AS 5013.18:2010 – Food microbiology Method 18: Examination for specific organisms—Vibrio parahaemolyticus. a-naphihol alpha.naphhol. 5% w/ in absolute alcohol
4.5 ReFerence cultures
4.5.1 AddilEonal on frol cuiiure3 optional
Sec Appendix C for procedures for the long-tcrni preservation of reference cultures.
5.1 Diluent
Peptone solution containing 30 g/L NaCI (4J.6) should be used to prepare dilutions of the test sample.
5.2 Procedure
I’hc procedure shall be as follows:
(a) Prepare the sample of food product for lcsting in accordance with the instructions en for that product iii 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.
6.1 Isolation of presumptise I purahuemolrtku.s
The procedure shall be as follows:
(a) Using the most probable numhcr (MPN) method (3-tube technique) described in AS 5013.3. inoculate tubes containing tO 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 tile reference culture(s) specified in Clause 4.5.
(c) Incubate tests and control at 36 ±2°C for 6 h to K h.
(d) Transfer I niL from each tube to a fresh tube of alkaline peptone water (4.2.1) (secondary enrichment). Streak a Ioopful of each primary enrichment culture (In to a prepared plate of lUllS agar (4,2,2) to obtain isolated colonies.
Each indisidtial 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 parahaernolviicus present.
(c) Incubate the secondary cnrichmcni and the TCHS agar plates at 36 ±2°C for IS ±2 h.
(1) Streak a loopful of each seconilars enrichment on to a prepared plate of TCHS agar (4.2.2) to obtain isolated colonies.
(g) Incubate the TUBS agar plates at 36 ±2°C for 18 ±2 h.
(h) At the end of their respective incubation periods, examine the plates for typical
colonies of V. parahaeinolyticus.
6.2 Confirmation of V. parahuernolylicus
The procedure shall he as follows:
(a) Select three typical V. parahaemo!vticus colonies from the TCBS agar plates streaked from each pair of primary and secondary enrichment cultures. Subculture each colony into a tube oftryptone water containing 30 gIL NaCI (4.3.1).
Typical colonies of V. parahaemolvticus are blue/green in colour and 3—5 mm in di am etc r.
It is not necessary to inoculate into tryptone broth prior to inoculation of biochemical tests if well isolated colonies on the selective agar plates are selected for confirmation. However, the nutrient agar containing 30 g/L NaCI plate inoculated at the time of biochemical confirmation shall be examined to confirm that the inocula were pure. Step (b) will not apply if this approach is used.
(b) Incubate at 36 ±2°C until faintly turbid, usually 2 h to 4 h.
(c) Inoculate each of the following with a drop of the culture from Step (b):
(I) Lysine, ornithine and arginine decarboxylase broths (4.2.4) and decarboxylase broth base (as a control).
(ii) Buffered glucose broth containing 30 gIL NaCI (4.3.2).
(iii) Bromcresol purple cellobiose broth containing 30 g/L NaCI (4.3.5).
(iv) Salt tolerance media (4.2.3).
(v) Tryptone water containing 30 gIL NaCI (4.3.1).
(d) Using the culture from Step (b), inoculate a slope of TSI agar containing 30 gIL NaCI (4.3.4) by stabbing the butt and streaking the slope with a straight wire.
(e) Using a loopful of the culture from Step (h), streak a prepared plate of nutrient agar containing 30 gIL NaCI (4.3.3) to obtain isolated colonies.
(f) Incubate the inoculated media from Steps (c), (d) and (e) as follows:
(i) Tryptone water containing 30 g/L NaCI 42 ±1°C for 18 h to 24 h.
(ii) Decarboxylase broths 36 ±2°C for 4 d.
(iii) Bromcresol purple cellobiose broth 36 ±2°C for 4 d.
(iv) Salt tolerance media 36 ±2°C for 48 ±2 h.
(v) All other media 36 ±2°C for 18 h to 24 Ii.
(g) Perform an oxidase test on the nutrient agar culture by removing sonic growth from the agar surface with a platinum loop and smearing it on a filter paper moistened with oxidase reagent (4.4.2).
NOTE: Development of a purple colour within 15 s is a positive reaction.
(h) After TSI slope has been incubated and the result recorded, perform ONPG test as folio w s:
(I) In a small sterile tube containing 0.25 mL physiological saline (4.2.5), emulsify a large loopful of culture growth from the TSI slope to form a heavy suspension.
(ii) Add I drop of toluene (4.4.3) to the tube and shake well to liberate enzyme.