JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1978, p. 181-1880095-1137/78/0008-0181$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 8, No. 2

Printed in U.S.A.

Serological Classification of Pseudomonas aeruginosa by a

Slide Agglutination TestHIDEO KUSAMA

Division of Laboratories and Research, New York State Department ofHealth, Albany, New York 12201

Received for publication 8 June 1978

Serological classification ofPseudomonas aeruginosa by the slide agglutinationtest with live organisms was studied, based on the O antigen schema adopted bythe international expert panel sponsored by the Subcommittee on Pseudomonasand Related Organisms of the International Committee on Systematic Bacteri-ology. The typing results obtained by the slide test with well-absorbed O sera

were identical to those obtained by the conventional tube agglutination test withautoclaved orgnsn. Most 0 antigens occur singly; but 02, 05, and 016 occur

in four combinations. Antigens 013 and 014 are closely related, as are 07 and 08,and it would be convenient to classify organisms possessing these antigenscollectively as 07,8 and 013,14.

Before 1970 a variety of serological classifica-tion systems for Pseudomonas aeruginosa wasused in different countries, such as those re-ported by Habs (4), Verder and Evans (15),Meitert (9), and Lânyi (8). To establish an inter-nationally acceptable serotyping schema, an in-ternational expert panel was organized in 1970under the auspices of the Subcommittee onPseudomonas and Related Organisms of theInternational Committee on Systematic Bacte-riology. The panel agreed to adopt 12 heat-stablesomatic antigens from Habs' schema and 5 moreantigens from other typing systems; these weredesignated by arabic numerals 1 through 17 (P.V. Liu, Chairman; personal communication).Many workers performed the slide agglutina-

tion test with live organisms (2, 6, 8-10, 13, 16,17) for the serotyping of P. aeruginosa becauseof its practical advantage over the conventionaltube agglutination test with autoclaved orga-isms. However, only LAnyi has reported de-

tailed procedures for the slide test (8), and heused his own serotyping system. Homma (5) andKodama and Ishimoto (7), who studied serotyp-ing of P. aeruginosa extensively with the slidetest, used their own classification system.We have investigated the performance of the

slide O agglutination test under the expert panelschema. Some basic findings which may contrib-ute to a better application of the new schema toPseudomonas serotyping are reported here.

MATERIALS AND MEMHODSBacteria. Seventeen strains of P. aeruginosa, se-

lected by the expert panel as representing specificheat-stable somatic antigens, were received from P. V.Liu of the University of Louisville, Louisville, Ky.

(Table 1). The first 12 are the original Habs' strains,which Liu received from M. Véron, Institut Pasteur,Paris. These reference strains were used to preparerabbit antisera and absorb cross-agglutinins. For con-venience, I shall refer to these strains by their Oantigens, rather than by their original designations.To evaluate the prepared typing sera, we used 98strains of P. aerugnsa isolated from patients inAlbany Medical Center Hoepital in 1964-65, lyophi-lized and maintained since then in our laboratory.These strains are designated by B followed by four-digit numbers.Some strains representative of Verder and Evans'

system (15) and Véron's subtype strains for types 2and 5 (16) were also examined. The former were sentto this laboratory by N. B. McCullough, NationalInstitute of Allergy and Infectious Diseases, Bethesda,Md.; the latter were sent by M. Véron.

Preparation ofimmune sera. Bacteria grown onTrypticase soy agar (Baltimore Biological Laboratory)for 18 to 20 h at 30°C were suspended in saline, heatedin a boiling-water bath for 2.5 h, washed with sterilesaline three times, and used to immunize rabbits afterthe density was properly adjusted. O antisera preparedby the procedure of Verder and Evans (15) were notpotent enough to be used for the slide test. Satisfactorysera were obtained by injecting a dense bacterial sus-pension in a shorter period of time, as described byMikkelsen (11, 12). A suspension containing 5 x 1o0<bacteria per ml was given intravenously in increasingdoses of 1.0, 2.0, and 4.0 ml at 4-day intervals. If therabbit had lost more than 10% of its initial weight bythe second and third injections, the dosage was re-duced. Three to four days after the third injection, therabbit was bled out. Sera produced by this methodgenerally gave strong reactions at 1:10 dilution orhigher in the slide test. The disadvantage, as men-tioned by Mikkelsen (11), was occasional los of arabbit due to the heavy dosage.A third method, tried later, was the use of the

chrome vaccine of Ando et al. (1). To a saline suspen-181

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J. CLIN. MICROBIOL.

TABLE 1. Reference strains of P. aeruginosa usedfor antiserum production

Anti- Original designation Origingen

0i 5933 (Véron) Habs, group 102 5934 (Véron) Habs, group 203 5935 (Véron) Habs, group 304 5936 (Véron) Habs, group 405 5937 (Véron) Habs, group 506 5939 (Véron) Habs, group 607 5938 (Véron) Habs, group 708 5940 (Véron) Habs, group 809 5941 (Véron) Habs, group 9010 5943 (Véron) Habs, group 10011 5944 (Véron) Habs, group 1i012 5945 (Véron) Habs, group 12013 6092 (Véron) Sandvik, group II014 1M-1 (Verder & Evans) Verder & Evans,

group V015 170022 (Lényi) Lànyi, group 12016 170003 (Lânyi) Lânyi, group 3017 Type 10 (Meitert) Meitert, type 10

sion containing 5 x 1010 bacteria per ml was added 1%thiomerosal solution at a final concentration of 1:5,000.Then a 1% solution of chromium potassium sulfatewhich had been filtered through a 450-nm membranefilter (Millipore Corp.) was added at 1/10 the volumeof the bacterial suspension. The mixture was kept at37°C for 3 to 4 days. Bacteria collected by centrifuga-tion were washed once with saline and resuspended tothe original volume in saline containing 0.01% thiom-erosal. Rabbits tolerated well this detoxified vaccine.Of the various immunization schedules tested, two 5-ml intravenous injections 5 days apart, followed bybleeding out after 5 days, evoked quick and highresponses. The titers of O sera obtained were compa-rable to those obtained by the method of Mikkelsen.Absorption of cross-reacting agglutinins. Bac-

teria grown for 20 h on Trypticase soy agar at 300Cwere suspended in saline, heated in a boiling-waterbath for 1 h, washed with saline three times, and usedfor absorption. The pellet from the last centrifugationwas mixed thoroughly with undiluted or diluted serum.Depending on the amount of cross-agglutinin, the ratioby volume of the pellet to the serum (adjusted for thedilution factor) varied from 1:1 to 1:4.Tube and slide O agglutination tests. For both

the tube and slide O agglutination tests, the O antigensconsisted of cultures grown on Trypticase soy agar at30°C for 20 h; for the tube test, they were also heatedat 120°C for 1 h. Otherwise, the tube test was per-

formed by the method of Verder and Evans (15).For the slide test, the surface of a glass slide was

divided into 8 to 10 compartments with a wax pencil.The pattern used by Finkelstein and Mukerjee (3) waspractical for quick mixing and conserving the reagents.One drop of a properly diluted antiserum was placedin each compartment. A small amount of bacterialgrowth was placed near the drop and mixed evenlywith the antiserum. The slide was rocked by handcontinuously, and the reaction was read at the end of1 min. An arbitrary scale from ++++ (a strong agglu-

tination appearing within a few seconds) to + (weak,but definitive agglutination by the end of 1 min) wasused. As observed by Wahba (17), loose, viscous ag-gregations occasionally appeared quickly but did notprogress further, giving an appearance different fromfull agglutination. They were disregarded because inruns with autoclaved cultures these reactions oftendisappeared and left negative results.

RESULTS

Cross-reactions in the tube and slide ag-glutination tests. Unabsorbed O sera, partic-ularly those produced by the method of Mikkel-sen (11, 12) or with the chrome vaccine (1),showed cross-reactions against all the auto-claved reference strains in the tube test due tothe presence of a common component, aspointed out by Lànyi (8). For instance, an anti-03 serum prepared by the method of Mikkelsenshowed various degrees of reaction against allthe autoclaved antigens (Table 2). After thisserum was absorbed with the 09 strain at apellet-to-serum ratio of 1:4, all the cross-reac-tions disappeared, resulting in the production ofa specific anti-03 serum. Absorption of this se-rum with only the 013 strain at a pellet-to-serumratio of 1:1, however, could not produce a specificserum in the tube test.When tested with live organisms on slides, the

unabsorbed serum did not react with manystrains at even a 1:5 dilution. In particular, anantigen like the 01 strain, which reactedstrongly with the unabsorbed serum. in the tubetest, caused no reaction in the slide test. Someof the antisera, such as anti-O11, anti-012, andanti-015, showed no cross-reaction at all onslides and could be used as specific O sera with-out absorption. In contract to the tube test,absorption of the anti-03 serum with the 09 or013 strain produced a specific O serum for theslide test.As long as potent antisera were employed,

there were no difficulties in carrying out theslide test with live organisms. The followingobservations were all made by the slide test.Occurrence of multiple antigens in a sin-

gle strain. Cross-reactions between 02 and 05organisms, between 07 and 08 organisms, andbetween their counterparts in other classifica-tion systems, such as those of Verder and Evans(15) and Linyi (8), have been frequently dis-cussed (2, 4, 6, 8, 12, 15-17). The complexity of02 and 05 antigens was analyzed by Véron (16),who introduced classification by substrains intogroups 02 and 05. Since a close relationshipbetween 02, 05, and 016 organisms has beenpointed out by Homma (5) and Terada et al.(14), we analyzed the antigenic makeup of thesethree strains.

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SEROTYPING OF P. AERUGINOSA 183

TABLE 2. Agglutinin titers of unabsorbed and absorbed anti-03 sera, tested with autoclaved (tube) and live(slide) P. aeruginosa

Titer of anti-03, K2400 serum

Strain Tube test Slide test

Unabsorbed Absorbed with Absorbed with Unabsorbed Absorbed Abworbed09 013 with 09 with 013

Q1 1,280 ba40 _b02 320 - - - - -

03 2,560 1,280 2,560 320 160 16004 160 - - 20 - -

05 160 - - -

06 640 - 20 5 -07 160 - - - -08 80 - - - -09 5,120 - 160 160 -010 640 - 20 10 -011 80 - - - -012 80 - - -

013 640 - - 10 -014 160 - - 20 -015 80 - - - -016 80 - - -

017 5,120 - 40 320 -

a Negative at 1:20 dilution.b Negative at 1:5 dilution.

An anti-02 serum, in the unabsorbed state,showed reactions against the 02, 05, and 016strains and also against the 09 and 017 strainsin the slide test (Table 3). The cross-reactionagainst the 09 and 017 strains was frequentlyobserved in other typing sera and was easilyremoved by absorbing sera with the 09 strain.This serum, after absorption with the 09 and016 strains, retained only the agglutinin againstthe homologous 02 antigen. Likewise, an anti-05 serum, which reacted with the 02, 05, and016 strains in the unabsorbed state, reacted onlyagainst the homologous 05 antigen after absorp-tion with the 016 strain.

In contrast, an anti-016 serum, which reactedwith the 02, 05, and 016 strains in the unab-sorbed state, completely lost the reactivityagainst the 02 and 05 strains and almost com-pletely against the 016 strain after absorptionwith the 02 strain. Absorption of this serumwith the 05 strain removed all reactivity againstthe 02 and 05 strains, leaving low reactivityagainst the 016 strain.These results demonstrate clearly that 016

antigen is common to the 02, 05, and 016strains, but it is a single major antigen in the016 strain. Besides 016 antigen, the referencestrains for 02 and 05 possess their own specific0 antigens. Factor sera for 02 and 05 can beprepared by proper absorption (Table 3). Anunabsorbed anti-016 serum can be used as afactor serum for 016.

With this set of factor sera it is possible todetermine the antigenic formulas of isolateswhose antigens are related to 02, 05, and 016.In the dozen isolates we have tested thus far,these three antigens occur in four combinations(see also Table 8). Our stock cultures happen toinclude Verder and Evans' group I and X strains(15) and Véron's substrains for 02 and 05 (16).Their antigenic formulas were easily determined(Table 3).Other striking cross-reactions constantly seen

in the reference strains were between the 07 and08 strains and between the 013 and 014 strains.Reciprocal absorption of each antiserum re-duced the homologous titer sharply in the slidetest, particularly with the anti-07 and anti-013sera (Table 4). For practical purposes it wouldbe advisable to classify these related organismscollectively and express their antigenic formulasas 07,8 and 013,14.Occurrence of cross-reacting agglutinins

which are not detected by the heterologousreference strains. If an antiserum shows astrong reaction against the homologous strainand no reaction against any of the 16 heterolo-gous reference strains at a proper serum dilution,it can generally be used as a specific 0 antiserum.But this is not always the case. An example isan anti-09 serum (K500) which, after being ab-sorbed with the reference 013 strain, seemed agood candidate for specific 09 antiserum in theslide test (Table 5). When this antiserum was

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TABLE 3. Antigenic analyses of P. aeruginosa strains related to antigens 02, 05, and 016 by the slideagglutination test

Anti-02 (J2512) 1:5 Anti-05 (K594) 1:10 Anti-016 (J5990) 1:5

Source and designation Absorbed Ab- Ab- Saline Antigenicof strain Unab- wth 09 & U - Frbd | bd formula

sorbed ]- 016 sorbed j-with 016 sorbed with 02 ~with 05 ____

Reference (Liu)5933:015934:025935:035936:045937:055939:065938:075940:085941:095943:0105944:0115945:0126092:0131M-1:014170022:015170003:016Meitert 10:017

IsolatesB1480B1759B1763B1766B1795B1807B1843B1852B1899

Verder and Evans2243:1a,lb,lc359:1a,lc,ldG2312:10,la

Véron623,0:2b624,0:2ab625,0:5d626,0:5cd

+++

+++

+++

++

++

++

++

+++

+++

++

+

02,16

05,16

016

015,1601601605,1602,1602,5,1602,5,1605,1605,16

02,1601605,16

02,5,1602,1605,1605,16

used to examine strains derived from patientisolates, however, some showed moderate tostrong reactions. Three 011 strains (B1774,B1845, and B1846) reacted to anti-09 as well asanti-011 serum. When the anti-09 serum wasabsorbed with both the 013 strain and B1846,these isolates showed no reaction. Similarly, thecross-agglutinin of an anti-07,8 serum to someisolates was easily eliminated by absorption witha cross-reacting isolate (Table 7).Pattern of wide-range cross-reactions.

Many of the anti-0 sera cross-reacted againstheterologous reference strains in the slide test.As expected, the extent and degree of cross-reactions varied greatly from one antiserum to

another. To formulate absorption schedules forpreparing the specific anti-0 sera, it was firstnecessary to examine each antiserum for reac-tions against all the reference strains. In thisprocess we observed a definite pattern. Cross-reactions among the 03, 06, 09, 010, 013, 014,and 017 strains occurred frequently; an antise-rum to any of these strains may not cross-reactwith all the others, but any cross-reactions whichoccur are limited to these strains (Table 6). The01, 04, and 011 strains cross-reacted less fre-quently. An antiserum (NW116) obtained byimmunizing rabbits with a mixture of equalamounts of 06 and 013 antigens exhibitedstrong to moderate reactions to all seven strains

-1 L-11 1

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SEROTYPING OF P. AERUGINOSA

TABLE 4. Agglutinin titers ofanti-07 and -08 sera and anti-013 and -014 sera ofP. aeruginosa in theslide test, before and after reciprocal absorptions

Anti-07 (NW127) Anti-08 (K3901) Anti-013 (J228) Anti-014 (K3929)

Strain Unab- Absorbed Unab- Absorbed Unab- Absorbed Unab- Abeorbed Saline

sorbed with 08 sorbed with07 sorbed with09 sorbed with 013&09 ~~& 01401 _02 - _ _ _ _ _ _ _03 - - - - - - - -

04 _ _ _05 - - - - - - - -

06 - - - _ 5 _ _ _07 320 20 40 - - - - -08 80 - 160 40 - - - -09 - - - - 80 - _ - _010 - - - - - - 5 - -

011 - - _ - - - -_012 - - - - - - - - _013 - - - - 320 10 40 - -014 - - - - 320 - 320 40 -015 - _ - - - - - -

016 - - - - -

017 - - 10 - 40 - - - -

a Negative at 1:5.

TABLE 5. Presence of a common antigen in the 09strain and some isolates ofP. aeruginosa as

revealed by the slide agglutination testAnti-09, K500 (1:10)

Strain Abo Absorbed Salineibt8ho013 with 013 &wt03 B1846

Reference01 - - -

02 - - -

03 - - -

04 - - -

05 - - -

06 - - -

07 - - -

08 - - -

09 ++++ ++++ _010 - _ _

011 - - _

012 _ _ _013 - _ _014 _ _ _015 - - -016 - -017 - - _

IsolatesB1763 (NTa) ++++ - -

B1769 (NT) ++++ - -B1774 (011) ++ - -B1845 (011) ++ - -B1846 (011) +++ - -a NT, Nontypable.

listed above. Few cross-reactions were seen withthe remaining strains.Use of polyvalent sera for routine exam-

nation. In routine examination of isolates withthe slide test, it is advisable to use polyvalentsera for the initial screening. However, whenmonovalent sera were pooled, the cumulativeeffect of minor agglutinin components, whichhad been neglected in each individual serum,often produced noticeable cross-reactions, ashad been pointed out by Lényi (8). Sometimesit was difficult to absorb out this kind of cross-reacting agglutinin. A pool (polyvalent serumI) of antisera against 03, 06, 09, 010, 013,014, and 017 often solved this problem, mini-miqing the necessity for absorption of the pooledsera.

Anti-02,5,16 and anti-07,8, which occasion-aily showed a few cross-reactions with otherrefence strains, comprise another group (poly-valent serum II). Anti-02,5,16 can best be pro-duced by immunizing rabbits with an 02,5,16strain (such as B1807, Table 3). Anti-07,8 canbe produced by immunization with either the07 or 08 strain; it should react strongly againstboth the 07 and 08 strains.The remaiIing antisera, anti-01, -04, -011,

-012, and -015, comprise another group (poly-valent serum 1).

Polyvalent, bivalent, and monovalent sera tobe used for routine serotyping are listed in Table7. Cross-reacting strains encountered in all theantisera prepared thus far are also given. Anindividual serum may not exhibit cross-reactionsagainst all the strains in the list. Although theselection of absorbing strains is largely depend-ent on the pattern of cross-reactions in the in-dividual antiserum, the strains which have so far

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TABLE 6. Distribution of cross-reacting antigen(s) among some reference strains of P. aeruginosa asrevealed by the slide agglutination test"

Anti-03 Anti-06 Anti-09 Anti-010 Anti-013 Anti-017 Anti-06,13 SalineStan (K2400) (K5425) (K5001) (K617) (J228) (K584) (NW116) n

01 - _ _ _ _ _ _ _

02 - - _ _ _ - _ _03 ++++ - + _ _ _ ++ _04 ++ - _ _ _ _ +05 - - - - - - - -

06 - ++++ - - - ++++ -

07 - - _ _ _ _ _ _08 - - - - - - - -

09 ++++ - ++++ +++ ++ ++ +++010 + ++ - ++++ - ++++011 - _ _ _ _ _ _ _012 - _ _ _ _013 ++ + - - ++++ - ++++014 + ++++ - + ++++ ++++015 - - - - - _ - _016 - - - - - - - -

017 +++ + ++ ++++ ++ ++++ +++

a All antisera were diluted to 1:10.

TABLE 7. Preparation of absorbed antisera for the routine serotyping of P. aeruginosa

.ntiseru.mmn .ts Strains against which cross-re- Strains which may be used forAntiserum Immunmuing strains actions may occur absorption

Polyvalent IAnti-01,4,11i 01,04 and 011 09,13,14,17 09+ 014Anti-012,15a 012 and 015 03,4,14,17 0

Polyvalent IIAnti-02,5,16 B1807(02,5,16) 09,17 09 + B1380(011)Anti-07,8 07 or 08 06,9,11,17

Polyvalent IIIAnti-03,9- 03 and 09 07,8,11,13,14,17Anti-06,13" 06 and 013 03,4,9,10,14,17 j 04 + B1846(Q11)Anti-0i0,17a 010 and 017 03,6,9,13,14 J

Anti-Qi 01 03,6,9,17 09Anti-02 02 05,9,16,17 09 + 016Anti-03 03 04,6,9,10,13,14,17 09 or 013 or 06 + 09Anti-04 04 03,6,9,11,13,14,15,17 09 + 011 + 013Anti-05 05 02,16 016Anti-06 06 03,9,10,13,14,17 010 + 013Anti-07,8 07 or 08 06,9,11,17 B1380(011)Anti-09 09 03,4,6,13,14,17 013 + B1846(011)Anti-O10 010 03,6,9,13,14,17 09Anti-Oll 011 03,4,17b NoneAnti-012 012 03,4b NoneAnti-013,14 013 or 014 03,4,6,9,10,17 09Anti-015 015 014,17b NoneAnti-016 016 02,5 NoneAnti-017 017 09 09

a Rabbits were immunized with a mixture of cultures.b Cross-reactions were low-grade, and no absorption was necessary.

been most successful for absorption are also isolated from patients in 1964-65 were examinedlisted. by the slide test. Our results (Table 8) resemble

Classification of isolated strains. Strains those of C. 0. Brokopp, R. Gomes-Lus, and J. J.

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SEROTYPING OF P. AERUGINOSA 187

Farmer III (Abstr. Annu. Meet. Am. Soc. Micro-biol. 1977, C105, p. 53), who typed recent isolatesby using the schema of the expert panel.

DISCUSSIONTwo major principles helped to shape the

proposed international schema. First, Habs'schema (4) has priority over any other systemcurrently available. This is one reason why ithas been adopted by the expert panel. Second,the panel decided to define the somatic antigens,not serotypes or serogroups. They did anticipatethe occurrence of strains with multiple antigens,but such strains can be easily identified by an-tigenic formulas (P. V. Liu, personal communi-cation). There is no need for creation of a newtype or group or for changes in designation, suchas Homma's recently proposed system with analphabetic designation (5).Our findings confirm that no major change is

necessary in the panel's schema at this time.What is needed is experience in applying theschema to actual serotyping practice.

Since Habs (4) reported that heating orga-nisms at 120°C for 2 h increases agglutinability,autoclaved organisms have been utilized for thetube test in many laboratories, but this time-consuming procedure has been an obstacle towide acceptance of serotyping. Moreover, ourresults (Table 2), as well as those reported byLànyi (8), clearly indicate that autoclaving un-covers an antigen which is common to all P.aeruginosa strains and is in a cryptic state inliving organisms. When autoclaved organismsare used in the agglutination test, the agglutinin

TABLE 8. Classification of 98 strains of P.aeruginosa isolated from patients in 1964-65

according to the proposed international schemaAntigenicformula

0102,1602,5,16030405,160607,809010011012013,14015016017Nontypable

No. ofstrains

1313515624o

31

17o

0

o

2O

8

13.31.03.15.1

15.36.1

24.5o

3.11.0

17.3o

o

o

2.0o

8.2

to the common antigen must be absorbed fromantisera. This procedure is not necessary whenlive organisms are used. Since using nonauto-claved organisms in the tube test is more dan-gerous and far more time-consuming than theslide test with live organisms, we recommendthe slide test for routine laboratory use.

Besides the common antigen, there are othercross-reacting antigens which are distributedamong a few heterologous strains. Antigens ofthis type participating in the tube test may notalways participate in the slide test, and viceversa. It is imperative that any set of antiserafor serotyping be checked as to whether it canbe used for the slide test, for the tube test, or forboth.To prepare specific O antisera, it was some-

times necessary to absorb the antisera withcross-reacting isolates. At the same time,properly absorbed O factor sera can reveal theexistence of multiple antigens in a single strain.Antigens 02, 05, and 016 were found to occurin four combinations. Homma (5) and Terada etal. (14) proposed a single group for all strainsassociated with these related antigens, but theuse of antigenic formulas seems more logical.Since these antigens are among those most fre-quently encountered in many countries (2, 4, 8,12, 15-17), a finer fingerprinting may help theepidemiological survey.

Antigens 013 and 014 are closely related, asare 07 and 08. We suggest that these antigensbe expressed collectively as 07,8 and 013,14,rather than creating new groups for them asproposed by Homma (5) and Terada et al. (14).It might be possible to analyze the patterns ofthese antigens, as we did with 02, 05, and 016,if enough isolates become available.When carefully absorbed sera with high po-

tencies are used for serotyping of isolates, theslide test with live organisms and the tube testwith autoclaved organisms have thus far givenidentical results. The isolates which could notbe typed by the slide test remained untyped bythe tube test. The simple, reliable slide tech-nique should be adopted as the routine proce-dure in clinical laboratories. Pyocine typing orphage typing is valuable as a supplementaryprocedure when a finer fingerprinting of strainsis needed.

ACKNOWLEDGMENTThe technical assistance of Dorothy M. Bolles is gratefully

acknowledged.

LITERATURE CITED1. Ando, K., H. Shimojo, and I. Tadokoro. 1952. A new

method of preparing bacterial vaccines by the use of

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188 KUSAMA J. CLIN. MICROBIOL.

chrome-salt (detoxified vaccine-chrome vaccine). Jpn.J. Exp. Med. 22:485-492.

2. Bergan, T. 1973. Epidemiological markers for Pseudom-onas aeruginosa. 1. Serogrouping, pyocine typing-andtheir interrelations. Acta Pathol. Microbiol. Scand.Sect. B 81:70-80.

3. Finkelstein, R. A., and S. Mukerjee. 1963. A rapidmethod for differentiating V. cholerae and El Torvibrios. Proc. Soc. Exp. Biol. Med. 112:355-359.

4. Habs, I. 1957. Untersuchungen uber die 0-Antigene vonPseudomonas aeruginosa. Z. Hyg. 144:218-228.

5. Homma, J. Y. 1976. A new antigenic schema and live-cellslide-agglutination procedure for the infrasubspecific,serologic classification of Pseudomonas aeruginosa.Jpn. J. Exp. Med. 46:329-336.

6. Kleinmaier, H. 1957. Die O-Gruppenbestimmung vonPseudomonas-Stâmmen mittels Objektrager-Aggluti-nation. Zentralbl. Bakteriol. Parasitenkd. Infektionskr.Hyg. Abt. 1 Orig. 170:570-583.

7. Kodama, IL, and M. Ishimoto. 1976. Comparison of testtyping sera for Pseudomonas aeruginosa. Jpn. J. Exp.Med. 46:383-391.

8. Lényi, B. 1966/67. Serological properties ofPseudomonasaeruginosa. I. Group-specific somatic antigens. ActaMicrobiol. Acad. Sci. Hung. 13:295-318.

9. Meitert, T. 1964. Contribution à l'étude de la structureantigénique des B. pyocyaniques (Pseudomonasaeruginosa). II. Individualisation des groupes sérolo-

giques au moyen des antigènes O. Arch. Roum. Pathol.Exp. Microbiol. 23:678-688.

10. Meitert, T., and E. Meitert. 1960. Contribution à l'étudede la structure antigénique des B. pyocyaniques (Pseu-domonas aeruginosa). I. Emploi des réactionsd'agglutination pour l'étude de 181 souches. Arch.Roum. Pathol. Exp. Microbiol. 19:623-634.

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