ISO 16649-1:2001 – Metodo orizzontale per il conteggio Beta-glucoronidase-positive E.coli ISO – 16649-2:2001 – ISO/TS 16649-3:2005
Horizontal method for the enumeration β-glucuronidase-positive Escherichia coli
(a) ISO 16649-1:2001
Colony–count technique at 44°c using membranes and 5-bromo-4-chloro-3-indolyl- β-D-glucoronic acid.
(b) ISO 16649-2:2001
Colony–count technique at 44°c using 5-bromo-4-chloro-3-indolyl- β-D-glucoronic acid.
(c) ISO/TS 16649-3:2005
Most Probable Number technique using 5-bromo-4-chloro-3-indolyl- β-D-glucoronide.
(a) ISO 16649-1:2001
Preparare il campione secondo quanto previsto dall’ International Standard ISO 16649-1:2001.
Filtrare una quantità precisa di campione da analizzare su delle membrane di cellulosa-acetato coprendola con mineral-modified glutamate agar (MMBA), incubare a 37°C per 4h.
Isolamento: porre le membrane sulla superficie del terreno agar triptone-bile glucuronide (TBX). Incubare a 44°C per 18-24h.
Verificare la presenza di Escherichia coli attraverso la prova, su ogni colonia, della produzione di indolo.
Conta: Calcolare ufc/g o mL di Eschericha coli, in base al numero di colonie blu.
(b) ISO 16649-2:2001
Isolamento: inoculare il TBX con le opportune diluizioni del campione.
Incubare le piastre per 18-24h a 44°C ed esaminare le piastre per verificare le colonie di Escherichia coli.
Conta: calcolare ufc/g o mL di Eschericha coli.
(c) ISO/TS 16649-3:2005
Inoculo di tre provette* di brodo selettivo (minerals modified glutamate medium) con il campione da analizzare, proseguire nelle medesime condizioni con le successive diluizioni.
Incubare le provette a 37°C per 24h.
Identificare provette che presentano produzione di acido, le quali indicano la fermentazione del lattosio.
Per ogni provetta che ha mostrato produzione di acido mettere un’aliquota di questa in tryptone bile glucoronide agar.
Incubare le piastre di tryptone bile glucoronide agar a 44°C per 20-24h. Verificare la presenza di colonie blu, che mostrano la presenza di Escherichia coli β-D-glucoronidase positivo.
Determinazione del numero più probabile (most probable number -. MPN) di presunti Escherichia coli grazie alle tabelle del MPN.* o cinque provette.
TESTO IN LINGUA INGLESE
Prepare the test sample in accordance with the specific International Standard appropriate to the product concerned. – ISO 16649-1:2001
Resuscitation: inoculation of a specified quantity of the test sample or initial suspension on to cellulose acetate membranes overlaid on mineral-modified glutamate agar (MMBA), then incubation at 37°C (or 30°C) for 4h.
Inoculation on additional same agar with the same conditions of decimal dilutions from the test sample or the initial suspension.
Isolation: transfer of membranes from the resuscitation stage on the mineral-modified glutamate agar to tryptone-bile glucuronide agar (TBX). Incubation at 44°C for 18-24h.
Enumeration: calculation of the number of CFU of presumptive Eschericha coli per gram or per millilitre of sample from the number of typical blue CFU.
ISO 16649-2:2001
The enumeration of presumptive Escherichia coli requires the successive stage after the preparation of initial suspension using as diluent peptone water (or the specific International Standard dealing with the product under examination).
Isolation: inoculation of chromogenic selective culture medium (TBX) with the specified quantity of the initial suspension and dilutions.
Inoculation, under the same conditions, using decimal dilutions of the test sample or of the initial suspension, with 2 plates per dilution.
Incubation of the dishes 18-24h at 44°C and examination to check for the presence of CFU which, from their characteristics, are considered to be Escherichia coli.
Enumeration: calculation of the number of colony-forming units (CFU) of presumptive Escherichia coli per gram or per millilitre of sample.
ISO/TS 16649-3:2005
Inoculation of three tubes* of liquid selective enrichment medium (minerals modified glutamate medium) with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the case of other products. Then, under the same conditions, inoculation of the medium with decimal dilutions of the test sample or the initial suspension
Incubation of the tubes of medium at 37°C for 24. Examination of the tubes for acid production, signifying lactose fermentation.
For each tube of medium showing acid production, subculture to tryptone bile glucuronide agar.
Incubation of the tryptone bile glucoronide agar at 44°C for 20-24h. Examination of the tryptone bile glucoronide agar for the presence of blue colonies, signifying the presence of β-D-glucoronidase positive Escherichia coli.
Determination of the most probable number of presumptive Escherichia coli by means of the MPN table, according to the number of tubes of medium the subcultures of which have produced blue colonies on tryptone bile glucoronide agar.*or five tubes.
