ASTM D7818-12(R2021) pdf free download

ASTM D7818-12(R2021) pdf free download.Standard Test Method for Enumeration of Proteolytic Bacteria in Fresh (Uncured) Hides and Skins
9. Preparation of Standard Plate Count Agar
9.1 Prepare the standard plate count agar per manufacturer label directions. 9.2 Autoclave the prepared agar for 15 min at 121°C. (See Note 1.) 9.3 Prepare a 10 % powdered skim milk mixture by adding 10 g powdered skim milk to 100 mL DI water, then stirring the mixture to dissolve it. Autoclave the mixture for 15 min at 121°C. (See Note 1.) 9.4 Cool the agar (9.2) to 45 6 1°C, then add 100 mL of the sterile 10 % powdered skim milk mixture (9.3) per litre ofagar. N OTE 2—Do not allow agar to solidify prior to pouring (9.5). 9.5 Pour the sterile agar into petri dishes. Replace the cover and swirl to evenly distribute the agar. Allow to solidify at room temperature on a flat surface. When solid, invert the petri dishes, with the cover on the bottom, leaving a slight opening to allow the plates to dry for 1 ⁄ 2 h.
10. Procedure
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g specimen in a sterile bag. Include both flesh and hair side. 10.2 Add 180 g of BPD (6.3) diluent into the same sterile bag (10.1). Stomach or hand-massage for 1 min. This provides a 1:10 dilution. 10.3 Prepare the following sample dilutions using 9mL dilution tubes (BPD): 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 (see Fig. 1). 10.3.1 Control Blank—In 10.9, incubate one of the petri dishes prepared in 9.5 as-is, with the sample plates. Example: To obtain a 10 -2 dilution, mix the 10 -1 dilution and pipet 1mL of that 10 -1 dilution into a 9 mL dilution tube. N OTE 3—When transferring the aliquots between the tubes, the analyst must use a different pipet or pipet tip for each transfer. 10.4 Pipet an appropriate portion (0.1mL or 0.2mL), of the 10 -2 dilution and place the liquid in the middle of a dried, skim milk agar plate.10.5 Flame sterilize a bent glass rod, or obtain a sterile, autoclaved bent glass rod. 10.6 Using the glass rod, spread the liquid evenly on the agar surface. 10.7 Replace the cover and allow the plate to dry at room temperature. 10.8 Repeat steps 10.4-10.7 for each dilution. 10.9 Invert all plates and incubate at 35 6 1°C for 48 6 3 h. 10.10 Following incubation, count only those plates that have 25–250 colonies. N OTE 4—If a plate shows confluent growth (i.e. bacterial growth covers the entire plate, making it impossible to determine the existence of discrete colonies), record that plate’s count as TNTC – “Too Numerous To Count”). See Figs. 2 and 3 for diagrams of a countable plate and a TNTC plate, respectively. N OTE 5—Count all the distinct colonies on the plate. If there are similar-appearing colonies growing in close proximity but not touching, count them as individual colonies, provided the distance between them is at least equal to the diameter of the smallest colony. Colonies that are impinging, and that differ in appearance, such as morphology or color, are counted as individual colonies. Colonies that are a cluster, and are similar in appearance, such as morphology or color, are counted as one colony (see arrow Fig. 4 – “7:00” position). There may also be “spreaders”: a chain of colonies not distinctly separated. Count as one colony if a chain of colonies appears to be caused by disintegration of a bacterial clump as agar and sample were mixed. Count as one colony ifa spreader developed as a film of growth between the agar and bottom of petri dish. Count as one colony ifa colony forms in a film ofwater at the edge or over the agar surface.Estimated counts can be made on plates with >250 colonies: report as estimated counts. In making such counts, the standard 15 × 100 mm petri dish is considered to have an area of about 56 cm 2 , therefore, use a factor of 56 when estimating the count. Example: 0.1 mL of a 10 -4 dilution was plated and the plate has an average count of 10 colonies per cm 2 . Therefore, the estimated count for that plate is 10 × 56 =560, and the estimated count for that dilution is 560 × 10 × 10,000 = 56,000,000. Estimated counts can also be made on plates with <25 colonies: report as estimated counts.