when medium surface is rough (resulting from bubbles in the
medium when it was poured). The following practices help minimize and/or contain aerosols.

1. Always perform operations such as transferring cultures and preparing microscope
smears for examination in a biological safety cabinet.

2. Perform any procedure which produces aerosols but cannot readily be done in a
safety cabinet (such as centrifuging in a large centrifuge, shaking broth cultures during
incubation, or using a high-speed blender) with safety equipment that will prevent the
dissemination of infectious aerosols if the primary container breaks or leaks.

3. Streak agar plates which have a smooth instead of rougli agar surface and thus
reduce aerosol formation by 99''r.

4. Insert a cool rather than hot inoculating loop into media. This will reduce aerosols
90%-95%.

5. .'Mlow the last drop in the pipette to drain out instead of blowing it out. This will
reduce aerosol formation 67%.

6. Mix cultures in a tube instead of a pipette and thus reduce droplet nuclei by
1 00':^.

7. Work over a disinfectant-soaked gauze sheet (rather than on the hard surface of a
bench top) to absorb falling drops of culture material and reduce aerosols by 90%.

8. Insert a needle wrapped with an alcohol pledget througli the top of a stoppered
vaccine bottle to release the pressure. This reduces aerosol production by 99%.

9. Leave screw caps on centrifuge tubes to allow contents to settle before opening
caps, and reduce aerosols considerably.

10. Centrifuge in safety cups to eliminate essentially all aerosol formation in the event
of tube breakage.

134

Laboratory Safety

B. Contaminated Materials

1. Perform any procedure that involves a potentially viable antigen very cautiously.

a. Fix impression smears of fresh tissue in lO'r Fonnalin.

b. A heat-fixed smear may still contain a viable organism; therefore discard
most stained microscope slides into a pan of disinfectant for autoclaving.

c. After staining, autoclave the rinse buffers and the towel onto which a
direct immunofluorescence slide is drained.

2. Decontaminate work surfaces after processing or otherwise handling potentially
infectious materials.

3. Immediately cover any spilled culture or specimen with Clorox® at 500 ppm.
Disinfect counter tops in work areas with an Environmental Protection Agency-
registered phenol-base disinfectant. If the culture contained a toxin. Hood with 1 N
NaOH to neutralize the toxin.

4. Autoclave all glassware, laboratory wastes, animal bedding cages, and other poten-
tially contaminated items before discarding or recycling.

Place discard material in a leak-proof pan with a loosely fitting cover; add approx-
imately I in of water to the pan and autoclave for I h. If bio-bags are used, fill them
only half-way, add a small amount of water, and leave tops open for air exchange
during autoclaving.

5. When an experimental animal is to be discarded, autoclave it before it is incin-
erated.

C. Lyopliilized Cultures

Most of the reference cultures obtained from the Center for Disease Control (CDC) are
lyopliilized and sealed under vacuum. Do not remove the top or release the vacuum before
reconstituting them. Perform work in a biological safety cabinet.

1. Reconstitute the lyophilized culture by aseptically injecting 1.0 ml of sterile
distilled water through the rubber stopper with a sterile needle and syringe.

2. Without removing the needle and syringe, swirl the vial to resuspend the contents.
Invert the vial, and withdraw the cell suspension while holding a cotton pledget at the
top of the vial to prevent an aerosol or puncture as the needle is withdrawn through
the stopper.

3. Transfer the cell suspension to a small sterile test tube. (Insert a disposable needle
through the top of the vial and autoclave before discarding.)

4. Treat the cell suspension with caution-these are viable organisms. Plate the sus-
pension immediately on the recommended agar media.

D. Fluorescence Equipment with Mercury Arc Lamps

1. Make certain that power packs for mercury arc lamps are electrically grounded.

2. Never open the lamp housing on mercury arc equipment while bulb is burning or
hot.

3. Always have a glass filter in place before looking into the microscope.

135

Laboratory Safety

4. If a mercury bulb explodes, quickly unplug the equipment and leave the room.
Do not allow anyone to enter the room for a minimum of 1 h while the mercury
vapors settle.

E. Personal Hygiene

1- A laboratory working with the LDB should be marked with a biohazard warning
sign: access should be controlled by the laboratory supervisor.

2. Use mechanical pipetting devices for transferring liquid materials. Mouth pipetting
is prohibited.

3. Examine glassware, and discard all pieces with rough or chipped edges that might
puncture the skin.

4. Wear gloves if there is any broken skin (cut, scratch, etc.) on the hands. Wear a
mask if th.ere is any chance of producing an aerosol.

5. Do not eat. drink, smoke, or store food in the laboratory.

6. Always wash hands after handling specimens and cultures, and before leaving the
laboratory area.

F. Medical Surveillance

Careful medical surveillance sliould be maintained for everyone working with the LDB
or in the same general area.

1. Obtain and store in a serum bank control or baseline sera from all personnel.

2. Carefully monitor all personnel for febrile illness which might indicate a labora-
tory-associated LDB infection.

SELECTED REFERENCES

1. Anderson, R.E., L. Stein, M.L. Moss, and N.H. Gross. 1952. Potential infectious hazards of common bac-
teriological techniques. J. Bact. 64:473.

2. Hanel, E. and R.L. Alg. 1955. Biological hazards of common laboratory procedures. II. The hypodermic
syringe and needle. Am. J. Med. Tech. 21:343.

3. Kenny, M.T. and F.L. Sabel. 1968. Particle size distribution o( Serraria marcescem aerosols created during
cominon laboratory procedures and simulated laboratory accidents. Appl. Microbiol. 16:1146.

4. Phillips, G.B. and M. Reitman. 1956. Biological hazards of common laboratory procedures. IV. The inoculat-
ing loop. Am. J. Med. Tech. 22:16.

5. Pike, R.M. 1976. Laboratory-associated infections: Summary and analysis of 3921 cases. Hltii. Lab. Sci.
13:105.

6. Reitman, M. and G.B. Phillips. 1955. Biological hazards of common laboratory procedures. 1. The pipette.
Am. J. Med. Tech. 21:338.

7. Reitman, M. and G.B. Phillips. 1956. Biological hazards of common laboratory procedures. III. The centri-
fuge. Am. J. Med. Tech. 22: 14.

8. Reitman, M. and A.G. Weduni. 1956. Microbiological safety. Pub. Hth. Reports. 71:659.

9. Stern, E.L., J.W. Johnson, D. Vesley, M.M. Halbert, L.E. Williams, and P. Blume. 1974. Aerosol production
associated with clinical laboratory procedures. Am. J. Clin. Path. 62:591.

10. Sullivan, J.F. and J.R. Songer. 1966. Role of differential air pressure zones in the control of aerosols in a
large animal isolation facility. Appl. Microbiol. 14:674.

11. Tomlinson, A.J.H. 1957. Infected air-borne particles liberated on opening screw-capped bottles. Brit. Med. J.
2:15.

12. Whitwell, F., P.J. Taylor, and A.J. Oliver. 1957. Hazards to laboratory staff in centrifuging screw-capped
containers. J. Clin. Path. 10:88.

136

II. LABORATORY SERVICES
A. Policy

The laboratories of CDC serve as a national reference facility and accept speci-
mens only from state public health laboratories and Federal facilities. Specimens which
cannot be adequately examined locally should be sent to the appropriate state labora-
tory where they will either be processed or. at the discretion of the state laboratory
director, forwarded to CDC Acceptance of diagnostic specimens by CDC directly from
private physicians or institutions and local health departments is authorized only under
special circumstances including prior agreement between CDC and the state laboratory
director.

^^ ^■^i>

c/yt/g/^Y^

Roslyn Q. Robinson, Ph.D.
Director, Bureau of Laboratories

B. Reference Material

1 . Cultures

Cultures of Legionnaires' disease bacteria (LDB) can be obtained by laboratories
for control purposes. However, before authorizing the distribution of a culture, CDC
requires that the laboratory director submit a written request which affirms that the
organism wUl be studied only under suitable containment conditions; that the cultures
will be handled by qualified microbiologists exercising appropriate care; and that no
subcultures of this organism will be distributed to other laboratories. These precautions
are necessary to insure the safety of all concerned until the mode of spread, habitat,
infectivity, and other characteristics of this organism are more clearly defined. The
letter of request should be sent to:

Center for Disease Control
Attention: Albert Balows, Ph.D.

Director, Bacteriology Division

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404)3 29-3711

When the requirements above are met, an LDB culture will be mailed. Open and
inspect the culture upon arrival. Most of our reference cultures are lyophilized and
sealed under vacuum, but some are currently mailed on agar slants. Stock culture
storage on agar media is discussed in the chapter by Feeley et al., a blood-freeze
method is detailed in the Appendix, and lyophilized cultures are discussed in the
section above on Laboratory Safety.

137

Laboratory Services

2. Reagents

The Legionnaires" disease direct and indirect fluorescent antibody research re-
agents listed below are available from:

Center for Disease Control

Attention: Biological Products Division

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 329-3355

Biological Products Requisition forms (see sample) should be used to order all
such reagents. Supplies of fonns can also be ordered from the above address.

At present there are no restrictions on distribution; however, when these reagents
become commercially available, they will be distributed in accordance with the estab-
lished reagent distribution policy stated in the next section.

Federal agencies are required to supply a purchase order with each requisition and
should contact the above address for a price quotation.

Catalog
Number

BE1578

BE1538

BA1629

BE1514
BA1630

BE1575
BA1631

BE1576
BA1632

BE1567

DIRECT FA PRODUCTS

Globulin FITC Polyvalent (Serogroups 1,2.3,4)

Globulin FITC Serogroup 1 , Knoxville Isolate
Antigen

Globulin FITC Serogroup 2.Togus 1 Isolate
Antigen

Globulin FITC Serogroup 3, Bloomington Isolate
Antigen

Globulin FITC Serogroup 4. Los Angeles I Isolate
Antigen

Globulin FITC Nomial

Packing
Unit

2 ml

2 ml

1 ml

2 ml

1 ml

2 ml

1 ml

2 ml
1 ml

1 ml

INDIRECT FA PRODUCTS

KK0003

BA1533
BA1515
BS1525
BG1566
BEI537

Reagent Set

Antigen Serogroup 1, Philadelphia 1 Isolate
Diluent Nomial Yolk Sac 37r
Globulin FITC Labeled Anti-Human
Serum Serogroup 1 Positive Control

Antigen Serogroup 1, Philadelphia 1 Isolate

Antigen Serogroup 2.Togus 1 Isolate

Serum Serogroup 2, Positive Control

Diluent Normal Yolk Sac 3%

Globulin FITC Labeled Anti-Human

2 ml

45 ml

4 ml

1 ml

2 ml

2 ml

1 ml

45 ml

2 ml

138

SAMPLE

BIOLOGICAL PRODUCTS REQUISITION
Ordering Agency Complete Bold-lined Section ONLY

REQUESTED BV

J.R. Smith

APPROVED BY

A.R. Jones

DATE

9/28/78

IF APPLICABLE

CDC ADDRESS

PHS GRANT NO

CENTER FOR DISEASE CONTROL

ATTN BIOLOGICAL PRODUCTS DIVISION

(NAME & ADDRESS OF CONSIGNEE)

Dr. Adam Smith

Microbiology Laboratory

State of Maine Health Department

Portland, Maine 78102

ATLANTA, GEORGIA 30333

CONSIGNEE

REIMB.REQUESTGR
CUSTOMER NO.

FED PC NO

CUSTOMER NC

1

rVPE OF SHIPMENT

^RGE
8. CONSIGNEE
REQUESTOR 4 NOT CONSIGNEE

CDC -CAN- NO

D{RN) NO CHt
n(RR) REIMB
D(RC) REIMB.

TP.EPHONPNo A|0|4 |.|2|9|2.| 3|3 6|8|

HlRM) RLMOVE FORM INVENTORY

REIMBURSABLE REQUESTOR (NOT CONSIGNEE)

REPLACEMENT FOR PREVIOUS SHIPMENT. D(
CREDIT DlW) WHO D(n)PAHO DfOOTHEf
REIMB: n(P) PC . CDC. WHO D(G) GRANT

Y) YES QlN) NO

DATE RECFIVED

INVOICE DATE

DATE SHIPPED

INVOICE NO TRAILER

PRODUCT NAME

< jU ANTITV
ORDEREEj

CATALU'j NO

LOT NO

LOCATION

QUANTITY
SHIPPED

' LD IFA Kit

1 Set

KK0003

LD Anti-human Globulin

1 Vial

BE 1537

10

12.

COMMENTS

CDC 3,40:^ REV 1 1 76

BIOLOGICAL PRODUCTS DIVISION COPY

139

Laboratory Services

a. Policies Governing the Distribution of CDC Products

The types of products produced and distributed by the Biological Products
Division, Bureau of Laboratories, CDC, are described below. Also included are a
listing and brief description of authorized recipients. Further inquiries should be
sent to:

Center for Disease Control

Attention: Biological Products Division

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 3 29-3355

( 1 ) Products

(a) In Vitro Diagnostic Reference

Reference products are provided to consumers so that they can
determine whether similar products prepared by commercial manufac-
turers or in noncommercial laboratories meet CDC specifications. For
this purpose, only small volumes, usually one packing unit, are distribu-
ted.

(b) In Vitro Diagnostic

When there is a public health need for certain diagnostic products
which can neither be purchased nor prepared in a noncommercial lab-
oratory, CDC may be able to supply quantities sufficient for diagnostic
use.

(c) Research and Investigational

When reagents are distributed for research and other investiga-
tions, the requester agrees to provide, upon request, information about
the specific use of products and reports of the results obtained. Some
of these products may not meet the requirements of published CDC
Specifications.

(2) Authorized Recipients

The following are authorized to receive products:

1. OTHER FEDERAL AGENCIES. These agencies receive products on
a reimbursable basis.

2. STATE, TERRITORIAL, AND LOCAL PUBLIC HEALTH AGEN-
CIES.

3. INTERNATIONAL PUBLIC HEALTH AGENCIES, such as the
World Health Organization (WHO) and the Pan American Health Organiza-
tion (PAHO).

4. PUBLIC HEALTH SERVICE GRANTEES. Grantees will be pro-
vided only those products needed for studies covered by their grants. They
are not eligible to receive other products.

140

Laboratory Services

5. COMMERCIAL PRODUCERS OF IN VITRO DIAGNOSTIC PROD-
UCTS. Manufacturers will be provided with CDC reference products for use
in evaluating lots of commercial products to determine whether they meet
the CDC Specifications.

6. COLLABORATING RESEARCHERS. This group includes private
investigators and investigators in universities, medical centers, and other
teaching and research facilities who are studying problems of interest to
CDC.

3 . Tissues

Control tissue for the modified Dieterle silver impregnation stain is available.

Sufficient lung tissue containing the Legionnaires' disease bacterium is not avail-
able for distribution. The control tissue distributed for this procedure contains a spiro-
chete. This material consists of a paraffin-embedded block and a matching Dieterle-
stained slide. A reference slide of Dieterle-stained lung tissue from a known case of
Legionnaires" disease will also be sent on request. Requests should be sent to;

Center for Disease Control
Attention: Mrs. Pat Greer

Control Tissue Respository

Pathology Division

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404)329-3623

4. Chemicals

Ferric pyrophosphate for Feeley-Gorman (F-G) and charcoal yeast extract (CYE)
agar media is available on request from;

Center for Disease Control

Attention; Biological Products Division

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 3 29-3353

141

Laboratory Services

C. Reference Diagnostic Services

1. Indirect Immunofluorescence (IF) Test

As a general rule, only paired (or serial) acute-phase and convalescent-phase sera
should be submitted. Ideally, the acute-phase serum should be collected during the first
week of illness, and the convalescent-phase serum should be obtained 3 wk after onset
of illness. Titers usually peak at approximately 5 wk after onset.

Place the acute-phase serum in a 1-dram screw-capped glass vial. Seal the vial with
waterproof tape, label for proper identification, and store at 4°C. Prepare a second vial
for the convalescent-phase senmi. When both sera are ready, tape the two vials together
to avoid separation in transit and mail them with CDC Fonn 3.203 to CDC. If you
subsequently receive additional convalescent-phase sera from the same patient, please
submit them to CDC with appropriate identification, a CDC Form 3.203, and a state-
ment that previous sera from this patient were submitted for the indirect IF test.

If we do not receive an acute-phase senim collected dunng the first week of
illness, we will perform the indirect IF test on a single serum collected at least 14 days
after onset of illness. However, because a single serum specimen obtained before this
time and submitted for indirect IF testing will not be evaluated without justification,
we strongly urge that every attempt be made to obtain a second sample. Sera from
blood samples obtained just before a patient dies or during autopsy will be tested, with
the understanding that we may not find a significant titer.

Address requests for indirect IF and other serologic tests to:

Center for Disease Control
Attention: Dr. Hazel W. Wilkinson

Special Immunology Laboratory

DASH-Unit 10

Bacterial Immunology Branch

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 329-3929

142

Laboratory Services

2. Isolation from Clinical Specimens

Portions of lung specimens submitted for isolation of possible LDB should be
carefully selected. To the extent possible, pathologists should select areas of dense
consolidation or areas with caseating necrosis, because such lung tissue is most likely to
contain LDB. Specimens should be packaged and mailed according to the Interstate
Quarantine Regulation (42 CFR Part 72) for shipping etiologic agents and should
be surrounded by enough cold-pack artificial ice to keep the material cold until it
arrives at CDC. Shipping specimens frozen or in dry ice is not necessaiy. Tissue fixed in
Fomialin or other fixatives is obviously unsuitable for isolation attempts but will be
examined with direct immunofluorescence.

Sputa or other respiratoiy tract fluid specimens, except pleural fluids, cannot be
accepted for isolation attempts unless authorized in advance.

Address requests and specimens to:

Center for Disease Control
Attention: Dr. Roger McKinney

Immunofluorescence Section

DASH -Unit 11

Analytical Bacteriology Branch

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404)329-3563

3. Direct Immunonuorescence (IF) Test

In addition to the two reference diagnostic services described above, the Bacteriol-
ogy Division provides reference diagnosis and consultation on the use of specific im-
munofluorescent conjugates for the direct staining of LDB in fresh or Fomialin-fixed
tissues and other clinical specimens. Neutral Formalin is the best fixative to use if the
tissue is to be examined with direct IF. Such tissues do not require refrigeration and
should not be frozen because they are then unsuitable for pathology studies.

Address requests and specimens to:

Center for Disease Control
Attention: Dr. Roger McKinney

Immunofluorescence Section
^ DASH-Unit 1 1

Analytical Bacteriology Branch

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 3 29-3563

143

Laboratory Services

4. Identification of Culture Isolates

Cultures suspected of being the LDB will be confimied or identified.
Address requests and specimens to:

Center for Disease Control
Attention: Dr. Robert E. Weaver

Special Bacteriology Section

DASH-Unit 17

Clinical Bacteriology Branch

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404)3 29-3248

5. Histopathological Examination

Formalin-fixed tissues from patients with suspected LD will be examined. Either
wet or paraffin-embedded tissues are acceptable.
Address requests and specimens to:

Center for Disease Control
Attention: Dr. Martin D. Hicklin

Pathology Division

DASH-Unit 09

Bureau of Laboratories
Atlanta, Georgia 30333
Telephone (404) 3 29-3 136

D. Submitting Reference Specimens

1. Infomiation Required

Specimens submitted to CDC for any or all of the above-listed reference diagnos-
tic services must be accompanied by the following patient information on CDC Fonn
3.203 (see below):

(a) Patient's name

(b) Patient's birth date

(c) Patient's sex

(d) Referring physician's clinical diagnosis

(e) Date of onset of illness

(f) Dates sera were drawn

(g) Whether illness was fatal

(h) Highest temperature recorded for patient

(i) Infomiation on other specimens from the patient which have been submitted
separately to a state health department laboratory or to CDC

2. Transporting

The reliability of laboratory test results largely depends upon the care and
thought used in the collection, handling, and shipment of diagnostic specimens.

144

U.S. DEPARTMENT OF HEALTH. EDUCATION, AND WELFARE

PUBLIC HEALTH SERVICE

CENTER fOR DISEASE CONTROL

Bureau of Laboratories

Atlanta, Georgia 30333

STATE HEALTH DEPARTMENT LABORATORY ADDRESS

STATE HEALTH
DEPT. NO.:

DATE SENT TO Month Day Year
CDC:

NAME AND ADDRESS OF PHYSICIAN OR ORGANIZATION:

PATIENT IDENTIFICATION Hospital No.

NAME: Last (18-37) First (38-47) Middle 1 nitial (48)

BIRTHDATE: (49-54)

Montti Day Year

1 1 1 1 1 1 1

SEX: (55)

(Below Ihis line for CDC Use Only)

CLINICAL DIAGNOSIS: (56-57)

! ■ (

CDC NUMBER

DATE RECEIVED (12-17)

ASSOCIATED ILLNESS: (58-59)

UNIT FY(3-4| NUMBER (510) SUFdU

1 1 1

Month Day Year
1 1 1 1 1

DATE OF ONSET (Mo. Da. Yr.) (60-65)

FATAL? (66)

n YES □ NO

REVERSE SIDE OF THIS FORM MUST BE COMPLETED

Type Specimen

"■ -•■ ~ ■ ^^

■ .fr'svVN^^. V ^-. ..^:^'

THIS FORM MUST BE EITHER PRINTED OR TYPED

PLEASE PREPARE A SEPARATE FORM FOR EACH SPECIMEN
(Consider paired sera one specimen)

DASH.

HSM 3.203-R
6-72

0 3

(12-13)
Comments:

Date Reported

Mo. Day Yr.

(1419)

(40-41)

D

6

5

(198-200)

CDC 3 203
REV 476

PLEASE DO NOT USE BLUE INK

145

LABORATORY EXAMINATIONISl REQUESTED (31 3bi

n ANiimicrobtal Susceptibility D IDentif ication D SErologv (Specify Test).
D Histology D isolation D OTher (Specify)

CATEGORY OF AGENT SUSPECTED (37J D Bacterial D Viral D Fungal D Rickettsial D Parasitic D Othei (Specify I .

SPECIFIC AGENT SUSPECTED

■ 136-401 I

OTHER ORGANISMISI FOUND

ISOLATION ATTEMPTED' (47| D Yes D No NO TIMES ISOLATED 148 49|

SPECIMEN SUBMITTED IS IS?): D Original Material D Puie Isolate D Mined Isolati

NO TIMES PASSED (50 511.

J_±_L

ORIGIN (59-60)

D HUman

n FOod

Dsoii

C ANimal (specify) .
D OTtier (Soecrlyl .

URine D CSF D HAir D SKin

I I I D Tissue (Specify)

I . I D OTher (Specify)

SOURCE OF SPECIMEN 161 6.-11 D BLood DgAshic D SErum D SPutum D
n wound (Site)

n STool n THroat

D Exudate (Site) .

SUBMITTED ON (63-64) D MEdium (Specify) ,
D ANimal (Specify) -

• I

D EGg n Tissue Culture (Type)
D OTher (Specify)

SERUM INFORMATION

(65-721
173-801
{81-881
(89-96)
(97-104)

D ACute

Cj convalescent

ns3
ns4

Dss

Mo.

Da.

1 1

Yr.

1,1,

1 1

1 i

L ^ 1

IMMUNIZATIONS:

.dZL

TREATMENT: Drugs Used D None (129) Dale Begun

Mo. Da. Y

Date Completed
Mo. Da. Yr,

l-ia3)
1-157)
l-Wl)

EPIDEMrOLOGICAL DATA: (172 173)

n single Case D Sporadic D COntact D EPidem.c D CArner

Famitv Illness (l '*»-

Communitv Illness _(176-

Travel and Residence (Location! . . —

n Foreign (178-183) I— --L_

D USA — ^1184.189) L_l_

1751 I

177)111

, Mo.

Animal Contacts (Species) (190-

Arthropod Contacts (192) D None D Exposure Only D Biie

Type of Arthropod (193-

Suspected Source ot Infection ( 195

,n~i

ig-i) l_

196)L

PREVIOUS LABORATORY RESULTS/OTHER CLINICAL INFORMATION:

CLINICAL TEST RESULTS

Sputurn and Histological Findings-
Blood Counts

■(12 13)1 0|2 I

Type Skin Tests Performed

tormeo
(14-211

f^flo.

Da.

1

Vr.

. 1

(23-301

. 1.1,1

(32-391

1 1 1 1 1 1

Strength Pos. Neg.

(22)
(31)
(40)

SIGNS AND SYMPTOMS

(48-49) D FEver

Maximum Temperature .
Duration Days

(56-57, DCH-lls <^^-"'

RASH:

(58-59) n MAculopapular

(60-61) D HEmofrhagic

(62 63) D VEsiCular

(64-65) D Erythema Nodosum

(66-67) D Erythema Marginatum

(68-69) D OTher

RESPIRATORY:

(70 71) D RHinitis

(72 73) n Pulmonary

(74-75) n PHaryngitis

(76 77) D CAIcifications

(78-79) n PNeumonia (type)

(80-81) D OTher

CARDIOVASCULAR:

(82-83) D MYocardilis
(84-85) Q PEricarditis
(86-87) n ENdocarditis
(88-89) D OTher

GASTROINTESTINAL:

(90-91 ) D Diarrhea
(92-93) D BLood
(94-95) n MUcOus
(96-97) n constipation
(98-99) n ABdominal pam

(100-101) D VOmiiing

(102-103) D OTher

CENTRAL NERVOUS SYSTEM:

(104-

05) GHEadache

(106-

07) D MEningismus

(108-

09) D Microcephalus

(110-

U) D Hydrocephalus

(112-

13) D SEizures

(114-

15) D CErebral Calcification

(116-

17) D CHorea

(I 18-

19) n PAralysiS

(120-

■?^)^\ nThPr

MISCE

LLANEOUS:

(122-

2 3) D JAundice

(124-

25) D MYalgia

(126-

127) D PLeurodynia

(128-

129) D conjunctivitis

( 1 so-

131) n CHorioretmitis

il 32-

133) D splenomegaly

(134-

135) n HEpatomegaly

(136-

137) n Liver Abscess

(138-

139) Q LYmphadenopathy

(140-

141) □ MUcous Membrane Lesions

(142-
STATE

1 1 1) n OThor

OF ILLNESS:

(144.

145) D SYmptomatic

(146-

147) Q Asymptomatic

(148-

149) D subacute

(150-

151) D CHronic

(152-

153) n Disseminated

(154-

155) D Localized

(156-

157) n INtraintestinal

(158-

159) D Extraintestinal

(160-

161) CI OTher ^ .

FOR CDC USE ONLY [Ojjjd? 13)

TYPE SERVICE: (14 15)
Ql-Relerence
02-Eoid, Aid
03-Proficiency Testing
04-Speciai Projects

L_

_l - Other

No Specimens (16-20)
LOCATION CODE: (26-27)

AR Mrgentma
AS Australia
AU Austria
BC BermuOa
BE Belgium
BH British Hon
BL Bolivia
BR Brazil
CA Canada
CB Cambodia

CM Cameroon

CO Colombia

C5 Costa Rica

CY Cyprus

DR Dominican I

EC Ecuador

ES El Salvador

ET Ethiopia

FR France

GE Germany

GQ Guam

GT Guatemala

HA Haiti

HO Honduras

IN India

IS Israel

IT Italy

IV Ivory Coast

JM Jamaica

MX MftxiCO

MV Malaysia

Nl Nigeria

No Tests (21-25)

SPECIMEN SUBMITTED BY: (28-30)

NU Nicaragua
NZ New Zelan
PA Paraguay

PN Panama
PP New/ Gull
RP Ptiilippines

gdorr

jerto Rico
Sierra Leone
Spam

TH Thailand

TVi/ Taiwan

UK United Kii

UR Soviet Un

UY Uruguay

VE Venezuela

VN Vietnam

VQ Virgin Islands

100-Health Dept.

200-CDC Clinic

202-Bioloqical Reagents

205-Proficiency Testing

214-S.E.P.

301-Army

302-Navy

303-Aii Force

307 V. A. Hosp.

310-U.S.D.A.
321-F.D.A.
323-lndian Hosp.
324-PHS Hosp.
550-Unlverslty
60e-Physlclan/Cllnic

CDC 3 203 (BACK)
REV 4-76

CDC Number L.

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147

Laboratory Services

The following are practical procedures which the CDC laboratory staff have found
insure viability of the infectious agent and provide maximum protection for those
handling the shipment, both in transit and after it arrives at the laboratoi^. These
procedures should be followed when specimens are shipped to CDC for testing.

a. Packing

Pack specimens so that they are protected in transit and so that the person-
nel who handle the packages are also protected.

(1) Enclose the specimen in a 2-ml or larger screw-capped tube or vial.
(This rule does not apply to bacterial cultures submitted for identification;
they should be submitted as slant or stab cultures.) Seal the tube or vial with
waterproof tape.

NEVER mail clinical specimens or cultures in petri plates!
NEVER enclose dry ice in hen)!etically sealed contaiiiers'
DO NOT use plastic rials for routi)ie shipment of specimens!

(2) Place the tube in a watertigiit shipping can. Pack absorbent cotton or
other suitable absorbent material around the tube to minimize shock and
contain any leakage. Do not use particulate material for this purpose. If
several tubes are placed in the same can, cushion them by wrapping each
individually in paper towels or cotton. Never wrap CDC Fomi 3.203 around
the tube containing the specimen; wrap it around the secondary container
(see Figure).

(3) In an outer cardboard shipping container, pack the secondary container
surrounded with crumpled newspaper or other shock-resisting insulating
material. CDC strongly recommends using a special commercially available
styrofoam-lined cardboard shipping container instead of ordinary cardboard
containers. Seal the outer shipping container securely and affix a properly
completed address label.

b. Shipping

(1) When CO2 is used as a refrigerant, mark the outer shipping container in
accordance with applicable Department of Transportation regulations and
Air Transport Association tariffs.

(2) Affix the Etiologic Agent/Biological Materials label to an exterior sur-
face of any container for etiologic agents (cultures, virus suspensions, bac-
terial toxins) in accordance with Title 42, Code of Federal Regulations,
Section 72.25.

(3) If you are shipping specimens for long distances, send tiiem by an
expedited package service to assure prompt arrival. (Avoid air freight because
it is often delayed.)

(4) With each specimen, submit a completed CDC Form 3.203. Furnish as
much of the information requested on the form as you possibly can, because
it facilitates faster and more reliable diagnostic service.

(5) When possible, ship specimens so that they will arrive at the laboratory
at the beginning or middle of the workweek and not just before or during a
weekend or holiday.

148

III. EPIDEMIOLOGIC SERVICES
A. Policy

TJie Center for Disease Coiifrol provides epidemiologic assistance to state health de-
partments at their request and has responsibility for interstate disease outbreaks. Consulta-
tion is freely provided to individual menbers of the health professions and lay public on
an ad hoc basis, although formal assistance is coordinated through state health departments.

Philip ^.^•t?ichmz.r[,y[D.
Director, Bureau of Epidemiology

B. Epidemiologic Consultation and Assistance

Legionnaires' disease may occur as sporadic cases or in outbreaks. Because some out-
breaks of Legionnaires" disease may be temiinated by identifying and eliminating a common
source of infection (e.g., a contaminated air-conditioning cooling tower or evaporative con-
denser), consultation may be desired concerning a cluster of cases of Legionnaires" disease.
This can be obtained directly from the state epidemiologist, whose name and telephone
number can be obtained from the department of health or comparable agency of the state
government, or from:

Center for Disease Control
Attention: Special Pathogens Branch

Bacterial Diseases Division

Bureau of Epidemiology
Atlanta. Georgia 30333
Telephone: (404)320-3687

The size of a cluster necessitating consultation will vary greatly depending on how
tightly the cases are clustered in time and space. Occasionally examination of tight clusters
as small as two to four cases may provide information of public health importance.

C. Clinical Consultation

Specific antibiotic therapy may decrease the case-fatality ratio associated with Legion-
naires' disease, so early clinical and laboratory diagnosis is of great importance. Consultation
on the clinical aspects of suspected cases of Legionnaires' disease is available from the state
epidemiologist, whose name and telephone number can be obtained from the department of
health or comparable agency of the state government, or from:

Center for Disease Control
Attention: Special Pathogens Branch

Bacterial Diseases Division

Bureau of Epidemiology
Atlanta, Georgia 30333
Telephone: (404) 329-3687

149

PART FIVE

Appendix

Appendix

Page
I. Chromogenic Cephalosporin Test for

i3-LactaiTiase Production 154

II. Cultivation of the Legionnaires' Disease

Bacterium in Yolk Sac of Embryonated Hens' Eggs 156

III. Detection of the Legionnaires' Disease Bacterium

in Clinical Specimens (Flow Chart) 157

IV. Blood-Freeze Storage of Bacteria 158

V. Immunotluorescence Buffers and Mounting Fluid 159

VI. Normal Chicken Yolk Sac (NYS) 160

VII. McFarland Nephelometer Density Standards 160

153

Appendix

I. CHROMOGENIC CEPHALOSPORIN TEST FOR (3-LACTAMASE PRODUCTION

Results of degradation studies and the chromogenic cephalosporin test have documented
that the Legionnaires' disease bacterium produces a /3-lactamase. The enzyme is more active on
cephalosporin than on penicillin, and is not at all active on cefoxitin or cefuroxime (newer
cephalosporins not yet approved in the United States but used in other countries).

The procedure for performing the chromogenic cephalosporin test for j3-lactamase pro-
duction follows.

Reagents

1 . Phosphate Buffer, pH 7.0

a. M/ 1 5 KH: PO4 9.07 g/liter distilled water

b. M/ 1 5 Na. HPO4 .... 9.46 g/liter distilled water

c. Mix 39.2 ml of the potassium phosphate solution with 60.8 ml of the sodium
phosphate solution. This buffer is stable for several weeks at room temperature.

2. Cephalosporin Substrate

a. Cephalosporin 87/312 is available only from:
Glaxo, Ltd.

Greenford, Middlesex UB6 OHE
England

b. Dissolve 10 mg of cephalosporin 87/312 in 1 ml of dimethylsulfoxide (DMSO).
Dilute with phosphate buffer, pH 7.0, to a concentration of 500 jUg/ml. The sub-
strate is yellow when viewed in a microdilution plate, but larger volumes may
appear more orange. However, the red reaction which constitutes a positive test
result is easily discerned. This substrate is stable at 4°-10°C for many weeks.

B. Procedure

1. In a Microdilution Plate or Small Tube:

a. Place 0.05 ml of cephalosporin substrate in a well of a microdilution plate (or in
a small tube).

b. Use a loop to remove several colonies of the test organism from the agar surface.
Make a turbid cell suspension in the cephalosporin substrate.

c. Mix the substrate and cells for 1 min. Observe for color change immediately,
after 10 min, and after 1 h.

d. If the test organism produces j3-lactamase, the color of the substrate will change
from yellow to red. |3-lactamase-producing Haemophilus influenzae or Neisseria
gonorrhoeae isolates usually turn the substrate red in less than 10 min. but
staphylococci may take an hour.

e. Run the test with a known |3-lactamase-producing strain and a non j3-lactamase-
producing strain of N. gonorrhoeae. H. influenzae, or Staphylococcus aureus.

154

Appendix

2. On an Agar Plate:

a. The agar plate should contain the test organism in pure culture.

b. Place a drop (approximately 0.05 ml) of the cephalosporin substrate on an area
of bacterial growth, and tilt the plate slightly so that the drop drains across the
surface of the growth.

c. If the test organism produces j3-lactamase, the substrate along the path will turn
red; if it is j3-lactamase negative, the substrate along the path will be yellow.

d. Although the red reaction on the plate is not as easily seen as that in the micro-
dilution well, it is generally quite obvious. If the analyst is unsure about the reac-
tion, the test should be repeated in a microdilution well or small tube.

e. Usually a positive reaction can be read immediately, but the plate should be
reexamined after 10 min.

C. Precautions

1. Primary isolation media (e.g., modified Thayer-Martin medium) may grow (3-lactamase-
producing bacteria in addition to the organism of interest (e.g.. Legionnaires' disease
bacterium), which may lead to false-positive results.

2. Clinical specimens (e.g.. pleural fluids) cannot be evaluated directly with the chro-
mogenic cephalosporin test.

155

Appendix

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157

Appendix

IV. BLOOD-FREEZE STORAGE OF BACTERIA

Some stock cultures should be maintained for checking each new lot of media and conjugate
and for staining along with each batch of unknowns.

These stock cultures can be obtained from positive clinical specimens or from a reference
laboratory. The stock cultures can be stored as described by Feeley et al. in this manual or by
the blood-freeze method described below.

A. Supplies

1 . Freeze tubes

The freezing tubes should be made of Pyrex glass (e.g.. Corning #9820, size
6 X 50 mm). The Pyrex will stand the sudden change in temperature to which it
will be subjected. New tubes should be boiled in three changes of distilled water to
remove any deleterious substance. These tubes are shaken thoroughly to remove the
water, dried, plugged, and sterilized in a hot air oven. The cultures can be identified
by using Va- X 1-in strips of waterproof adhesive tape on which the name or number
of the culture has been typed or printed in India ink.

2. Storage boxes

Arrange for some type of storage boxes that will fit into the available freezer
space and in which the cultures can be systematically filed. Storage boxes can be
constructed of 3/8-in marine waterproof plywood, plastic, or cardboard.

B. Procedure

Transfer the stock cultures to charcoal yeast extract agar slants, and incubate them
at 35°C for 48-72 h. or to Feeley-Gorman agar slants, and incubate for 3-5 days at 35°C
in air plus 2.5% CO2 .

1 . Freezing

After growth is obtained, add 1 to 2 ml of sterile defibrinated rabbit or sheep
blood aseptically to each slant. Use capillary pipettes to suspend the growth in the
blood, then aspirate and deliver approximately 0.2 ml to each of the previously
numbered freezing tubes. If cotton plugged tubes are used, cut off excess cotton
and flame the Hp. Transfer the tubes to their respective numerical slots in the
storage box. Store at — 50°C.

2. Recultivation

Do not thaw and refreeze frozen suspensions. To recover the organisms, remove
one tube from the freezer, and thaw the contents rapidly by holding the tube
tightly in the palm of the hand or by placing the tube in a 35°C water bath. Draw
off the thawed suspension with a capillary pipette and transfer to a suitable culture
medium.

158

Appendix

V. IMMUNOFLUORESCENCE BUFFERS AND MOUNTING FLUIDS

Some immunofluorescence techniques utilize pliosphate buffers and others call for carbon-
ate buffers. Both systems are recommended in this manual and presented below.

A. Phosphate Reagents

1 . Phosphate Buffered Saline (PBS), pH 7.6, 0.01 M:

a. Concentrated Stock Solution (pH will not be 7.6);

Naj HPO4 (anhydrous; reagent grade) 12.36 gm

NaH, PO4 -H, O (reagent grade) 1 .80 gm

NaCl (reagent grade) 85.00 gm

Distilled water to make final volume 1 liter

Dibasic salt dissolves much more readily in water at 37°C
than at :5°C.

b. Working Solution (pH 7.6; 0.01 M buffer; 0.85% NaCl):

Concentrated stock solution 100 ml

Distilled water to make final volume 1 liter

:. Phosphate Buffered Saline (PBS), pH 7.2:

0.15 M KH, PO4 24 ml

0.15 MNa2HP04 76 ml

0.85% NaCl 100 ml

3. Buffered Glycerol Mounting Fluid, approx. pH 9.0:

Glycerol, reagent grade 90 ml

0.2 M Na. HPO4 10 ml

B. Carbonate Reagents

1 . Carbonate-Bicarbonate Buffer, pH 9.0, 0.5 M:

a. 0.5MNa2CO3 5.3 g/ 100 ml of distilled water

b. 0.5 M NaHCOj 4.2 g/100 ml of distilled water

c. Mix 4.4 ml of solution (a) with the 100 ml of solution (b). Theoretically, a pH
of 9.0 should result from this mixture; however, as much as 17.0 ml of solution
(a) may have to be added to the 100 ml of solution (b).

2. Glycerol Mounting Fluid:

a. 0.5 M Carbonate-bicarbonate buffer, pH 9.0 1 part

b. Neutral glycerol, reagent grade 9 parts

c. Combine and mix by stirring (do not shake).

159

Appendix

VI. IMORMAL CHICKEN YOLK SAC (NYS)

A normal cliicken egg yolk sac suspension is used in the indirect immunonuorescence test
for antibody to the Legionnaires' disease bacterium (see chapter by Wilkinson et al.). The sus-
pension is needed for the preparation of working dilutions of the bacterial antigens and for the
initial dilution in titration of sera.

1. Harvest the yolk sac from 12- to 14-day-old embryonated hens' eggs. Separate as much
of the yolk material from the sac as possible. The average yield of yolk sac per egg is 4 g.

2. Place the yolk sacs in an equal volume of 0.02 M phosphate buffered saline. pH 7.2
(PBS), containing 0.05% sodium azide.

3. Homogenize in an Ominimixer or Waring blender, and filter through two layers of gauze.

4. Dilute the suspension with PBS containing 0.05% sodium azide to 0.5% (w/v) for antigen
preparation or 3% for use as a diluent in the FA test.

VII. McFARLAND IMEPHELOMETER DENSITY STANDARDS

Add 1% barium chloride and 1% sulfuric solutions to 10 uniform tubes in the proportions
shown below. Seal to prevent any evaporation, and label the tubes. Before using standards, shake
tubes well to resuspend the precipitate.

McFarland
standard

#

1% BaCh

solution

(ml)

1%H2S04

solution

(ml)

Appx. density of
bacteria rep.
(million/ml)

International
Units (I.U.)
of Opacity

1

O.I

9.9

300

3

2

0.2

9.8

600

7

3

0.3

9.7

900

10

4

0.4

9.6

1200

12

5

0.5

9.5

1500

15

6

0.6

9.4

1800

-

7

0.7

9.3

2100

20

8

0.8

9.2

2400

-

9

0.9

9.1

2700

-

10

1.0

9.0

3000

30

160

PART SIX

Bibliography

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