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New Media for the Isolation of Vibrio cholerae
Zbl. Bakt. Hyg., I.Abt. Orig. A 247, 71-73 (1980)
Department of Microbiology and Parasitology, Faculty of Medicine, University of Ankara
New Media for the Isolation of Vibrio cholerae Ein neuer Nahrboden zur Isolierung von Vibrio cholerae K. OZSAN and F. MERCANGOZ
Abstract The new media are recommended for the isolation of cholera vibrios. The medium for Vibrio El Tor contains only starch, gelatin polymyxin B and natrium chloride and has no nutrient or inhibitor substances such as peptone, meat extract, yeast extract, blood, natrium taurocholate and bile salts. The medium for Vibrio cholerae contains starch, gelatin, natrium taurocholate and potassium tellurite, but has no peptone, meat extract, yeast extract or polymyxin B.
Zusammenfassung In der vorliegenden Arbeit wird von Versuchen mit zwei neuen Nahrboden berichtet, welche fiit die Isolierung von Vibrio cholerae besonders geeignet sind. Der Nahrboden fiir Vibrio EI Tor enthalt nur Gelatine, Starke, Polymyxin B und Kochsalz und keinen Nahrstoff oder Inhibitor wie Pep ton, Hefeextrakt, Fleischextrakt oder Blut, sowie auch nicht Natriumtaurocholat oder Salze der Gallensaure. Der Nahrboden fiir Vibrio cholerae enthalt Gelatine, Starke, Kaliumtellurit, Natrium taurocholicum, aber nicht Pepton, Hefeextrakt, Fleischextrakt oder Polymyxin B. The epidemics of El Tor vibrios have recently become a major problem for all parts of the world. In the Middle-East the situation is particularly critical in view of the ongoing wars in this area and the multitude of people making pilgrimage to Mecca. These epidemics have led to increased efforts toward isolation in the cholera laboratories during the past two decades, and new media have been introduced by some research workers (1,2,3,4). We are proposing two selective media:
Medium 1: Starch . . . . . Gelatin (Difco) Natrium chloride Agar-agar . . . .
10 g 30 g 5g 20 g
72
K. Ozsan and F. Mercangoz
Polymyxin B Distilled water .
50 Y g/ml 1 liter
Final pH: 8.5 Autoclave at 110°C for 15 min
Medium 2: Starch . . . . . Gelatin . . . . . Natrium chloride Agar-agar . . . . Natrium taurocholate. Distilled water. . . .
109 30 g Sg 20 g Sg 1 liter
Final pH: 8.5 Autoclave for 10 min in 110°C, cool to 4S-50 °c and add: 2.S ml Potassium tellurite sol. 1% Dispense the medium into sterile petri dishes and conserve at refrigerator. Observations Cultivation of Vibrio El Tor on medium 1 for 12-1S hours at 37°C gave rise to creamy-white colonies having a diameter about 1-2 mm surrounded with a clear zone and a zone of opacity, whereas E. coli, Alcaligenes and pathogenic colon bacteria did not grow within 24 hours or even 48 hours, except Proteus which does not produce any zone of clearing On medium 2, the visible colonies can be seen within 10-12 hours having black centers surrounded with clear zone, a zone of opacity. The vibrios change starch into sugar and then produce acid, while colon organisms and pathogenic colon bacteria cause no further action. The effect on starch can be detected by a drop of lugol solution on one of the colonies. This causes the vibrio colony and the surrounded area to become yellow, while the orthe side of the medium develops a dark blue color. The zone of opacity at the surrounded area of the vibrio colonies is a result of gelatinase activity of the organism. Discussion The method of cultivating cholera suspect stools on solid media was originally recommended by Koch (1884), and he described in great detail the growth appearance of Vibrio cholerae on the gelatin plate. Subsequent workers have taken advantage of the fact that Vibrio cholerae develops a characteristic halo (3). As early as 1901 Eijkman recorded that cholera and cholera-like vibrios in contrast to E. coli, produce a halo around their colonies when grown on agar plates containing rice or arrowroot-starch. Media containing starch have been recommended for selective cultivation of Vibrio cholerae by many authors (S). Comparative studies made by different authors have shown that the media containing polymyxin B (SO yg/ml) can separate Vibrio el Tor from Vibrio cholerae, while Vibrio El Tor thrives well in this media, Vibrio cholerae does not (2).
New Media for the Isolation of Vibrio cholerae
73
We are recommending medium I for the isolation of Vibrio EI Tor and medium two for Vibrio cholerae. The first medium contains only starch, gelatin, polymyxin B, and natrium chloride and has not any nutritient or inhibitor substances such as peptone, meat extract, yeast extract blood, natrium taurocholate or bile salts. The second medium contains starch, gelatin, natrium taurocholate and potassium tellurite, but has no peptone, meat extract, yeast extract, blood or polymyxin B. The studies with artificially infected stools and field studies with stools of pilgrims at quarantine have shown that these media can be recommended for the isolation of cholera vibrios. While cholera and El Tor vibrios invariably grow well, other organisms such as E. coli and pathogenic colon bacteria did not grow within 24 hours or even 48 hours, except Proteus bacteria which do not produce any zone of clearing. The slide agglutination tests with the vibrio growing on these media gave always good results. References 1.
N.: Vibrio cholerae'nin izolasyonu i<;in yeni besiyeri. Turk. Ij. tecr. Biyo!. Derg, 26 (1966) 144-146 2. Gancarosa, E. J.: Differentiation between Vibrio cholerae and Vibrio cholerae biotype el tor by polymyxin B disc test. Comparative result with T. C.B. 5., Monsur's MullerHinton and Nutritient agar media. Bul!. WId Hlth Org. 36 (1967) 987-990 3. Monsur, H. A.: Bacterial diagnosis of cholera under field conditions. Bul!. WId Hlth Org.28 (1963) 387-389 4. Qzsan, K., E. Mercangoz: Kolera vibryonu izolmaninda yeni besiyerleri uzerinde <;ali~ malar. Turk. Ij. tecr. Biyo!. Derg. 33 (1973) 21-26 5. Poliitzer, R.: Cholera. WId Hlth Org., Palais des Nations, Geneva (1959) Alkl~,
Prof. Dr. Kemal Qzsan, A. O. TIp Fakultesi, Mikrobiyoloji ve Parazitoloji Kursusu, Morfoloji Binasl, Slhhlye, Ankara, Tiirkei
Department of Microbiology and Parasitology, Faculty of Medicine, University of Ankara
New Media for the Isolation of Vibrio cholerae Ein neuer Nahrboden zur Isolierung von Vibrio cholerae K. OZSAN and F. MERCANGOZ
Abstract The new media are recommended for the isolation of cholera vibrios. The medium for Vibrio El Tor contains only starch, gelatin polymyxin B and natrium chloride and has no nutrient or inhibitor substances such as peptone, meat extract, yeast extract, blood, natrium taurocholate and bile salts. The medium for Vibrio cholerae contains starch, gelatin, natrium taurocholate and potassium tellurite, but has no peptone, meat extract, yeast extract or polymyxin B.
Zusammenfassung In der vorliegenden Arbeit wird von Versuchen mit zwei neuen Nahrboden berichtet, welche fiit die Isolierung von Vibrio cholerae besonders geeignet sind. Der Nahrboden fiir Vibrio EI Tor enthalt nur Gelatine, Starke, Polymyxin B und Kochsalz und keinen Nahrstoff oder Inhibitor wie Pep ton, Hefeextrakt, Fleischextrakt oder Blut, sowie auch nicht Natriumtaurocholat oder Salze der Gallensaure. Der Nahrboden fiir Vibrio cholerae enthalt Gelatine, Starke, Kaliumtellurit, Natrium taurocholicum, aber nicht Pepton, Hefeextrakt, Fleischextrakt oder Polymyxin B. The epidemics of El Tor vibrios have recently become a major problem for all parts of the world. In the Middle-East the situation is particularly critical in view of the ongoing wars in this area and the multitude of people making pilgrimage to Mecca. These epidemics have led to increased efforts toward isolation in the cholera laboratories during the past two decades, and new media have been introduced by some research workers (1,2,3,4). We are proposing two selective media:
Medium 1: Starch . . . . . Gelatin (Difco) Natrium chloride Agar-agar . . . .
10 g 30 g 5g 20 g
72
K. Ozsan and F. Mercangoz
Polymyxin B Distilled water .
50 Y g/ml 1 liter
Final pH: 8.5 Autoclave at 110°C for 15 min
Medium 2: Starch . . . . . Gelatin . . . . . Natrium chloride Agar-agar . . . . Natrium taurocholate. Distilled water. . . .
109 30 g Sg 20 g Sg 1 liter
Final pH: 8.5 Autoclave for 10 min in 110°C, cool to 4S-50 °c and add: 2.S ml Potassium tellurite sol. 1% Dispense the medium into sterile petri dishes and conserve at refrigerator. Observations Cultivation of Vibrio El Tor on medium 1 for 12-1S hours at 37°C gave rise to creamy-white colonies having a diameter about 1-2 mm surrounded with a clear zone and a zone of opacity, whereas E. coli, Alcaligenes and pathogenic colon bacteria did not grow within 24 hours or even 48 hours, except Proteus which does not produce any zone of clearing On medium 2, the visible colonies can be seen within 10-12 hours having black centers surrounded with clear zone, a zone of opacity. The vibrios change starch into sugar and then produce acid, while colon organisms and pathogenic colon bacteria cause no further action. The effect on starch can be detected by a drop of lugol solution on one of the colonies. This causes the vibrio colony and the surrounded area to become yellow, while the orthe side of the medium develops a dark blue color. The zone of opacity at the surrounded area of the vibrio colonies is a result of gelatinase activity of the organism. Discussion The method of cultivating cholera suspect stools on solid media was originally recommended by Koch (1884), and he described in great detail the growth appearance of Vibrio cholerae on the gelatin plate. Subsequent workers have taken advantage of the fact that Vibrio cholerae develops a characteristic halo (3). As early as 1901 Eijkman recorded that cholera and cholera-like vibrios in contrast to E. coli, produce a halo around their colonies when grown on agar plates containing rice or arrowroot-starch. Media containing starch have been recommended for selective cultivation of Vibrio cholerae by many authors (S). Comparative studies made by different authors have shown that the media containing polymyxin B (SO yg/ml) can separate Vibrio el Tor from Vibrio cholerae, while Vibrio El Tor thrives well in this media, Vibrio cholerae does not (2).
New Media for the Isolation of Vibrio cholerae
73
We are recommending medium I for the isolation of Vibrio EI Tor and medium two for Vibrio cholerae. The first medium contains only starch, gelatin, polymyxin B, and natrium chloride and has not any nutritient or inhibitor substances such as peptone, meat extract, yeast extract blood, natrium taurocholate or bile salts. The second medium contains starch, gelatin, natrium taurocholate and potassium tellurite, but has no peptone, meat extract, yeast extract, blood or polymyxin B. The studies with artificially infected stools and field studies with stools of pilgrims at quarantine have shown that these media can be recommended for the isolation of cholera vibrios. While cholera and El Tor vibrios invariably grow well, other organisms such as E. coli and pathogenic colon bacteria did not grow within 24 hours or even 48 hours, except Proteus bacteria which do not produce any zone of clearing. The slide agglutination tests with the vibrio growing on these media gave always good results. References 1.
N.: Vibrio cholerae'nin izolasyonu i<;in yeni besiyeri. Turk. Ij. tecr. Biyo!. Derg, 26 (1966) 144-146 2. Gancarosa, E. J.: Differentiation between Vibrio cholerae and Vibrio cholerae biotype el tor by polymyxin B disc test. Comparative result with T. C.B. 5., Monsur's MullerHinton and Nutritient agar media. Bul!. WId Hlth Org. 36 (1967) 987-990 3. Monsur, H. A.: Bacterial diagnosis of cholera under field conditions. Bul!. WId Hlth Org.28 (1963) 387-389 4. Qzsan, K., E. Mercangoz: Kolera vibryonu izolmaninda yeni besiyerleri uzerinde <;ali~ malar. Turk. Ij. tecr. Biyo!. Derg. 33 (1973) 21-26 5. Poliitzer, R.: Cholera. WId Hlth Org., Palais des Nations, Geneva (1959) Alkl~,
Prof. Dr. Kemal Qzsan, A. O. TIp Fakultesi, Mikrobiyoloji ve Parazitoloji Kursusu, Morfoloji Binasl, Slhhlye, Ankara, Tiirkei