1、Designation: D7818 12 (Reapproved 2016)Standard Test Method forEnumeration of Proteolytic Bacteria in Fresh (Uncured)Hides and Skins1This standard is issued under the fixed designation D7818; the number immediately following the designation indicates the year oforiginal adoption or, in the case of r
2、evision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the enumeration of bacteria thatcan hydrolyze protein/collagen in fresh (
3、uncured) hides andskins. This test method is applicable to uncured hides andskins.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated wi
4、th its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D6715 Practice for Sampling and Preparation of Fresh orSalt-Preser
5、ved (Cured) Hides and Skins for Chemical andPhysical TestsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE177 Practice for Use of the Terms Precision and Bias inASTM Test Methods3. Summary of Test Method3.1 Samples of uncured hides and skins are seria
6、lly dilutedand plated on agar containing casein from skim milk. Theplates are incubated under aerobic conditions at 35C for 48 h.After incubation, to determine bacteria that can hydrolyzeprotein (proteolytic), the plates are flooded with dilute acid andthe colonies showing a halo are counted.4. Sign
7、ificance and Use4.1 This test method enumerates proteolytic bacteria. Pro-teolytic bacteria have been known to cause damage to hidesand skins.5. Apparatus5.1 Incubator, 35 6 1C.5.2 Colony counter(not mandatory, but highly recom-mended).5.3 Sterile pipets.5.4 Bent glass rods, sterile.5.5 Stomacher, f
8、or mixing initial dilution. (If stomacher isunavailable, hand-mix.)5.6 Balance.5.7 Sterile petri dishes.5.8 Autoclave (sterilizer). (Check the effectiveness of ster-ilization weekly. For example, place spore suspensions or stripsof Bacillus stearothermophilus (commercially available) insideglassware
9、 for a full autoclave cycle. Follow manufacturersdirections for sterilization of specific media.)5.9 Stomacher bags, or sterile, sealable quart plastic bag(e.g. food storage type, sterile bag).5.10 Cutting tool, sterile (e.g. scalpel blade and forcep, asneeded for cutting fresh hides and skins).5.11
10、 Vortex mixer, for mixing dilution tubes (optional).5.12 pH meter.5.13 Waterbath, 45 6 1C.5.14 Autoclave thermometer.6. Reagents and Materials6.1 5 % acetic acid.6.2 Butterfields Phosphate Stock Solution: Dissolve 34 gKH2PO4(Potassium Phosphate monobasic) in 500 mL DIwater. Adjust the pH to 7.2 6 0.
11、1 with 1N 6N NaOH. Bringvolume to 1 L with DI water. Sterilize for 15 min at 121C.NOTE 1Typical autoclave setting is 120 124C. (See 5.8.)1This test method is under the jurisdiction ofASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee D31.02 on Wet Blue.Current edition appr
12、oved Sept. 1, 2016. Published October 2016. Originallyapproved in 2012. Last previous edition approved in 2012 as D7818 12. DOI:10.1520/D7818-12R16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandar
13、ds volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.3 Butterfields Phosphate Diluent (BPD): Take 1.25 mLofButterfields Phosphate Stock solution (6.2)
14、 and bring to 1 Lwith DI water. Dispense into 1-L bottles and 9-mL dilutiontubes. Sterilize for 15 min at 121C. (See Note 1.)6.4 Standard plate count agar containing 100 mL of 10 %powdered skim milk solution per litre of agar.6.5 Alcohol (for flame sterilizing), e.g. 70 % Isopropylalcohol.6.6 Bent g
15、lass rod (“hockey-stick”).6.7 Powdered skim milk.6.8 Distilled or deionized water.6.9 Bacillus stearothermophilus spore suspensions or strips(commercially available), or equivalent.6.10 1N 6N NaOH.7. Hazards7.1 All reagents and chemicals should be handled with care.Before using any chemical, read an
16、d follow all safety precau-tions and instructions on the manufacturers label or MSDS(Material Safety Data Sheet).8. Sampling8.1 The specimen shall be sampled in accordance withPractice D6715, and placed in sterile containers.9. Preparation of Standard Plate Count Agar9.1 Prepare the standard plate c
17、ount agar per manufacturerlabel directions.9.2 Autoclave the prepared agar for 15 min at 121C. (SeeNote 1.)9.3 Prepare a 10 % powdered skim milk mixture by adding10 g powdered skim milk to 100 mL DI water, then stirring themixture to dissolve it. Autoclave the mixture for 15 min at121C. (See Note 1.
18、)9.4 Cool the agar (9.2)to456 1C, then add 100 mL of thesterile 10 % powdered skim milk mixture (9.3) per litre of agar.NOTE 2Do not allow agar to solidify prior to pouring (9.5).9.5 Pour the sterile agar into petri dishes. Replace the coverand swirl to evenly distribute the agar. Allow to solidify
19、atroom temperature on a flat surface. When solid, invert the petridishes, with the cover on the bottom, leaving a slight openingto allow the plates to dry for12 h.10. Procedure10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 gspecimen in a sterile bag. Include both flesh and hair side.10.2
20、 Add 180 g of BPD (6.3) diluent into the same sterilebag (10.1). Stomach or hand-massage for 1 min. This providesa 1:10 dilution.10.3 Prepare the following sample dilutions using 9-mLdilution tubes (BPD): 10-2,10-3,10-4,10-5,10-6, and 10-7(seeFig. 1).10.3.1 Control BlankIn 10.9, incubate one of the
21、petridishes prepared in 9.5 as-is, with the sample plates.Example: To obtain a 10-2dilution, mix the 10-1dilution andpipet 1 mL of that 10-1dilution into a 9-mL dilution tube.NOTE 3When transferring the aliquots between the tubes, the analystmust use a different pipet or pipet tip for each transfer.
22、10.4 Pipet an appropriate portion (0.1 mL or 0.2 mL), of the10-2dilution and place the liquid in the middle of a dried, skimmilk agar plate.FIG. 1 PlatingD7818 12 (2016)210.5 Flame sterilize a bent glass rod, or obtain a sterile,autoclaved bent glass rod.10.6 Using the glass rod, spread the liquid e
23、venly on theagar surface.10.7 Replace the cover and allow the plate to dry at roomtemperature.10.8 Repeat steps 10.4 10.7 for each dilution.10.9 Invert all plates and incubate at 35 6 1C for 48 6 3h.10.10 Following incubation, count only those plates thathave 25 250 colonies.NOTE 4If a plate shows c
24、onfluent growth (i.e. bacterial growth coversthe entire plate, making it impossible to determine the existence ofdiscrete colonies), record that plates count as TNTC “Too Numerous ToCount”). See Figs. 2 and 3 for diagrams of a countable plate and a TNTCplate, respectively.NOTE 5Count all the distinc
25、t colonies on the plate. If there aresimilar-appearing colonies growing in close proximity but not touching,count them as individual colonies, provided the distance between them isat least equal to the diameter of the smallest colony. Colonies that areimpinging, and that differ in appearance, such a
26、s morphology or color, arecounted as individual colonies. Colonies that are a cluster, and are similarin appearance, such as morphology or color, are counted as one colony(see arrow Fig. 4 “7:00” position). There may also be “spreaders”: achain of colonies not distinctly separated. Count as one colo
27、ny if a chainof colonies appears to be caused by disintegration of a bacterial clump asagar and sample were mixed. Count as one colony if a spreader developedas a film of growth between the agar and bottom of petri dish. Count asone colony if a colony forms in a film of water at the edge or over the
28、 agarsurface.Estimated counts can be made on plates with 250 colonies: report asestimated counts. In making such counts, the standard 15 100 mm petridish is considered to have an area of about 56 cm2, therefore, use a factorof 56 when estimating the count. Example: 0.1 mL of a 10-4dilution wasplated
29、 and the plate has an average count of 10 colonies per cm2.Therefore, the estimated count for that plate is 10 56 = 560, and theestimated count for that dilution is 560 10 10,000 = 56,000,000.Estimated counts can also be made on plates with 25 colonies: report asestimated counts.10.11 Record each pl
30、ates dilution and count on the work-sheet. This initial count is the aerobic plate count (A).10.12 For the same plate(s) counted in 10.10, flood theplate(s) with 5 % acetic acid.NOTE 6Use enough of the 5 % acetic acid to fully cover the surfaceof the plate.10.13 Pour off the acetic acid and immediat
31、ely count onlythose colonies completely surrounded by a semi-clear zone.NOTE 7Proteolytic ColoniesDo not count all the colonies inside thezone; only count the colonies that cause/affect the shape of the zone.Whenidentifying the semi-clear zone(s), it is sometimes helpful to pick up theplate and chan
32、ge the angle of view slightly to determine if it is a true circleor if it has another protruberance off its edge. (For weak semi-clear zones,it is helpful to rotate the plate in the light to be sure about the clearing).Look for a circle of clearing around a centrally located colony. If theclearing i
33、s an elongated shape such as in the bottom right quadrant of Fig.5 (“5:30” position), break the cleared zone into separate circles of clearing(circles can be of varying sizes) and count the colonies located in thecenters of each of the circles. When there are multiple colonies in the sameclear zone,
34、 look to see if the zone is a uniform circle (caused by onecentrally located colony), or if it appears to have circular bumpsprotruding from the edges of the central circle, each bump being caused bya separate colony.NOTE 8Colonies on the edge of the zone that are not fully encom-passed or do not af
35、fect the shape of the zone should not be counted.10.14 Record each plates dilution and count on the work-sheet. This second count is the proteolytic count (B).11. Calculation of Results for Aerobic Plate Count andProteolytic Bacteria Count11.1 Calculate the result for aerobic plate bacteria by using
36、the following formula:Aerobic plate count 5 A 3 D 3 E (1)where:A = number of colonies counted in step 10.11,D = plating factor (see Table 1), andE = dilution factor (see Table 2).FIG. 2 Diagram of a Countable PlateD7818 12 (2016)311.2 Calculate the result for proteolytic bacteria by using thefollowi
37、ng formula:Proteolytic bacteria 5 B 3 D 3 E (2)where:B = number of colonies counted in step 10.14,D = plating factor (see Table 1), andE = dilution factor (see Table 2).12. Report12.1 Report the results from 11.1 as Aerobic Plate Count(spread plate method) per gram of sample and the result from11.2
38、as Proteolytic bacteria per gram of sample.12.2 Report the condition of the specimen (hair-on ordehaired).13. Precision and Bias13.1 The precision of this test method is based on aninterlaboratory study of D7818, conducted in 2010. Twolaboratories participated in this study. Each of the two labora-t
39、ories reported seven replicate test results for both the numberof colony forming units per gram, and the Log proteolyticbacteria, of a single uncured hide material. Every “test result”reported represents an individual determination. Except for theuse of only two laboratories, and a single material,
40、PracticeE691 was followed for the design and analysis of the data; thedetails are given in an ASTM Research Report.313.1.1 Repeatability Limit (r)Two test results obtainedwithin one laboratory shall be judged not equivalent if theydiffer by more than the “r” value for that material; “r”istheinterval
41、 representing the critical difference between two testresults for the same material, obtained by the same operatorusing the same equipment on the same day in the samelaboratory.13.1.1.1 Repeatability limits are listed in Tables 3 and 4.13.1.2 Reproducibility Limit (R)Two test results shall bejudged
42、not equivalent if they differ by more than the “R” valuefor that material; “R” is the interval representing the criticaldifference between two test results for the same material,obtained by different operators using different equipment indifferent laboratories.13.1.2.1 Reproducibility limits are lis
43、ted in Tables 3 and 4.13.1.3 The above terms (repeatability limit and reproduc-ibility limit) are used as specified in Practice E177.13.1.4 Any judgment in accordance with statements 13.1.1and 13.1.2 would normally have an approximate 95 % prob-ability of being correct, however the precision statist
44、ics ob-tained in this ILS must not be treated as exact mathematicalquantities which are applicable to all circumstances and uses.The limited number of materials tested and laboratories report-ing results guarantees that there will be times when differencesgreater than predicted by the ILS results wi
45、ll arise, sometimeswith considerably greater or smaller frequency than the 95 %probability limit would imply. The repeatability limit and thereproducibility limit should be considered as general guides,and the associated probability of 95 % as only a rough indicatorof what can be expected.13.2 BiasA
46、t the time of the study, there was no acceptedreference material suitable for determining the bias for this testmethod, therefore no statement on bias is being made.13.3 The precision statement was determined through sta-tistical examination of 28 test results, from two laboratories, ona single uncu
47、red hide material.14. Keywords14.1 bacteria; collagen; hides; proteolytic; skins3Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D31-1017.FIG. 3 Diagram of Confluent Growth (TNTC)D7818 12 (2016)4FIG. 4 Example of a Plate Before F
48、looding It with the Acid SolutionD7818 12 (2016)5NOTE 1The colonies noted with black circles are considered proteolytic colonies. The colonies noted with black squares are also consideredproteolytic colonies, although their semi-clear zones are weak compared to those noted with black circles.FIG. 5
49、Example of the Same Plate After Flooding It with the Acid SolutionTABLE 1 Plating FactormL Plated (10.4) Plating Factor0.1 mL 100.2 mL 5TABLE 2 Dilution FactorTest Tube Plated (10.4) Dilution Factor10-210010-31,00010-410,00010-5100,00010-61,000,00010-710,000,000TABLE 3 Colony Forming Units per gramAvgARepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibilityLimitxsrsRrRFresh(uncured)hide15286 9868 9868 27631 27631AThe average of the laboratories calculated averages.D7818 12 (2016)
