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RI Issued: 6/16!87 MICRO SOP-P19 PrepAration of Sample Dilutions o Initiate analysis as soon as possible after collection to minimize changes in the bacterial population. Typically, samples should be asasyed within 6 h of collection if unrefrigerated and within 30 h if refrigerated. Samples should not be frozen. o Mark each dilution tube with the appropriate dilution value. o Thoroughly mix all samples by vortexing or by rapidly hand shaking. o Dilute the sample (usually 10-fold and 100-fold are prepared but other dilutions may be appropriate) to obtain countable plates (30-300 colony forming units (CFU) per plate) as follows: Using the Pipetman® or a pipet and pipet filler, add either: a. 1.0 mL of the sample to a aerile 9.0 mL phosphate buffered dilution blank (1:10 or 10� diiution), t b. 0.1 mL of the sample to is Stirile 9.9 mL phosphate buffered dilution blank (1:1O0 or 10dilution), or k c. an appropriate dilution of eampl4 with phosphate buffer to A obtain ccvnts in the 3D-170 CFU range per plate. . o A new sterile pipet o: tip ;Aust by used for each trarssfer, and each dilution must be thoroughly mixed bafore removing an aliquot for subsequent dilution. When an aliquot is removed, the.pipet tip should be inserted jue} below the surface of the ligtid (e g., no more than approximately 2.5 am below the eurface). o To minimize bacterial d3nsity changes in the samples, do not prepare any mora samples then can be diluted and plated in 20-25 min. Plating the Diluted Sample b Mark each plate witheample number, dilution value, or date and any other necessary information before examination. . (If desired, replicate f plates can be prepared for each Sample or dilation examined.) a Using a Pipetman® 6r pipet and pipet filler, place 3.1 mL of the ;> thoroughly mixed sample or dilutions on a TSA plate or other appropriate , medium. a Pines the inoculated plate an the petzi dish turntable. o Remove a bent_11©ss rod from the ethanol and flame it. CARE MUST BE TAKEN TO HOLD !HE ROD DOWNWARD SUCH THAT THE FLAMING ALCOHOL WILL NOT RUN TOWARD THE OPERATOR'S HAND AND CAUSE A BURN. ` 3 - 1.