AS 4276.17.2:2016 pdf free download – Water microbiology Method 17.2: Spores of Clostridium perfringens—Estimation of most probable number (MPN) using the multiple tube dilution technique

AS 4276.17.2:2016 pdf free download – Water microbiology Method 17.2: Spores of Clostridium perfringens—Estimation of most probable number (MPN) using the multiple tube dilution technique.
5.1.1 Diffèrential reinforced clostridial medium (DRCM)
5.1.4 columbia blood cigar or other suitable nutrient-rich cigar
5.1.5 Columbia blood agar with neomvcin (0.01%)
5.1.6 Acid pliosphatase reagent
5.2 Reference cultures
5.2.1 Positive culture
C. perfringens ATCC 13124, NCTC 8237, WDCM 00007 or a culture traceable to any of these strains.
5.2.2 Negative culture
C. sporogenes ATCC 19404, WDCM 00008 or a culture traceable to any of these strains.
5.2.3 the of re/crence cultures
The reference cultures are used in media quality control and analytical quality control procedures relevant to this method. The purpose of the reference cultures is to demonstrate and ensure that typical growth characteristics and test reactions are exhibited by the cultures on the media and in the tests used in this method.
When testing a sample of water by this standard method, a culture of the positive reference microorganism (5.2.1) shall be submitted to the test procedures at the same time to demonstrate and ensure that typical growth characteristics and test reactions are exhibited. The negative reference microoganism (5.2.2) is used in addition to the positive reference microorganism for the acid phosphatase test.
If the reference cultures do not give appropriate results, then an investigation shall he undertaken. Test results shall be regarded as invalid unless the investigation reveals reasons that invalidate the quality control procedures, e.g. prepared suspension shown not to contain target bacteria.
NOTE:
ATCC— American Type Culture Collection
NCTC- National Collection of Typc Culiurcs
WDC M —World Data Ccntrc for Microorganisms
6 APPARATUS
6.1 Screw cap containers
Suitable for the volume of water to be tested.
B7 ACID PHOSPHATASF. REAGENT
Reference:
STANDING COMMITTEE OF ANALYSTS, The Microbiology of Drinking Water (2010)
— Part 6 — Methods for the isolation and enumeration of suiphite-reducing clostridia and Clostridium perfringens by membrane filtration. Methods for the Examination of Waters and Associated Materials. Environment Agency. England & Wales. 2010.
l-naphthylphosphate disodium salt 0.4 g
Fast blue 13 salt [o-dianisidine bis (diazotized) zinc double salt] 0.8 g
Acetate buffer (pH 4.6 ±0.2) 20.0 mL
Prepare the acetate buffer by dissolving 0.3 mL glacial acetic acid and 0.4 g sodium acetate in deionized water and make up to 100 mL. The final p11 should be 4.6 ±0.2.
Dissolve the ingredients in the acetate buffer and allow to stand for 60 ±5 mm at 5 ±3°C to allow to precipitate. Then filter the solution through a fluted filter to remove the precipitate. Store prepared solution at 5 ±3°C for no longer than two weeks. If precipitation occurs filter once more before use.
NOTE: Instead of I.naphthylphosphate disodium salt. l-naphthylphosphate monosodium salt may be used.
CAUTION: FAST BLUE B SALT IS TOXIC AND MAY CAUSE CANCER -TAKE
APPROPRIATE PRECAUTIONS WHEN WEIGHING OUT. PREPARIN(i AND
DISCARDING THE REAGENT.