J. appl. Bact. 1976,40,213-216
SHO RT CO MMUNl C A T l 0NS Colony Morphology of Vibrio cholerae on SV medium C. A. SALLES, S. VOROS, E. C. MARBELL AND L. AMENUVOR Public Health Reference laboratory, Ministry of Health, P.O. Box 300, AccralGhana Received 15 May 1975 and accepted 29 October 1975
COLONY VARIANTS of Vibrio cholerae have been described (Pollitzer, 1959). Finkelstein 8z Gomez (1963) were able to correlate colony morphology with cultural characteristics (haemolysis). No correlation has been established between agglutinability of biotype and colonial morphology. Colonial variation is a practical problem in the characterization of V. cholerae strains by serology, bio- and phage-typing since the variants may have widely different characteristics. It is also important in primary isolation where the possibility of a mixed culture of two or more vibrios must be considered. We have observed that when vibrio cultures are plated on SV medium (described below) a great diversity of colonies may appear. These vary in size, shape, texture, colour and in the production of a red precipitate in the medium. To the aggregate of these characteristics the term morphotype is proposed and a classification of vibrio colonies into morphotypes is suggested.
Materials and Methods Organisms Vibrio cholerae biotype eltor and V.parahaemolyticus, freshly isolated in this laboratory were used. Also included were collection strains of V. cholerae biotype classical (Inaba 569B, Inaba 35, Ogawa 41); non-agglutinable (NAG) vibrios from Algeria, Brahein, Czechoslovakia, Ghana, Italy, Nigeria, Pakistan, Philippines, Sweden, Thailand, Taiwan; and V. parahaemolyticus, strains T-1, T-2, K-33, K-56, K-12 from Japan. The fresh isolates were obtained by placing rectal swabs in alkaline peptone water, incubating for 6 h at 31" and plating directly on to SV medium. The collection strains were kept in soft agar, grown in alkaline peptone water and plated on SV medium. Except where indicated all tests were done with smooth colonies. Collection cultures were derived from a single well-isolated smooth colony on nutrient agar after two subcultures to check purity and routine identification tests. Medium The SV medium contained (g/l): sodium citrate (Merck fur analyse) 10.0; NaZC03 (Merck fur analyse) 2.6; NaCl (Merck fur Analyse) 10.0; ferric citrate (Hopkins 12131
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and Williams) 10.0; ox bile (Oxoid) 8.0; yeast extract (Difco) 5.0; sucrose (BDH analytical) 20.0; agar (BDH) 15.0; pH 8.5. The medium was melted by steaming and cooled to 56". Twenty ml of human blood was added and the flask shaken to complete haemolysis. The medium was not autoclaved. Incubation Plates were incubated at 34" for 16 h and kept for 24 h at 25". Reading Morphotype classification was made with a stereomicroscope. Incident light was about 30" above the plane of the stage, and the lamp filament focused on the agar surface. The total magnification was 10 x . Notation Five main morphotypes were recognized. S series. Smooth ellipsoid discs, e.g.: SOR, opaque dull red; SOY, opaque uniform yellow; SO W Y , opaque white border and yellow centre; STY, translucent borders and yellow centre.
K series. Circinated, conical, e.g.: KOW, opaque white; KOY, opaque yellow; KOR, opaque with red centre; KTG, translucent, grey. C series. As in K series but smaller and more compact. Q series. Flat disc with large uniform coloured centre, white or translucent borders.
F series. Conical, mixed translucent and opaque material (fluffy) e.g. : FOG, opaque grey, FOY, opaque yellow; two other characters were noted: d character: a raised deep pigmented dot in the centre of the colony. V character: a red precipitate in the agar. Other morphotypes were seen on SV medium but their status is not clear at present. They seem to represent defective vibrios in various stages of roughness.
Results and Discussion Twenty-nine freshly isolated strains of V. cholerae eltor had S, 15 freshly isolated and 5 collection strains of V ,parahaemolyticus had K and 3 collection strains of V. cholerae classical had F morphotypes. From 23 collection cultures of NAG vibrios 10, 5, 5 and 3 cultures had morphotypes C, K, Q and S, respectively. Freshly isolated NAG vibrios from 2 batches of Volta clam (Egeria radiuta), 10 clams per batch, yielded mixed cultures of 4 morphotypes: COR V, COY, QV and Q. NAG vibrios from humans or water are extremely rare and freshly isolated strains were not available. It is thus possible to differentiate V. cholerae serogroup 01 from V.parahaemolyticus and perhaps from the 2 biotypes of V. cholerae serogroup 01. Since the number of classical cultures was small and from collections this point needs confirmation in areas where classical V. cholerae occurs. The 2 biotypes of V. cholerae serogroup 01
VIBRIO CHOLERAE ON SV MEDIUM
215
differ from the majority of NAG vibrio morphotypes and this makes it possible to differentiate in primary isolation the 2 types of vibrio in about 85 of cases. Certain old cultures of V. cholerae serogroup 01 and of NAG vibrios dissociate and produce on SV medium several morphotypes as shown in Table 1. The V character seems to correlate with changes in the Voges-Proskauer reaction. Plating rough cultures of V. cholerae serogroup 01 and NAG vibrios on SV medium showed that there is a correlation between the d character and roughness. The reverse is not true since rough colonies may be found without the d character. The dissociation process as shown on SV medium is not restricted to old cultures. After a few subcultures some eltor strains produce SV and later K morphotypes. In the process of isolating smooth classical strains for antigen preparation a new morphotype (T), smooth but with low antigenicity for rabbit was found. These atypical morphotypes TABLE1 Variants of Vibrio cholerae isolated on SV medium
Strain
Morphotypes found in SV medium
Observations
~
C/2318 (NAG)
C/410/74 (haba)
COR, V COG SOR, V SOY SOWY SOY FOG KOR, V
KTG KOY C/321/75 (NAG)
Q COR, V Qd
NAG,Haemolytic, Heiberg I, VPNAG,Haemolytic, Heiberg V, VP+ Serogroup 01, VPSerogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VPNAG,Heiberg V, VP+ NAG,Heiberg I, VP+ NAG,VP+ NAG,VPRough, VP+
were not seen in primary isolation (eltor) or after serial intraperitoneal passage in mice (classical). The formula of the SV medium presented here is one among several possible combinations based on the association human blood-bile-carbohydrate and may be modified for specific purposes. The addition of peptone makes the medium richer but the discrimination of certain morphotypes is affected. If the critical discrimination is not required a simplified SV medium may be obtained by adding 2% human blood to melted TCBS medium (Oxoid). We are grateful to Dr Y. Watanabe, World Health Organization, Geneva, to Dr J. Gallut of The Institute Pasteur, Paris; Dr S. Otatsume, Fukushima Medical School, Japan and Drs A. Furniss and J. Lee, Public Health Laboratory, Maidstone, England, for the supply of collection strains and other invaluable help. We acknowledge permission from the Director of Medical Services, Ministry of Health, Accra, to publish this note. 16
216
C . A. SALLES ET AL.
References FINKELSTEIN, R. A. & GOMEZ, C. Z . (1963). Comparison of methods for the rapid recognition of cholera vibrios. BuN. Wld Hlth Org. 28, 327. POLLITZER, R. (1959). Cholera. Monograph Series No. 43. Geneva: World Health Organization.
SHO RT CO MMUNl C A T l 0NS Colony Morphology of Vibrio cholerae on SV medium C. A. SALLES, S. VOROS, E. C. MARBELL AND L. AMENUVOR Public Health Reference laboratory, Ministry of Health, P.O. Box 300, AccralGhana Received 15 May 1975 and accepted 29 October 1975
COLONY VARIANTS of Vibrio cholerae have been described (Pollitzer, 1959). Finkelstein 8z Gomez (1963) were able to correlate colony morphology with cultural characteristics (haemolysis). No correlation has been established between agglutinability of biotype and colonial morphology. Colonial variation is a practical problem in the characterization of V. cholerae strains by serology, bio- and phage-typing since the variants may have widely different characteristics. It is also important in primary isolation where the possibility of a mixed culture of two or more vibrios must be considered. We have observed that when vibrio cultures are plated on SV medium (described below) a great diversity of colonies may appear. These vary in size, shape, texture, colour and in the production of a red precipitate in the medium. To the aggregate of these characteristics the term morphotype is proposed and a classification of vibrio colonies into morphotypes is suggested.
Materials and Methods Organisms Vibrio cholerae biotype eltor and V.parahaemolyticus, freshly isolated in this laboratory were used. Also included were collection strains of V. cholerae biotype classical (Inaba 569B, Inaba 35, Ogawa 41); non-agglutinable (NAG) vibrios from Algeria, Brahein, Czechoslovakia, Ghana, Italy, Nigeria, Pakistan, Philippines, Sweden, Thailand, Taiwan; and V. parahaemolyticus, strains T-1, T-2, K-33, K-56, K-12 from Japan. The fresh isolates were obtained by placing rectal swabs in alkaline peptone water, incubating for 6 h at 31" and plating directly on to SV medium. The collection strains were kept in soft agar, grown in alkaline peptone water and plated on SV medium. Except where indicated all tests were done with smooth colonies. Collection cultures were derived from a single well-isolated smooth colony on nutrient agar after two subcultures to check purity and routine identification tests. Medium The SV medium contained (g/l): sodium citrate (Merck fur analyse) 10.0; NaZC03 (Merck fur analyse) 2.6; NaCl (Merck fur Analyse) 10.0; ferric citrate (Hopkins 12131
214
C . A. SALLES ET AL.
and Williams) 10.0; ox bile (Oxoid) 8.0; yeast extract (Difco) 5.0; sucrose (BDH analytical) 20.0; agar (BDH) 15.0; pH 8.5. The medium was melted by steaming and cooled to 56". Twenty ml of human blood was added and the flask shaken to complete haemolysis. The medium was not autoclaved. Incubation Plates were incubated at 34" for 16 h and kept for 24 h at 25". Reading Morphotype classification was made with a stereomicroscope. Incident light was about 30" above the plane of the stage, and the lamp filament focused on the agar surface. The total magnification was 10 x . Notation Five main morphotypes were recognized. S series. Smooth ellipsoid discs, e.g.: SOR, opaque dull red; SOY, opaque uniform yellow; SO W Y , opaque white border and yellow centre; STY, translucent borders and yellow centre.
K series. Circinated, conical, e.g.: KOW, opaque white; KOY, opaque yellow; KOR, opaque with red centre; KTG, translucent, grey. C series. As in K series but smaller and more compact. Q series. Flat disc with large uniform coloured centre, white or translucent borders.
F series. Conical, mixed translucent and opaque material (fluffy) e.g. : FOG, opaque grey, FOY, opaque yellow; two other characters were noted: d character: a raised deep pigmented dot in the centre of the colony. V character: a red precipitate in the agar. Other morphotypes were seen on SV medium but their status is not clear at present. They seem to represent defective vibrios in various stages of roughness.
Results and Discussion Twenty-nine freshly isolated strains of V. cholerae eltor had S, 15 freshly isolated and 5 collection strains of V ,parahaemolyticus had K and 3 collection strains of V. cholerae classical had F morphotypes. From 23 collection cultures of NAG vibrios 10, 5, 5 and 3 cultures had morphotypes C, K, Q and S, respectively. Freshly isolated NAG vibrios from 2 batches of Volta clam (Egeria radiuta), 10 clams per batch, yielded mixed cultures of 4 morphotypes: COR V, COY, QV and Q. NAG vibrios from humans or water are extremely rare and freshly isolated strains were not available. It is thus possible to differentiate V. cholerae serogroup 01 from V.parahaemolyticus and perhaps from the 2 biotypes of V. cholerae serogroup 01. Since the number of classical cultures was small and from collections this point needs confirmation in areas where classical V. cholerae occurs. The 2 biotypes of V. cholerae serogroup 01
VIBRIO CHOLERAE ON SV MEDIUM
215
differ from the majority of NAG vibrio morphotypes and this makes it possible to differentiate in primary isolation the 2 types of vibrio in about 85 of cases. Certain old cultures of V. cholerae serogroup 01 and of NAG vibrios dissociate and produce on SV medium several morphotypes as shown in Table 1. The V character seems to correlate with changes in the Voges-Proskauer reaction. Plating rough cultures of V. cholerae serogroup 01 and NAG vibrios on SV medium showed that there is a correlation between the d character and roughness. The reverse is not true since rough colonies may be found without the d character. The dissociation process as shown on SV medium is not restricted to old cultures. After a few subcultures some eltor strains produce SV and later K morphotypes. In the process of isolating smooth classical strains for antigen preparation a new morphotype (T), smooth but with low antigenicity for rabbit was found. These atypical morphotypes TABLE1 Variants of Vibrio cholerae isolated on SV medium
Strain
Morphotypes found in SV medium
Observations
~
C/2318 (NAG)
C/410/74 (haba)
COR, V COG SOR, V SOY SOWY SOY FOG KOR, V
KTG KOY C/321/75 (NAG)
Q COR, V Qd
NAG,Haemolytic, Heiberg I, VPNAG,Haemolytic, Heiberg V, VP+ Serogroup 01, VPSerogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VP+ Serogroup 01, VPNAG,Heiberg V, VP+ NAG,Heiberg I, VP+ NAG,VP+ NAG,VPRough, VP+
were not seen in primary isolation (eltor) or after serial intraperitoneal passage in mice (classical). The formula of the SV medium presented here is one among several possible combinations based on the association human blood-bile-carbohydrate and may be modified for specific purposes. The addition of peptone makes the medium richer but the discrimination of certain morphotypes is affected. If the critical discrimination is not required a simplified SV medium may be obtained by adding 2% human blood to melted TCBS medium (Oxoid). We are grateful to Dr Y. Watanabe, World Health Organization, Geneva, to Dr J. Gallut of The Institute Pasteur, Paris; Dr S. Otatsume, Fukushima Medical School, Japan and Drs A. Furniss and J. Lee, Public Health Laboratory, Maidstone, England, for the supply of collection strains and other invaluable help. We acknowledge permission from the Director of Medical Services, Ministry of Health, Accra, to publish this note. 16
216
C . A. SALLES ET AL.
References FINKELSTEIN, R. A. & GOMEZ, C. Z . (1963). Comparison of methods for the rapid recognition of cholera vibrios. BuN. Wld Hlth Org. 28, 327. POLLITZER, R. (1959). Cholera. Monograph Series No. 43. Geneva: World Health Organization.
