AS 5013.18:2010 pdf – Food microbiology Method 18: Examination for specific organisms—Vibrio parahaemolyticus

AS 5013.18:2010 pdf – Food microbiology Method 18: Examination for specific organisms—Vibrio parahaemolyticus.
4.5.1 Additional control cultures—optional
V. chokrae (non-Ol) NCTC 4711, ATCC 14730
V. vu1nfieus ATCC 27562.
See Appendix C for procedures for the long-term preser’a1ion 01 relerence cultures.
5 PRFPARATION OF I)I1.UTIONS OF Fool) PROI)LICT
5.1 Diluent
Peptonc solution containing 30 gIL NaCI (4.3.6) should be used to prepare dilutions of the test sample.
5.2 Procedure
The procedure shall be as follows:
(a) Prepare the sample of food product for testing in accordance with the instructions given for that product in the appropriate AS 5013 Standard.
(b) Prepare the required decimal dilutions of the prepared sample by the procedure described in AS 5013, Methods 11.1 and 11.3, as appropriate.
6 TEST PROCEDURES
6.1 Isolation of presumplic F’ puruhaenwl,’ticu.s
The procedure shall be as follows:
(a) Using the most probable number MPN) method (3-tube technique) described in AS 5013.3, inoculate tubes containing 10 ml. of alkaline peptone water (4.2.1) with appropriate dilutions of the test sample (primary enrichment).
(b) Prepare a set of controls for each series of tests by inoculating tubes of alkaline peptone water (4.2.1) with the reference culture(s) specified in Clause 4.5.
(c) Incubate tests and control at 36 ±2°C for 6 h to 8 h.
(d) Transfer I mL from each lube to a fresh tube of alkaline peptone water (4.2.1) (secondary enrichment). Streak a loopful of each primary enrichment culture on to a prepared plate of TCBS agar (4.2.2) to obtain isolated colonies.
Each individual primary and secondary enrichment culture shall carry a unique identification code in order to determine which secondary enrichment corresponds to each primary’ enrichment culture when selecting colonies for confirmation and when calculating the most probable number of V. parahuernolviwus present.
(e) Incubate the secondary enrichments and the TUBS agar plates at 36 ±2°C for 18 ±2 Ii,
(f) Streak a loopful of each secondary enrichment on to a prepared plate of TUBS agar (4.2.2) to obtain isolated colonies.
(g) Incubate the TCBS agar plates at 36±2°C for 18±2 h.
(a) Select three typical V. parcdiaeino!vlicus colonies from the TCBS agar plates streaked from each pair of primary and secondary enrichment cultures. Subculture each colony into a tube of ryptone ater containing 30 gIL NaUI (4.3.1).
Typical colonies of V. parahaeinolviicus are blue/green in colour and 3—5 mm in diameter.
It is not necessary to inoculate into Iryplone broth prior to inoculation of biochemical tests if well isolated colonies on the selective agar plates are selected for confirmation. Howeer. the nutrient agar containing 30 gIL NaCI plate inoculated at the time of biochemical confirmation shall he examined to confirm that the inocula were pure. Step (b) will not apply if ihis approach is used.
(b) Incubate at 36 ±2°C until faintly turbid, usually 2 h to 4 h.