JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1978, p. 320-325 0095-1137/78/0008-0320$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 8, No. 3 Printed in U.S.A.
Primary Isolation Media for Legionnaires Disease Bacterium JAMES C. FEELEY,J* GEORGE W. GORMAN,' ROBERT E. WEAVER,2 DON C. MACKEL,l AND HOLLIS W. SMITH' Epidemiologic Investigations Laboratory Branch, Bacterial Diseases Division, Bureau of Epidemiology,' and Special Bacteriology Laboratory Section, Bacteriology Division, Bureau of Laboratories,2 Center for Disease Control, Atlanta, Georgia 30333 Received for publication 21 June 1978
Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% IsoVitaleX and 1% hemoglobin (MHIH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the IsoVitaleX reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different culture conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35°C under 2.5% C02 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH. were
and liquid media, including Bordet-Gengou, cysteineglucose blood, heart infusion (HI), HI with 5% egg yolk, HI with 5% rabbit blood, Mueller-Hinton (MH), MH with 5% Fildes enrichment (6), MH with 5% fetal calf serum, MH with 1% hemoglobin and 1% IsoVitaleX (MH-IH) agars; and Ellinghausen, Fletcher, HI, nutrient, tryptone (2%), Trypticase soy, and ToddHewitt broths. All cultures were incubated aerobically at 35°C and under 2.5, 5, and 10% C02 and in a candle extinction jar. Subsequently, yolk sac suspensions of the organisms were inoculated onto MH agar containing 0.025% iron phosphate scales (soluble [15] ferric phosphate, manufactured by Mallinckrodt Chemical Works approximately 50 years ago) and 0.04% L-cysteine hydrochloride adjusted to pH 6.9. Because the soluble ferric phosphate is no longer produced, soluble ferric pyrophosphate (lot. ELG 25; Mallinckrodt [5528]) was substituted in a combination designated F-G agar, which contained, in 1.0 liter of distilled water: casein (acid hydrolysis), 17.5 g; beef extractives, 3 g; L-cysteine hydrochloride H20, 0.4 g; soluble ferric pyrophosphate, 0.25 g; starch, 1.5 g; and agar, 17 g. MH agar (lot 105621; Baltimore Biological Laboratory) contains all the ingredients of F-G agar except L-cysteine hydrochloride and soluble ferric pyrophosphate and served as a source of these ingredients. This lot contained 300 ml of beef infusion for beef extractives. Other lots of MH agar may vary in formulation and in suitability for use in F-G agar. A 38-g quantity of this agar base was dissolved by boiling in 980 ml of distilled water, autoclaved at 1210C for 15 min, and then cooled to 50°C in a water bath. Fresh separate solutions of L-cysteine hydrochloride (0.40 g in 10 ml of distilled water) and soluble ferric pyrophosphate (0.25 g in 10 ml of distilled water) were prepared and filter sterilized separately before adding
The term Legionnaires disease was first used in the summer of July 1976 to describe an outbreak of acute respiratory illness that occurred in Pennsylvania, predominantly among members of the American Legion attending a statewide convention in Philadelphia. A gram-negative pleomorphic bacterium was ultimately shown to be the etiological agent of the epidemic in Philadelphia (5, 7, 13), as well as of four previously unsolved or undetected outbreaks (1-3). This paper described (i) the bacteriological methods first used to grow the Legionnaires disease bacterium (LDB) on artificial medium by one of the authors (R.E.W.), (ii) experiments elucidating growth requirements of the LDB, and (iii) a transparent primary isolation medium that supports growth of the LDB from clinical specimens. MATERIALS AND METHODS Strains. Four strains of LDB were used in this study. Philadelphia 1 and 2 were isolated from fatal cases from the 1976 outbreak in Philadelphia (7, 11). Pontiac 1 was isolated from tissues of guinea pigs exposed to air in a building in Pontiac, Mich. (2). These strains were obtained from yolk sac suspensions harvested from infected embryonated chicken eggs (11). The fourth isolate (Flint 1) had been isolated directly on artificial medium from pleural fluid from a patient with pneumonia by Morris Dumoff, McLaren General Hospital, Flint, Mich. (4). Gas-liquid chromatography studies showed that all of these isolates had essentially the same fatty acid composition (14). Primary isolations. The yolk sac suspensions of the Philadelphia strains were inoculated to both solid 320
VOL. 8, 1978 to the sterile medium base. The medium was adjusted to pH 6.6 while at 50°C by adding 3.5 ml of 1.0 N HCl; the medium was poured in 20-ml quantities into sterile petri dishes. After cooling, the medium in one plate was checked for the required final pH of 6.9 by placing either a surface electrode on the surface of an agar plate or a combination electrode in emulsified agar in distilled water.
Growth conditions. Optimum pH, temperature, and oxygen requirements were determined by inoculating test media with suspensions of LDB in sterile phosphate-buffered saline adjusted to a turbidity of the McFarland 2 standard (9). (i) pH. Plates of MH-IH agar were prepared at 0.5pH-unit increments from pH 5.0 to 11.0 and inoculated with stock cultures of the four LDB strains. Growth was observed between pH 6.5 and 7.5. MH-IH agar was then prepared at pH 6.5 to 7.5 in approximately 0.1-pH-unit increments. (ii) Oxygen tension. The four LDB strains were incubated onto MH-IH agar for 6 days at 35°C in atmospheres of 20, 10, 5, and 2.5%02 and anaerobically. Reduced O2 tension was achieved by evacuating air from modified pressure cookers to a pressure of -38.1 cm of Hg and replacing it with 100% N2 to a pressure of 1 atmosphere. One exchange reduced the 02 tension by half, to 10%; two exchanges produced 5% O2; and three exchanges produced 2.5% 02. (iii) Temperature. LDB strains were inoculated onto MH-IH agar and incubated aerobically at 25, 29, 35, and 42°C for 6 days. Growth factors. MH-IH agar contains three components: MH agar base, hemoglobin, and IsoVitaleX. Each was analyzed to determine its specific contribution to the growth of the LDB. IsoVitaleX is composed of 12 chemicals, including the following, in grams per liter: adenine, 1.0; p-aminobenzoic acid, 0.013; cocarboxylase, 0.1; L-cysteine hydrochloride, 25.9; L-cysteine, 1.1; dextrose, 100.0; diphosphopyridine nucleotide (oxidized), 0.25; ferric nitrate, 0.02; glutamine hydrochloride, 10.0; guanine hydrochloride, 0.03; thiamine hydrochloride, 0.003; and vitamin B12, 0.01. To determine which chemical or chemicals were necessary for the growth of the LDB, two sets of MH agar plates with 1% hemoglobin were made. One set was supplemented with one of each of the ingredients of IsoVitaleX. The other set was supplemented with each possible combination of all but one of these ingredients. Later, a series of MH-IH agars was prepared with concentrations of either IsoVitaleX or Lcysteine hydrochloride ranging up to five times that originally prepared in MH-IH agar. In an effort to determine whether L-cysteine hydrochloride could be replaced by other chemicals, plates of MH agar with 1% hemoglobin were prepared. A core of agar (6-mm diameter) was removed from the center of each plate, and the remaining agar surface was seeded heavily with a standardized inoculum of Philadelphia strain 1. Solutions (1%) of mercaptoethanol, L-serine, and L-methionine were placed into the wells. Hemoglobin experiment. To determine whether hemoglobin could be replaced by a simpler ingredient, MH agar supplemented with 1% IsoVitaleX was in-
LEGIONNAIRES DISEASE MEDIA
321
oculated with a standard inoculum of Philadelphia strain 1 after a 6-mm-diameter core of agar was removed from the center of the plates. Solutions at 1% concentration of the following compounds or chemicals were placed into the wells: Fildes enrichment (6), ferric nitrate, ferrous sulfate, ferric chloride, ferric ammonium sulfate, soluble ferric phosphate, hematin, hemin, and laked sheep blood. MH agars supplemented with 0.026% L-cysteine hydrochloride were made, incorporating ferric nitrate and soluble ferric phosphate in concentrations ranging from 0.01 to 1.0% to determine their usefulness as a metabolic source of iron. F-G cysteine-iron agar. A new agar medium, F-G agar, was prepared substituting L-cysteine hydrochloride and soluble ferric pyrophosphate for IsoVitaleX and hemoglobin, respectively. Ferric pyrophosphate at concentrations of from 0.001 to 1.0% was tested at two concentrations of L-cysteine hydrochloride (0.026 and 0.052%). Soluble ferric pyrophosphate (0.001 to 1.0%) was also evaluated in a broth medium with an L-cysteine hydrochloride concentration of 0.4%. For the purpose of testing the sensitivity of the new medium, F-G agar and MH-IH were inoculated with guinea pig spleen tissue infected with LDB. MH agar base experiment. MH agar (lot 105621; Baltimore Biological Laboratory) is composed of acidicase, beef infusion, starch, and agar. Each of these components except beef extract (substituted for beef infusion) was incorporated singly and in combination with one another into 1 liter of basal medium containing 5.0 g of NaCl, 1.5 g of soluble starch, 17.0 g of agar, and 980 ml of distilled water and supplemented with 0.40 g of L-cysteine hydrochloride, and 0.25 g of soluble ferric pyrophosphate. A number of other peptones (1%, wt/vol) were examined in this medium for enhancement of growth, including biosate, gelysate, myosate, phytone, Trypticase, Casamino Acids, Bactopeptone, Bacto-peptone no. 2, Bacto-peptone no. 3, neopeptone, Bactone-tryptone, and Bacto-tryptose. Two possible activities of starch in the MH-IH medium are those of detoxifier or of substrate. Charcoal was substituted for starch in some media to determine whether the action was detoxifying. To determine whether the starch was a substrate and was being digested, a solution of iodine was flooded over some plates (MH supplemented with 1% IsoVitaleX and a 5% peptic digest of blood) which had a heavy growth of LDB.
RESULTS Initially MH-IH agar was the only medium found that supported growth of the LDB from yolk sac suspensions. Growth first appeared on this medium within 3 to 5 days at the site where a drop of the inoculum was placed and left unstreaked. Later (5 to 7 days), small colonies appeared in the streaked areas on the plates. Subsequently the LDB was grown from the infected yolk sac suspensions on F-G agar. The comparison studies of F-G agar and MH-IH agar in which infected guinea pig spleen tissue was used showed that the LDB grew more rapidly and in greater numbers on F-G agar than on
322
FEELEY ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Macroscopic colony counts of LDB Philadelphia I from infected guinea pig spleen on F-G and MH-IH agars adjusted to various pH levels Medium
F-G agar
pHduof medium
7.31
3 oa
O 0
0b 0b 0b 0b 0b 0b 0 0 0
Colony counts after the following days of incubation: 4 5 6 7
o O 1 il 71 62 67 117 14 0 0 0
O O 1 14 82 71 101 145 86 0 0 0
O 0 1 14 82 71 140 159 138 100 0 0
il
O 0 1 25 98 78 171 168 163 123 0 0
O O 3 52 130 124 241 199 205 169 35 0
MH-IH agar
s6.42 0 0 0 0 0 0 6.50 0 0 0 0 1 1 6.58 0 0 0 0 17 17c 6.68 0 0 0 0 17 17c 6.80 0 0 0 0 0 0 6.90 0 0 0 0 1 1 7.00 0 0 0 0 1 1 -7.10 0 0 0 0 0 0 a Average of three plates rounded to nearest whole number. The inoculum consisted of a 10% tissue suspension diluted 1:10 in sterile phosphate-buffered saline. Each plate was inoculated with 0.05 ml of the 1:10 dilution. b Colonies were easily observable with a dissecting microscope. c A total of 30 to 50 suspect colonies were seen with a dissecting microscope.
TABLE 2. Growth of the LDB on MH agar supplemented with 1% hemoglobin and various constituents of IsoVitaleX Growth of LDB Strainsb IsoVitaleX chemical deleted or added
Concna (g/liter)
Adenine p-Aminobenzoic acid Cocarboxylase L-Cysteine hydrochloride L-Cystine Dextrose Diphosphopyridine nucleotide (oxidized) Ferric nitrate Glutamine hydrochloride Guanine hydrochloride Thiamine hydrochloride Vitamin B12
0.01 0.00013 0.001 0.0259 0.001 1.000 0.0025 0.0002 0.100 0.0003 0.00003 0.0001
Chemical deleted
Pi 4 3 4
0 4 4 4 4 4 4 4 3
P2 4 4 4
0 4 4 4 4 4 4 4 4
Chemical added
Fl 3
Pol 4
4
3
3 0 4 4
4
4 4
3 4 3 4
Pi
P2
0 0 0
0 0 0 4 0 0 0 0 0 0 0 0
0
4
4 4 4 4 4 4 4 4
0 0 0 0 0 0 0 0
Fl 0 0 0
Pol
0 0 0
4
4
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
All chemicals 0 0 4 0 4 4 0 4 a Concentration normally present in medium. b Growth rated at 6 days on a scale of 0 to 4. Philadelphia 1; P2, Philadelphia 2; Fl, Flint 1; Pol, Pontiac 1. C Medium contained all of IsoVitaleX chemicals except chemical indicated. d Medium supplemented only with IsoVitaleX chemical indicated. -
Pi,
MH-IH agar. Within 3 days, colonies could be observed on the F-G agar with a dissecting microscope, and by 4 days they were macroscopically visible as pinpoint colonies, which enlarged
with longer incubation (Table 1). The colonies of LDB characteristically appeared to have a cut-glass appearance when observed microscopically with the light illumination from one side
LEGIONNAIRES DISEASE MEDIA
VOL. 8, 1978
at a 100 angle. In areas of heavy growth of the LDB, especially in subcultures, a distinctive brownish darkening of the LDB colonies and surrounding agar occurred on both the F-G and MH-IH agars. In addition, growth of the LDB on F-G agar produced a fluorescent substance in the colonies and surrounding agar that could be detected by long-wave (366 nm) UV light. Studies of the composition of IsoVitaleX showed that LDB would not grow only when Lcysteine hydrochloride was deleted from the medium (Table 2). Increase of either IsoVitaleX or L-cysteine hydrochloride to two times normal concentrations in MH agar with hemoglobin enhanced the growth of the LDB. Higher concentrations of these chemicals did not increase growth. However, fluorescence and browning were influenced. Browning was maximum with the original concentrations of L-Cysteine hydrochloride and decreased with the increase to two times the original concentration. The opposite was true for fluorescence. Browning was stable, whereas fluorescence was maximum at about 4 days and decreased with time; 0.40% L-cysteine hydrochloride gave good browning and fluorescence. Soluble ferric pyrophosphate and ferric nitrate enhanced growth of the LDB better than did hemoglobin and the other iron-containing substances that were evaluated. Soluble ferric pyrophosphate stimulated the growth of the LDBorga organism sm better than did the discontinued
323
growth of the LDB from stock cultures as well as did the beef infusion and acidicase contained in the lot of MH agar used in these studies. Other peptones either supported poorer growth or did not support growth at all (Table 4). The organism grew best aerobically with 2.5% TABLE 4. Growthai LDB on F-G cystene-iron agar prepared wi' h var'oup Growth ofLDB strainsb Company Peptone P2 F1 Pol Pl Baltimore Bio-
Acidicase
logical Laboratory
Beef extract
0
0
0
0
Acidicase + beef 4
4
4
4
Biosated
4
4
4
4
2 0 1 0
0 0
Beef extract
2 0 0 0
0
1 0 1 0
Casamino Acids Bacto-peptone
0
0 ±
0 ±
0
±
Gelysate Myosate Phytone
2
Trypticase Difco Labora-
2
2
0
2
tories
no.2 Bacto-peptone no. 3 Neopeptone
3
3
3
3
0
Bacto-tryptose Bcotyts
i
0 3
0 4
3
0
Acidicase and beef extract were at the same concentrabsetermthan (Th dbeconTiT' tons as m MH agar. Other peptones were at 1% (wt/vol)
LB
fi h h f ui TT
LDB grew less well on MH agar supplemented concentrations. b Growth rated at 6 days on a scale of 0 to 4. ± =
Vol. 8, No. 3 Printed in U.S.A.
Primary Isolation Media for Legionnaires Disease Bacterium JAMES C. FEELEY,J* GEORGE W. GORMAN,' ROBERT E. WEAVER,2 DON C. MACKEL,l AND HOLLIS W. SMITH' Epidemiologic Investigations Laboratory Branch, Bacterial Diseases Division, Bureau of Epidemiology,' and Special Bacteriology Laboratory Section, Bacteriology Division, Bureau of Laboratories,2 Center for Disease Control, Atlanta, Georgia 30333 Received for publication 21 June 1978
Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% IsoVitaleX and 1% hemoglobin (MHIH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the IsoVitaleX reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different culture conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35°C under 2.5% C02 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH. were
and liquid media, including Bordet-Gengou, cysteineglucose blood, heart infusion (HI), HI with 5% egg yolk, HI with 5% rabbit blood, Mueller-Hinton (MH), MH with 5% Fildes enrichment (6), MH with 5% fetal calf serum, MH with 1% hemoglobin and 1% IsoVitaleX (MH-IH) agars; and Ellinghausen, Fletcher, HI, nutrient, tryptone (2%), Trypticase soy, and ToddHewitt broths. All cultures were incubated aerobically at 35°C and under 2.5, 5, and 10% C02 and in a candle extinction jar. Subsequently, yolk sac suspensions of the organisms were inoculated onto MH agar containing 0.025% iron phosphate scales (soluble [15] ferric phosphate, manufactured by Mallinckrodt Chemical Works approximately 50 years ago) and 0.04% L-cysteine hydrochloride adjusted to pH 6.9. Because the soluble ferric phosphate is no longer produced, soluble ferric pyrophosphate (lot. ELG 25; Mallinckrodt [5528]) was substituted in a combination designated F-G agar, which contained, in 1.0 liter of distilled water: casein (acid hydrolysis), 17.5 g; beef extractives, 3 g; L-cysteine hydrochloride H20, 0.4 g; soluble ferric pyrophosphate, 0.25 g; starch, 1.5 g; and agar, 17 g. MH agar (lot 105621; Baltimore Biological Laboratory) contains all the ingredients of F-G agar except L-cysteine hydrochloride and soluble ferric pyrophosphate and served as a source of these ingredients. This lot contained 300 ml of beef infusion for beef extractives. Other lots of MH agar may vary in formulation and in suitability for use in F-G agar. A 38-g quantity of this agar base was dissolved by boiling in 980 ml of distilled water, autoclaved at 1210C for 15 min, and then cooled to 50°C in a water bath. Fresh separate solutions of L-cysteine hydrochloride (0.40 g in 10 ml of distilled water) and soluble ferric pyrophosphate (0.25 g in 10 ml of distilled water) were prepared and filter sterilized separately before adding
The term Legionnaires disease was first used in the summer of July 1976 to describe an outbreak of acute respiratory illness that occurred in Pennsylvania, predominantly among members of the American Legion attending a statewide convention in Philadelphia. A gram-negative pleomorphic bacterium was ultimately shown to be the etiological agent of the epidemic in Philadelphia (5, 7, 13), as well as of four previously unsolved or undetected outbreaks (1-3). This paper described (i) the bacteriological methods first used to grow the Legionnaires disease bacterium (LDB) on artificial medium by one of the authors (R.E.W.), (ii) experiments elucidating growth requirements of the LDB, and (iii) a transparent primary isolation medium that supports growth of the LDB from clinical specimens. MATERIALS AND METHODS Strains. Four strains of LDB were used in this study. Philadelphia 1 and 2 were isolated from fatal cases from the 1976 outbreak in Philadelphia (7, 11). Pontiac 1 was isolated from tissues of guinea pigs exposed to air in a building in Pontiac, Mich. (2). These strains were obtained from yolk sac suspensions harvested from infected embryonated chicken eggs (11). The fourth isolate (Flint 1) had been isolated directly on artificial medium from pleural fluid from a patient with pneumonia by Morris Dumoff, McLaren General Hospital, Flint, Mich. (4). Gas-liquid chromatography studies showed that all of these isolates had essentially the same fatty acid composition (14). Primary isolations. The yolk sac suspensions of the Philadelphia strains were inoculated to both solid 320
VOL. 8, 1978 to the sterile medium base. The medium was adjusted to pH 6.6 while at 50°C by adding 3.5 ml of 1.0 N HCl; the medium was poured in 20-ml quantities into sterile petri dishes. After cooling, the medium in one plate was checked for the required final pH of 6.9 by placing either a surface electrode on the surface of an agar plate or a combination electrode in emulsified agar in distilled water.
Growth conditions. Optimum pH, temperature, and oxygen requirements were determined by inoculating test media with suspensions of LDB in sterile phosphate-buffered saline adjusted to a turbidity of the McFarland 2 standard (9). (i) pH. Plates of MH-IH agar were prepared at 0.5pH-unit increments from pH 5.0 to 11.0 and inoculated with stock cultures of the four LDB strains. Growth was observed between pH 6.5 and 7.5. MH-IH agar was then prepared at pH 6.5 to 7.5 in approximately 0.1-pH-unit increments. (ii) Oxygen tension. The four LDB strains were incubated onto MH-IH agar for 6 days at 35°C in atmospheres of 20, 10, 5, and 2.5%02 and anaerobically. Reduced O2 tension was achieved by evacuating air from modified pressure cookers to a pressure of -38.1 cm of Hg and replacing it with 100% N2 to a pressure of 1 atmosphere. One exchange reduced the 02 tension by half, to 10%; two exchanges produced 5% O2; and three exchanges produced 2.5% 02. (iii) Temperature. LDB strains were inoculated onto MH-IH agar and incubated aerobically at 25, 29, 35, and 42°C for 6 days. Growth factors. MH-IH agar contains three components: MH agar base, hemoglobin, and IsoVitaleX. Each was analyzed to determine its specific contribution to the growth of the LDB. IsoVitaleX is composed of 12 chemicals, including the following, in grams per liter: adenine, 1.0; p-aminobenzoic acid, 0.013; cocarboxylase, 0.1; L-cysteine hydrochloride, 25.9; L-cysteine, 1.1; dextrose, 100.0; diphosphopyridine nucleotide (oxidized), 0.25; ferric nitrate, 0.02; glutamine hydrochloride, 10.0; guanine hydrochloride, 0.03; thiamine hydrochloride, 0.003; and vitamin B12, 0.01. To determine which chemical or chemicals were necessary for the growth of the LDB, two sets of MH agar plates with 1% hemoglobin were made. One set was supplemented with one of each of the ingredients of IsoVitaleX. The other set was supplemented with each possible combination of all but one of these ingredients. Later, a series of MH-IH agars was prepared with concentrations of either IsoVitaleX or Lcysteine hydrochloride ranging up to five times that originally prepared in MH-IH agar. In an effort to determine whether L-cysteine hydrochloride could be replaced by other chemicals, plates of MH agar with 1% hemoglobin were prepared. A core of agar (6-mm diameter) was removed from the center of each plate, and the remaining agar surface was seeded heavily with a standardized inoculum of Philadelphia strain 1. Solutions (1%) of mercaptoethanol, L-serine, and L-methionine were placed into the wells. Hemoglobin experiment. To determine whether hemoglobin could be replaced by a simpler ingredient, MH agar supplemented with 1% IsoVitaleX was in-
LEGIONNAIRES DISEASE MEDIA
321
oculated with a standard inoculum of Philadelphia strain 1 after a 6-mm-diameter core of agar was removed from the center of the plates. Solutions at 1% concentration of the following compounds or chemicals were placed into the wells: Fildes enrichment (6), ferric nitrate, ferrous sulfate, ferric chloride, ferric ammonium sulfate, soluble ferric phosphate, hematin, hemin, and laked sheep blood. MH agars supplemented with 0.026% L-cysteine hydrochloride were made, incorporating ferric nitrate and soluble ferric phosphate in concentrations ranging from 0.01 to 1.0% to determine their usefulness as a metabolic source of iron. F-G cysteine-iron agar. A new agar medium, F-G agar, was prepared substituting L-cysteine hydrochloride and soluble ferric pyrophosphate for IsoVitaleX and hemoglobin, respectively. Ferric pyrophosphate at concentrations of from 0.001 to 1.0% was tested at two concentrations of L-cysteine hydrochloride (0.026 and 0.052%). Soluble ferric pyrophosphate (0.001 to 1.0%) was also evaluated in a broth medium with an L-cysteine hydrochloride concentration of 0.4%. For the purpose of testing the sensitivity of the new medium, F-G agar and MH-IH were inoculated with guinea pig spleen tissue infected with LDB. MH agar base experiment. MH agar (lot 105621; Baltimore Biological Laboratory) is composed of acidicase, beef infusion, starch, and agar. Each of these components except beef extract (substituted for beef infusion) was incorporated singly and in combination with one another into 1 liter of basal medium containing 5.0 g of NaCl, 1.5 g of soluble starch, 17.0 g of agar, and 980 ml of distilled water and supplemented with 0.40 g of L-cysteine hydrochloride, and 0.25 g of soluble ferric pyrophosphate. A number of other peptones (1%, wt/vol) were examined in this medium for enhancement of growth, including biosate, gelysate, myosate, phytone, Trypticase, Casamino Acids, Bactopeptone, Bacto-peptone no. 2, Bacto-peptone no. 3, neopeptone, Bactone-tryptone, and Bacto-tryptose. Two possible activities of starch in the MH-IH medium are those of detoxifier or of substrate. Charcoal was substituted for starch in some media to determine whether the action was detoxifying. To determine whether the starch was a substrate and was being digested, a solution of iodine was flooded over some plates (MH supplemented with 1% IsoVitaleX and a 5% peptic digest of blood) which had a heavy growth of LDB.
RESULTS Initially MH-IH agar was the only medium found that supported growth of the LDB from yolk sac suspensions. Growth first appeared on this medium within 3 to 5 days at the site where a drop of the inoculum was placed and left unstreaked. Later (5 to 7 days), small colonies appeared in the streaked areas on the plates. Subsequently the LDB was grown from the infected yolk sac suspensions on F-G agar. The comparison studies of F-G agar and MH-IH agar in which infected guinea pig spleen tissue was used showed that the LDB grew more rapidly and in greater numbers on F-G agar than on
322
FEELEY ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Macroscopic colony counts of LDB Philadelphia I from infected guinea pig spleen on F-G and MH-IH agars adjusted to various pH levels Medium
F-G agar
pHduof medium
7.31
3 oa
O 0
0b 0b 0b 0b 0b 0b 0 0 0
Colony counts after the following days of incubation: 4 5 6 7
o O 1 il 71 62 67 117 14 0 0 0
O O 1 14 82 71 101 145 86 0 0 0
O 0 1 14 82 71 140 159 138 100 0 0
il
O 0 1 25 98 78 171 168 163 123 0 0
O O 3 52 130 124 241 199 205 169 35 0
MH-IH agar
s6.42 0 0 0 0 0 0 6.50 0 0 0 0 1 1 6.58 0 0 0 0 17 17c 6.68 0 0 0 0 17 17c 6.80 0 0 0 0 0 0 6.90 0 0 0 0 1 1 7.00 0 0 0 0 1 1 -7.10 0 0 0 0 0 0 a Average of three plates rounded to nearest whole number. The inoculum consisted of a 10% tissue suspension diluted 1:10 in sterile phosphate-buffered saline. Each plate was inoculated with 0.05 ml of the 1:10 dilution. b Colonies were easily observable with a dissecting microscope. c A total of 30 to 50 suspect colonies were seen with a dissecting microscope.
TABLE 2. Growth of the LDB on MH agar supplemented with 1% hemoglobin and various constituents of IsoVitaleX Growth of LDB Strainsb IsoVitaleX chemical deleted or added
Concna (g/liter)
Adenine p-Aminobenzoic acid Cocarboxylase L-Cysteine hydrochloride L-Cystine Dextrose Diphosphopyridine nucleotide (oxidized) Ferric nitrate Glutamine hydrochloride Guanine hydrochloride Thiamine hydrochloride Vitamin B12
0.01 0.00013 0.001 0.0259 0.001 1.000 0.0025 0.0002 0.100 0.0003 0.00003 0.0001
Chemical deleted
Pi 4 3 4
0 4 4 4 4 4 4 4 3
P2 4 4 4
0 4 4 4 4 4 4 4 4
Chemical added
Fl 3
Pol 4
4
3
3 0 4 4
4
4 4
3 4 3 4
Pi
P2
0 0 0
0 0 0 4 0 0 0 0 0 0 0 0
0
4
4 4 4 4 4 4 4 4
0 0 0 0 0 0 0 0
Fl 0 0 0
Pol
0 0 0
4
4
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
All chemicals 0 0 4 0 4 4 0 4 a Concentration normally present in medium. b Growth rated at 6 days on a scale of 0 to 4. Philadelphia 1; P2, Philadelphia 2; Fl, Flint 1; Pol, Pontiac 1. C Medium contained all of IsoVitaleX chemicals except chemical indicated. d Medium supplemented only with IsoVitaleX chemical indicated. -
Pi,
MH-IH agar. Within 3 days, colonies could be observed on the F-G agar with a dissecting microscope, and by 4 days they were macroscopically visible as pinpoint colonies, which enlarged
with longer incubation (Table 1). The colonies of LDB characteristically appeared to have a cut-glass appearance when observed microscopically with the light illumination from one side
LEGIONNAIRES DISEASE MEDIA
VOL. 8, 1978
at a 100 angle. In areas of heavy growth of the LDB, especially in subcultures, a distinctive brownish darkening of the LDB colonies and surrounding agar occurred on both the F-G and MH-IH agars. In addition, growth of the LDB on F-G agar produced a fluorescent substance in the colonies and surrounding agar that could be detected by long-wave (366 nm) UV light. Studies of the composition of IsoVitaleX showed that LDB would not grow only when Lcysteine hydrochloride was deleted from the medium (Table 2). Increase of either IsoVitaleX or L-cysteine hydrochloride to two times normal concentrations in MH agar with hemoglobin enhanced the growth of the LDB. Higher concentrations of these chemicals did not increase growth. However, fluorescence and browning were influenced. Browning was maximum with the original concentrations of L-Cysteine hydrochloride and decreased with the increase to two times the original concentration. The opposite was true for fluorescence. Browning was stable, whereas fluorescence was maximum at about 4 days and decreased with time; 0.40% L-cysteine hydrochloride gave good browning and fluorescence. Soluble ferric pyrophosphate and ferric nitrate enhanced growth of the LDB better than did hemoglobin and the other iron-containing substances that were evaluated. Soluble ferric pyrophosphate stimulated the growth of the LDBorga organism sm better than did the discontinued
323
growth of the LDB from stock cultures as well as did the beef infusion and acidicase contained in the lot of MH agar used in these studies. Other peptones either supported poorer growth or did not support growth at all (Table 4). The organism grew best aerobically with 2.5% TABLE 4. Growthai LDB on F-G cystene-iron agar prepared wi' h var'oup Growth ofLDB strainsb Company Peptone P2 F1 Pol Pl Baltimore Bio-
Acidicase
logical Laboratory
Beef extract
0
0
0
0
Acidicase + beef 4
4
4
4
Biosated
4
4
4
4
2 0 1 0
0 0
Beef extract
2 0 0 0
0
1 0 1 0
Casamino Acids Bacto-peptone
0
0 ±
0 ±
0
±
Gelysate Myosate Phytone
2
Trypticase Difco Labora-
2
2
0
2
tories
no.2 Bacto-peptone no. 3 Neopeptone
3
3
3
3
0
Bacto-tryptose Bcotyts
i
0 3
0 4
3
0
Acidicase and beef extract were at the same concentrabsetermthan (Th dbeconTiT' tons as m MH agar. Other peptones were at 1% (wt/vol)
LB
fi h h f ui TT
LDB grew less well on MH agar supplemented concentrations. b Growth rated at 6 days on a scale of 0 to 4. ± =
