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    Preparation and use of reference materials containing bacteriophages

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    Authors
    Mooijman KA
    Bahar M
    Havelaar AH
    Type
    Report
    Language
    en
    
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    Title
    Preparation and use of reference materials containing bacteriophages
    Translated Title
    Bereiding en gebruik van referentiematerialen met bacteriofagen
    Publiekssamenvatting
    Tijdens het EU-project 'Bacteriofagen in zwemwater' (januari 1996 - juni 1999) werd onderzoek uitgevoerd naar de ontwikkeling van referentie materialen voor de implementatie van methoden voor de bepaling van bacteriofagen in water. Verschillende partijen referentie materialen (RMs) met reincultures van fagen, alsmede ook natuurlijk besmette standaard monsters met mengsels van (natuurlijk voorkomende) fagen en achtergrond flora, werden bereid. Voor alle drie de typen bacteriofagen welke tijdens dit project van belang waren, werden homogene en stabiele referentie materialen met reincultures van fagen bereid: somatische colifagen (SOMCPH), F-specifieke RNA fagen (FRNAPH; welke het verschil is tussen het totaal aantal F-specifieke fagen (FTOTPH) en het aantal F-specifieke DNA fagen (FDNAPH)) en fagen van Bacteroides fragilis (BFRPH). De faag RMs werden bereid door faag suspensies met glycerol te mengen (tot een eindconcentratie van 5% v/v) waarna de materialen bij -70 graden C werden opgeslagen. De gebruikte standaard fagen waren: ?X174 voor SOMCPH, MS2 voor FRNAPH, B40-8 voor BFRPH met gastheer HSP40 en B56-1 voor BFRPH met gastheer RYC2056. De faag RMs hebben hun nut bewezen voor kwaliteitscontrole doeleinden door middel van bereiding van controlekaarten. De natuurlijk besmette standaard monsters werden bereid door rioolwater te mengen met pepton fysiologische zoutoplossing en glycerol. Deze materialen waren echter minder homogeen dan de RMs met faag reincultures. De standaard monsters waren minimaal 4 maanden stabiel bij opslag bij (-70 ong. 10) graden C en 1-2 maanden indien opgeslagen bij (-20 ong. 5) graden C. Bij temperaturen boven 0 graden C was de stabilitiet slecht.<br>
    During the EU-project 'Bacteriophages in bathing water' (January 1996 - June 1999) research was carried out on the development of reference materials for the implementation of methods for the determination of bacteriophages in water. Several batches of reference materials (RMs) containing pure phage cultures, as well as naturally polluted standard samples, containing a mixture of (naturally occurring) phages and background flora, were prepared. Homogeneous and stable RMs containing pure phage cultures could be prepared for all three types of bacteriophages considered during the project: somatic coliphages (SOMCPH), F-specific RNA phages (FRNAPH; which is the difference between the total number of F-specific phages (FTOTPH) and the number of F-specific DNA phages (FDNAPH)) and phages of Bacteroides fragilis (BFRPH). The RMs were prepared by mixing phage suspensions with glycerol (to a final concentration of 5% v/v) and storing of the final materials at (-70 about 10) degrees C. The standard phages used were ?X174 for SOMCPH, MS2 for FRNAPH, B40-8 for BFRPH with host HSP40 and B56-1 for BFRPH with host RYC 2056. The phage RMs were shown to be useful for quality control purposes in the preparation of control charts. The naturally polluted standard samples were prepared by mixing sewage with peptone saline solution and glycerol. These materials were less homogeneous than the pure culture phage RMs. The standard samples were stable when stored at (-70 about 10) degrees C for at least 4 months and at (-20 about 5) degrees C for a period of 1-2 months. At temperatures above 0 degrees C the stability was poor.<br>
    Publisher
    Rijksinstituut voor Volksgezondheid en Milieu RIVM
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