J. clin. Path., 1975, 28, 33-36
Titres and cross reactions of commercial antisera for the capsular typing of Klebsiella species M. W. CASEWELL From the Department of Microbiology, St Thomas's Hospital Medical School, London
Ninety commercially supplied sera for the capsular typing of Klebsiella species were tested in a full chequerboard against 72 type strains. Only four of 144 homologous reactions could not be detected. Of the 6336 possible heterologous reactions, 91 actual cross reactions were found. All positive reactions were titrated and working dilutions deduced. This provides essential information for the successful typing of unknown strains with these sera.
SYNOPSIS
An earlier report (Casewell, 1972) described a technique for demonstrating the quellung reaction with Klebsiella species using the then newly available commercial (Difco) antisera, and suggested that these sera provided a much needed practical means of typing klebsiella strains. Although the manufacturer's instructions suggest that the sera may be used at dilutions of 1: 8 or 1: 16 we have found that homologous reactions could not always be detected at these dilutions. Furthermore, if used undiluted, several sera gave cross reactions which cause confusion when typing unknown strains. Henriksen (1954) has demonstrated differences in the reactions of klebsiella typing sera produced in different laboratories, and subsequently Edwards Received for publication 23 September 1974.
and Fife (1955) emphasized the importance of fully evaluating the cross reactions and titres of any new set of klebsiella typing sera. Accordingly, each of the 18 pooled and 72 specific Difco antisera was tested against 72 klebsiella capsular type strains to determine cross reactions, titres, and appropriate working dilutions. Materials and Methods The detailed methods for culture, suspension, and demonstration of positive quellung reactions are described elsewhere (Casewell, 1972). Known capsular type strains were cultured on Worfel-Ferguson agar for 18 hours at 20°C. Suspensions, somewhat lighter than previously described, were made of each culture to give approximately 107 organisms/ml
Pool No.
Containing Specific Antisera
Type Strains Giving Positive Quellung Reactions (Titres
1 2 3 4 5 6 7 8 9 10
1 (32), 4 (4), 8 (64), 13 (16),
12 13 14 15 16 17
3, 24 6, 7 10 25 15, 16 19, 20 21, 22, 23, 11 26, 27, 28, 30 12, 29, 40, 41, 31, 32, 43, 44 33, 34, 35, 36 37, 38, 39,42 45, 46 47 48, 49, 50, 51 52, 53, 54, 55 56, 57, 58, 59 60, 61, 62, 63 64, 65, 66, 67, 68, 69
18
70, 71, 72
11
1, 2 4. 5, 8 9, 13, 14, 17, 18,
-,
21 26 12 31 33
in
Parentheses)
2 (16), 3 (8), 24 (8), 13 (16), 68 (16). 69 (4) 5 (4), 6 (2), 7 (2), 1 (4) 9 (8), 10 (8), 25 (16), 7 (4) 14 (8), 15 (8), 16 (2) 18 (8), -, 20 (4), 44 (2) (8), 22 (4). 23 (8), 11 (2), 37 (2), 41 (4) (8), 27 (8), 28 (32), 30 (2), 46 (2), 69 (2) (4), 29 (8), 40 (8), 41 (4) (4), 32 (8), 43 (4), 44 (16) (2), 34 (8), 35 (4), (8), 38 (8), 39 (2), 42 (8), 22 (32) (2), 46 (16), 47 (2), 27 (4), 28 (2) (4), 49 (2), 50 (4), 51 (2) (2), 53 (4), 54 (2), 55 (2), 17 (2), 47 (2) (8), 57 (8), 58 (2), 59 (8) (8!, 61 (2), 62 (8), 63 (2), 10 (4) (4), 65 (4), 66 (8), 67 (16), 68 (8), 69 (8), 3 (8), 14 (8), 26 (2),
37 45 48 52 56 60 64 30 (2)
70(16), 71 (2), 72 (4)
Table I Quellung reactions and titres for 18 pooled sera tested against 72 type strains 33
34 Specific Serum
M. W. Casewell Type Strains Giving Positive Quellung Reactions (Titres in Parentheses)'
Chosen Working Dilution
Type Strains Giving Posirive Reactions with Diluted Serum
2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 26 27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
62
63 64 65 66 67 68 69 70 71 73
1 (4) 2 (32), 13 (2), 69 (4) 3 (8), 68 (4) 4 (64), 29 (2) 5 (2) 6 (4) 7 (16), 10 (2), 56 (4) 8 (8) 9 (32) 10 (8), 7 (4) 1 1(32), 21 (1 6), 26 (2), 12 (8), 29 (8), 41 (16), 13 (16), 2 (2), 30 (2) 14 (16), 64 (8) 15 (64) 16 (16) 17 (128), 47 (2) 18 (64), 44 (16), 60 (2) 19 (16) 20 (2) 21 (64), 11 (8), 26 (2) 22 (8), 23 (4), 26 (2), 23 (32) 41 (8) 24 (32) 25 (2) 26 (8) 28 (32) -, 28 (64), 8 (4), 46 (4) 29 (16), 12 (8), 42 (8) 30(16), 13 (8), 59 (2) 31 (64) 60 (4) 32 (16) 33(16) 11 (2) 21 (8), 34 (32) 35 (4), 34 (4) 36 (8) 37 (32), 22 (2) 38 (16), 41 (4) 39 (4), 40 (2) 40 (16), 17 (2) 41 (16), 12 (4) 42 (32) 12 (8), 19 (4), 43 (4) 44 (32), 18 (8) 45 (4) 46 (16), 5 (2), 6 (4), 47 (32), 53 (2) 48 (128), 17 (4) 49 (2), 48 (8) 50 (2) 51 (64) 55 (4) 52 (2) 53 (16), 15 (4), 17 (4), 54 (2) 55 (16) 56 (32) 57 (16) 58 (8) 59 (8) 60 (32) 61 (8), 7 (4). 10 (4) 62 (16) 63 (8) 64 (16), 14 (16), 65 (4) 65 (16) 66 (8), 14 (4), 64 (4) 67 (16) 68 (16), 3 (8) 69 (16), 2 (8), 26 (2), 70 (16), 72 (2) 71 (64) 72 (16)
33 (4) 42 (2)
37 (8), 41 (4)
69 (2) 35 (2)
29 (16)
27 (8), 28 (8)
47 (8), 62 (2)
30 (4)
Neat 1:4 Neat 1:4 Neat Neat 1:4 1 2 1:4 1:2 1:8 1:2 1:4 1:4 1:8 4 1:16 1:8 1:2 Neat 1:8 Neat 1:4 1:4 Neat Neat Neat 1:8 1 :4 1:2 1:8 1:4 1:4 1:4 Neat Neat 1:8 1:2 1:2 1:2 1:2 1:4 Neat 1-8 Neat 1:4 1:2 1:16 Neat Neat 1:4 Neaat 1:2 Neat 1:2 1:4 1:2 Neat 1:2 1:2 1:2 1:2 Neat 1:4 1:2 1:2 1:2 1 4 1:2 1:2 1:4 1:2
2
3, 68 4 5 6 7 8 9
10, 7 11, 21 12, 29 41 13 14, 64 15 16 17 18, 44 19 20 21
22, 23, 26, 37, 41 23, 41 24 25 26 -, 28 28 29, 12 42 30, 13 31 32 33, 21 34 35, 34 36 37 38, 41 39 40 41, 12 42, 12, 29 43 44 45 46, 27, 28 47 48 49, 48 50 51 52 53, 15, 17. 47 54 55 56 57 58 59 60 61, 7, 10 62 63 64, 14 65 66, 14, o4 67
68, 3 69, 2, 30 70 71 72
Table II Quellung reactions, titres, and working dilutions for 72 specific sera tested against 72 type strains 'In contrast to the working dilutions, the 'titres' include an extra dilution factor of 2 to allow for the volume of the
suspension of orginismns.
Titres and cross reactions of commercial antisera for the capsular typing of Klebsiella species or 1-5 organisms per high-power field with a lO/1/-30 objective. A small loopful of suspension was mixed with an equal volume of serum and examined, under a coverslip, by oil-immersion phase-contrast microscopy. Positive quellung reactions were clearly indicated by a dark line delineating the periphery of the capsule. Each of the 18 pooled and 72 specific sera was tested against 72 capsular type strains, a total of 6480 reactions. Each positive reaction was then titrated by a micromethod. Using one row of a microtitre plate (Sterilin M25 A) doubling dilutions of serum were made in sterile water, pH 7 0 to give a final volume in each well of 004 ml. An equal volume of klebsiella suspension (approximately 107 organisms/ml in 10% formol phosphate-buffered saline, pH 7-3) was added to each dilution and mixed. After approximately 10 minutes one loopful of the contents of each well was examined in the usual way for a positive quellung reaction. The endpoint was taken as the reciprocal of the greatest dilution at which a positive reaction could still be clearly detected. To allow for the volume of added suspension the final titre was taken as twice the endpoint.
Results POOLED SERA
Table I shows the homologous reactions, cross reactions, and titres of the 18 pools tested against 72 type strains. The only strains that did not give a positive reaction with their homologous undiluted pools were types 17, 19, and 36. In general the homologous titres with pooled sera were not high and it seemed that when typing, the pools should be used undiluted in order to avoid missing positive reactions with poorly capsulated strains. SPECIFIC SERA
The quellung titres obtained by testing each of the 72 specific sera against 72 known type strains are summarized in table II. Only specific serum 27 failed to give any reaction with its homologous type strain. Several sera cross reacted with heterologous strains, but the titre of such reactions was often lower than the homologous reaction. Thus it was often possible to determine a working dilution at which the cross reactions were diluted out whilst leaving a reasonably strong homologous reaction. The chosen working dilutions, and remaining cross reactions at these dilutions, are also shown in table II.
Discussion This set of typing
sera is
adequate for typing
un-
35
known strains of Klebsiella species, although it is necessary to be aware of the cross reactions and employ appropriate working dilutions. There are few sera that give false-negative results when tested against homologous type strains. The failure of type strains, 17, 19, and 36 to give positive reactions with their homologous pools necessitates the addition of the corresponding specific sera to the set of pooled sera for the screening of unknown strains. The only specific serum failing to give a detectable quellung reaction was serum 27. Although the specificity of these sera is not ideal, when viewed in the context of the total number of possible reactions, cross reactions are relatively infrequent. Of 1224 possible heterologous pool reactions, only 20 were positive and only 71 out of 5112 possible heterologous reactions with specific sera were positive. Kauffmann (1949) and 0rskov (1 955a) have pointed out that titres obtained for capsular reactions are sensitive to antigen excess. If care was taken to control, as far as possible, the density of suspension, the titres obtained here were reproducible, with an occasional twofold difference. Serum with different batch numbers gave the same results on several occasions. The working dilutions effectively eliminated about one half of the specific serum cross reactions, and also reduced the amount, and thus cost, of serum used. Unknown strains can usually be typed by using a set of undiluted pooled sera and a set of specific sera at working dilution. Where cross reactions give an indeterminate answer the mosaic of cross reactions obtained with undiluted sera permits the strain to be matched with a known type strain. Where two-way cross reactions occur (for example, type strains 3 and 68) titrations may occasionally have to be performed on the unknown strain. Rarely, unknown strains may not match precisely any of the known type strains, and it is known that intermediate types do occur (Orskov, 1955a, 1955b; Edwards and Fife, 1955). We have used these sera for typing several thousand strains in an epidemiological survey, with a typing rate of about 90%. Two experienced workers can type about 10 strains per hour, at a cost in serum of about 20p per strain. I am indebted to Professor Ian Phillips for his valuable advice and encouragement, to Dr Ida 0rskov for providing the type strains, and to Mrs Valerie Dawes and Miss Usha Lakhani for helping with particularly painstaking technical work. This research forms part of a project supported by the Medical Research Council.
36 6. W. Casewell References Casewell, M. W. (1972). Experiences in the use of commercial antisera for the typing of klebsiella species. J. clin. Path., 25, 734-737. Edwards, P. R., and Fife, M. A. (1955). Studies on the klebsiellaaerobacter group of bacteria. J. Bact., 70, 382-390. Henriksen, S. D. (1954). Studies on the klebsiella group (Kauffmann). 1. Sero-types of a collection of strains from human sources and from water. Acta pat/i. niicrobiol. scand.. 34, 249-258.
Kauffmann, F. (1949). On the serology of the klebsiella group. Acta path. microbiol. scand., 26, 381-406. 0rskov, I. (1955a). Serological investigations in the klebsiella group. 3. Occurrence of klebsiella strains in the faeces of normal infants. Acta path. nmicrobiol. scand., 36, 461-470. 0rskov, I. (1955b). Serological investigations in the klebsiella group. 2. Occurrence of klebsiella in sputa. Acta path. mticrobiol. scanid., 36, 454-460.
The December 1974 Issue THE DECEMBER 1974 ISSUE CONTAINS THE FOLLOWING PAPERS
Quality control in cervical cytology 0. A. N. HUSAIN, E. BLANCHE BUTLER, D. M. D. EVANS, J. ELIZABETH MACGREGOR, AND R. YULE
The pre-albumin fraction: A useful parameter in the interpretation of routine protein electrophoresis R. I. HARRIS AND J. KOHN
nOSiS
An evaluation of low voltage counterimmunoelectrophoresis for the detection of hepatitis-B antigen (HB Ag) E. 0. CAUL AND P. C. ROBERTS
Comparative cytogenetical and morphological studies
A case of acute measles meningoencephalitis with virus isolation D. R. PURDHAM AND P. F. BATTY
Observer variation and quality control of cytodiagD. M. D. EVANS, GLENYS SHELLEY, B. CLEARY, AND YVONNE BALDWIN
in ovarian dysgenesis
MAGDOLNA GAAL, J. LASZLO,
AND P. BOSZE
A functioning black adenoma of the adrenal cortex: A clinico-pathological entity J. W. VISSER, J. K. BOEIJINGA, AND C. V.D. MEER
Bacterial flora of the appendix fossa in appendicitis and postoperative wound infection D. A. LEIGH, KATE SIMMONS, AND EDELGARD NORMAN
Progress towards agreement in reports of antibiotic sensitivity R. H. GEORGE
Tietze's disease H. U. CAMERON AND V. L. FORNASIER
Nodular, non-cirrhotic liver associated with portal hypertension in a patient with rheumatoid arthritis
Implementation of a British computer system for laboratory data handling C. H. GRAY, A. D. HIRST,
M. HARRIS, R. M. RASH, AND I. W. DYMOCK
P. J. N. HOWORTH, T. P. LOCKE, B. MELLOR, AND M. WALTER
Leukaemic reticuloendotheliosis: A morphological and immunological study of four cases D. G.
Training and career appointments in the pathological sciences in the United Kingdom D. N.
HAEGERT, J. C. CAWLEY, R. D. COLLINS, R. J. FLEMANS, AND J. L. SMITH
BARON
Morphological abnormalities in lymphocyte mitochondria associated with iron-deficiency anaemia
Micro method for manual analysis of true glucose in plasma without deproteinization B. B. BAUMINGER
J. H. JARVIS AND A. JACOBS
The effect of age upon the coagulation system P. J. HAMILTON, MARY ALLARDYCE, D. OGSTON, AUDREY A. DAWSON, AND A. S. DOUGLAS
Technical method Assessment of amoebiasis: serodiagnosis by immunofluorescence with lyophilized entamoebae J. C. COX AND R. C. NAIRN
The elution of 99Tcm from red cells and its effect on red-cell volume measurement A. FERRANT, S. M.
Book reviews
LEWIS, AND L. SZUR
Notices
Copies are still available and may be obtained from the PUBLISHING MANAGER, BRITISH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, LONDON, WCIH 9JR, price £1 -05
Titres and cross reactions of commercial antisera for the capsular typing of Klebsiella species M. W. CASEWELL From the Department of Microbiology, St Thomas's Hospital Medical School, London
Ninety commercially supplied sera for the capsular typing of Klebsiella species were tested in a full chequerboard against 72 type strains. Only four of 144 homologous reactions could not be detected. Of the 6336 possible heterologous reactions, 91 actual cross reactions were found. All positive reactions were titrated and working dilutions deduced. This provides essential information for the successful typing of unknown strains with these sera.
SYNOPSIS
An earlier report (Casewell, 1972) described a technique for demonstrating the quellung reaction with Klebsiella species using the then newly available commercial (Difco) antisera, and suggested that these sera provided a much needed practical means of typing klebsiella strains. Although the manufacturer's instructions suggest that the sera may be used at dilutions of 1: 8 or 1: 16 we have found that homologous reactions could not always be detected at these dilutions. Furthermore, if used undiluted, several sera gave cross reactions which cause confusion when typing unknown strains. Henriksen (1954) has demonstrated differences in the reactions of klebsiella typing sera produced in different laboratories, and subsequently Edwards Received for publication 23 September 1974.
and Fife (1955) emphasized the importance of fully evaluating the cross reactions and titres of any new set of klebsiella typing sera. Accordingly, each of the 18 pooled and 72 specific Difco antisera was tested against 72 klebsiella capsular type strains to determine cross reactions, titres, and appropriate working dilutions. Materials and Methods The detailed methods for culture, suspension, and demonstration of positive quellung reactions are described elsewhere (Casewell, 1972). Known capsular type strains were cultured on Worfel-Ferguson agar for 18 hours at 20°C. Suspensions, somewhat lighter than previously described, were made of each culture to give approximately 107 organisms/ml
Pool No.
Containing Specific Antisera
Type Strains Giving Positive Quellung Reactions (Titres
1 2 3 4 5 6 7 8 9 10
1 (32), 4 (4), 8 (64), 13 (16),
12 13 14 15 16 17
3, 24 6, 7 10 25 15, 16 19, 20 21, 22, 23, 11 26, 27, 28, 30 12, 29, 40, 41, 31, 32, 43, 44 33, 34, 35, 36 37, 38, 39,42 45, 46 47 48, 49, 50, 51 52, 53, 54, 55 56, 57, 58, 59 60, 61, 62, 63 64, 65, 66, 67, 68, 69
18
70, 71, 72
11
1, 2 4. 5, 8 9, 13, 14, 17, 18,
-,
21 26 12 31 33
in
Parentheses)
2 (16), 3 (8), 24 (8), 13 (16), 68 (16). 69 (4) 5 (4), 6 (2), 7 (2), 1 (4) 9 (8), 10 (8), 25 (16), 7 (4) 14 (8), 15 (8), 16 (2) 18 (8), -, 20 (4), 44 (2) (8), 22 (4). 23 (8), 11 (2), 37 (2), 41 (4) (8), 27 (8), 28 (32), 30 (2), 46 (2), 69 (2) (4), 29 (8), 40 (8), 41 (4) (4), 32 (8), 43 (4), 44 (16) (2), 34 (8), 35 (4), (8), 38 (8), 39 (2), 42 (8), 22 (32) (2), 46 (16), 47 (2), 27 (4), 28 (2) (4), 49 (2), 50 (4), 51 (2) (2), 53 (4), 54 (2), 55 (2), 17 (2), 47 (2) (8), 57 (8), 58 (2), 59 (8) (8!, 61 (2), 62 (8), 63 (2), 10 (4) (4), 65 (4), 66 (8), 67 (16), 68 (8), 69 (8), 3 (8), 14 (8), 26 (2),
37 45 48 52 56 60 64 30 (2)
70(16), 71 (2), 72 (4)
Table I Quellung reactions and titres for 18 pooled sera tested against 72 type strains 33
34 Specific Serum
M. W. Casewell Type Strains Giving Positive Quellung Reactions (Titres in Parentheses)'
Chosen Working Dilution
Type Strains Giving Posirive Reactions with Diluted Serum
2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 26 27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
62
63 64 65 66 67 68 69 70 71 73
1 (4) 2 (32), 13 (2), 69 (4) 3 (8), 68 (4) 4 (64), 29 (2) 5 (2) 6 (4) 7 (16), 10 (2), 56 (4) 8 (8) 9 (32) 10 (8), 7 (4) 1 1(32), 21 (1 6), 26 (2), 12 (8), 29 (8), 41 (16), 13 (16), 2 (2), 30 (2) 14 (16), 64 (8) 15 (64) 16 (16) 17 (128), 47 (2) 18 (64), 44 (16), 60 (2) 19 (16) 20 (2) 21 (64), 11 (8), 26 (2) 22 (8), 23 (4), 26 (2), 23 (32) 41 (8) 24 (32) 25 (2) 26 (8) 28 (32) -, 28 (64), 8 (4), 46 (4) 29 (16), 12 (8), 42 (8) 30(16), 13 (8), 59 (2) 31 (64) 60 (4) 32 (16) 33(16) 11 (2) 21 (8), 34 (32) 35 (4), 34 (4) 36 (8) 37 (32), 22 (2) 38 (16), 41 (4) 39 (4), 40 (2) 40 (16), 17 (2) 41 (16), 12 (4) 42 (32) 12 (8), 19 (4), 43 (4) 44 (32), 18 (8) 45 (4) 46 (16), 5 (2), 6 (4), 47 (32), 53 (2) 48 (128), 17 (4) 49 (2), 48 (8) 50 (2) 51 (64) 55 (4) 52 (2) 53 (16), 15 (4), 17 (4), 54 (2) 55 (16) 56 (32) 57 (16) 58 (8) 59 (8) 60 (32) 61 (8), 7 (4). 10 (4) 62 (16) 63 (8) 64 (16), 14 (16), 65 (4) 65 (16) 66 (8), 14 (4), 64 (4) 67 (16) 68 (16), 3 (8) 69 (16), 2 (8), 26 (2), 70 (16), 72 (2) 71 (64) 72 (16)
33 (4) 42 (2)
37 (8), 41 (4)
69 (2) 35 (2)
29 (16)
27 (8), 28 (8)
47 (8), 62 (2)
30 (4)
Neat 1:4 Neat 1:4 Neat Neat 1:4 1 2 1:4 1:2 1:8 1:2 1:4 1:4 1:8 4 1:16 1:8 1:2 Neat 1:8 Neat 1:4 1:4 Neat Neat Neat 1:8 1 :4 1:2 1:8 1:4 1:4 1:4 Neat Neat 1:8 1:2 1:2 1:2 1:2 1:4 Neat 1-8 Neat 1:4 1:2 1:16 Neat Neat 1:4 Neaat 1:2 Neat 1:2 1:4 1:2 Neat 1:2 1:2 1:2 1:2 Neat 1:4 1:2 1:2 1:2 1 4 1:2 1:2 1:4 1:2
2
3, 68 4 5 6 7 8 9
10, 7 11, 21 12, 29 41 13 14, 64 15 16 17 18, 44 19 20 21
22, 23, 26, 37, 41 23, 41 24 25 26 -, 28 28 29, 12 42 30, 13 31 32 33, 21 34 35, 34 36 37 38, 41 39 40 41, 12 42, 12, 29 43 44 45 46, 27, 28 47 48 49, 48 50 51 52 53, 15, 17. 47 54 55 56 57 58 59 60 61, 7, 10 62 63 64, 14 65 66, 14, o4 67
68, 3 69, 2, 30 70 71 72
Table II Quellung reactions, titres, and working dilutions for 72 specific sera tested against 72 type strains 'In contrast to the working dilutions, the 'titres' include an extra dilution factor of 2 to allow for the volume of the
suspension of orginismns.
Titres and cross reactions of commercial antisera for the capsular typing of Klebsiella species or 1-5 organisms per high-power field with a lO/1/-30 objective. A small loopful of suspension was mixed with an equal volume of serum and examined, under a coverslip, by oil-immersion phase-contrast microscopy. Positive quellung reactions were clearly indicated by a dark line delineating the periphery of the capsule. Each of the 18 pooled and 72 specific sera was tested against 72 capsular type strains, a total of 6480 reactions. Each positive reaction was then titrated by a micromethod. Using one row of a microtitre plate (Sterilin M25 A) doubling dilutions of serum were made in sterile water, pH 7 0 to give a final volume in each well of 004 ml. An equal volume of klebsiella suspension (approximately 107 organisms/ml in 10% formol phosphate-buffered saline, pH 7-3) was added to each dilution and mixed. After approximately 10 minutes one loopful of the contents of each well was examined in the usual way for a positive quellung reaction. The endpoint was taken as the reciprocal of the greatest dilution at which a positive reaction could still be clearly detected. To allow for the volume of added suspension the final titre was taken as twice the endpoint.
Results POOLED SERA
Table I shows the homologous reactions, cross reactions, and titres of the 18 pools tested against 72 type strains. The only strains that did not give a positive reaction with their homologous undiluted pools were types 17, 19, and 36. In general the homologous titres with pooled sera were not high and it seemed that when typing, the pools should be used undiluted in order to avoid missing positive reactions with poorly capsulated strains. SPECIFIC SERA
The quellung titres obtained by testing each of the 72 specific sera against 72 known type strains are summarized in table II. Only specific serum 27 failed to give any reaction with its homologous type strain. Several sera cross reacted with heterologous strains, but the titre of such reactions was often lower than the homologous reaction. Thus it was often possible to determine a working dilution at which the cross reactions were diluted out whilst leaving a reasonably strong homologous reaction. The chosen working dilutions, and remaining cross reactions at these dilutions, are also shown in table II.
Discussion This set of typing
sera is
adequate for typing
un-
35
known strains of Klebsiella species, although it is necessary to be aware of the cross reactions and employ appropriate working dilutions. There are few sera that give false-negative results when tested against homologous type strains. The failure of type strains, 17, 19, and 36 to give positive reactions with their homologous pools necessitates the addition of the corresponding specific sera to the set of pooled sera for the screening of unknown strains. The only specific serum failing to give a detectable quellung reaction was serum 27. Although the specificity of these sera is not ideal, when viewed in the context of the total number of possible reactions, cross reactions are relatively infrequent. Of 1224 possible heterologous pool reactions, only 20 were positive and only 71 out of 5112 possible heterologous reactions with specific sera were positive. Kauffmann (1949) and 0rskov (1 955a) have pointed out that titres obtained for capsular reactions are sensitive to antigen excess. If care was taken to control, as far as possible, the density of suspension, the titres obtained here were reproducible, with an occasional twofold difference. Serum with different batch numbers gave the same results on several occasions. The working dilutions effectively eliminated about one half of the specific serum cross reactions, and also reduced the amount, and thus cost, of serum used. Unknown strains can usually be typed by using a set of undiluted pooled sera and a set of specific sera at working dilution. Where cross reactions give an indeterminate answer the mosaic of cross reactions obtained with undiluted sera permits the strain to be matched with a known type strain. Where two-way cross reactions occur (for example, type strains 3 and 68) titrations may occasionally have to be performed on the unknown strain. Rarely, unknown strains may not match precisely any of the known type strains, and it is known that intermediate types do occur (Orskov, 1955a, 1955b; Edwards and Fife, 1955). We have used these sera for typing several thousand strains in an epidemiological survey, with a typing rate of about 90%. Two experienced workers can type about 10 strains per hour, at a cost in serum of about 20p per strain. I am indebted to Professor Ian Phillips for his valuable advice and encouragement, to Dr Ida 0rskov for providing the type strains, and to Mrs Valerie Dawes and Miss Usha Lakhani for helping with particularly painstaking technical work. This research forms part of a project supported by the Medical Research Council.
36 6. W. Casewell References Casewell, M. W. (1972). Experiences in the use of commercial antisera for the typing of klebsiella species. J. clin. Path., 25, 734-737. Edwards, P. R., and Fife, M. A. (1955). Studies on the klebsiellaaerobacter group of bacteria. J. Bact., 70, 382-390. Henriksen, S. D. (1954). Studies on the klebsiella group (Kauffmann). 1. Sero-types of a collection of strains from human sources and from water. Acta pat/i. niicrobiol. scand.. 34, 249-258.
Kauffmann, F. (1949). On the serology of the klebsiella group. Acta path. microbiol. scand., 26, 381-406. 0rskov, I. (1955a). Serological investigations in the klebsiella group. 3. Occurrence of klebsiella strains in the faeces of normal infants. Acta path. nmicrobiol. scand., 36, 461-470. 0rskov, I. (1955b). Serological investigations in the klebsiella group. 2. Occurrence of klebsiella in sputa. Acta path. mticrobiol. scanid., 36, 454-460.
The December 1974 Issue THE DECEMBER 1974 ISSUE CONTAINS THE FOLLOWING PAPERS
Quality control in cervical cytology 0. A. N. HUSAIN, E. BLANCHE BUTLER, D. M. D. EVANS, J. ELIZABETH MACGREGOR, AND R. YULE
The pre-albumin fraction: A useful parameter in the interpretation of routine protein electrophoresis R. I. HARRIS AND J. KOHN
nOSiS
An evaluation of low voltage counterimmunoelectrophoresis for the detection of hepatitis-B antigen (HB Ag) E. 0. CAUL AND P. C. ROBERTS
Comparative cytogenetical and morphological studies
A case of acute measles meningoencephalitis with virus isolation D. R. PURDHAM AND P. F. BATTY
Observer variation and quality control of cytodiagD. M. D. EVANS, GLENYS SHELLEY, B. CLEARY, AND YVONNE BALDWIN
in ovarian dysgenesis
MAGDOLNA GAAL, J. LASZLO,
AND P. BOSZE
A functioning black adenoma of the adrenal cortex: A clinico-pathological entity J. W. VISSER, J. K. BOEIJINGA, AND C. V.D. MEER
Bacterial flora of the appendix fossa in appendicitis and postoperative wound infection D. A. LEIGH, KATE SIMMONS, AND EDELGARD NORMAN
Progress towards agreement in reports of antibiotic sensitivity R. H. GEORGE
Tietze's disease H. U. CAMERON AND V. L. FORNASIER
Nodular, non-cirrhotic liver associated with portal hypertension in a patient with rheumatoid arthritis
Implementation of a British computer system for laboratory data handling C. H. GRAY, A. D. HIRST,
M. HARRIS, R. M. RASH, AND I. W. DYMOCK
P. J. N. HOWORTH, T. P. LOCKE, B. MELLOR, AND M. WALTER
Leukaemic reticuloendotheliosis: A morphological and immunological study of four cases D. G.
Training and career appointments in the pathological sciences in the United Kingdom D. N.
HAEGERT, J. C. CAWLEY, R. D. COLLINS, R. J. FLEMANS, AND J. L. SMITH
BARON
Morphological abnormalities in lymphocyte mitochondria associated with iron-deficiency anaemia
Micro method for manual analysis of true glucose in plasma without deproteinization B. B. BAUMINGER
J. H. JARVIS AND A. JACOBS
The effect of age upon the coagulation system P. J. HAMILTON, MARY ALLARDYCE, D. OGSTON, AUDREY A. DAWSON, AND A. S. DOUGLAS
Technical method Assessment of amoebiasis: serodiagnosis by immunofluorescence with lyophilized entamoebae J. C. COX AND R. C. NAIRN
The elution of 99Tcm from red cells and its effect on red-cell volume measurement A. FERRANT, S. M.
Book reviews
LEWIS, AND L. SZUR
Notices
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