1、Designation: D7816 12Standard Test Method forEnumeration of Halophilic and Proteolytic Bacteria inRaceway Brine, Brine-Cured Hides and Skins1This standard is issued under the fixed designation D7816; the number immediately following the designation indicates the year oforiginal adoption or, in the c
2、ase of revision, 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 tolerate high salt concentrat
3、ions or can hydrolyze protein/collagen, or both. This test method is applicable to racewaybrine, brine-cured hides and skins, and pre-charge racewayliquor.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 doe
4、s not purport to address all of thesafety concerns, if any, associated with 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 Stan
5、dards:2D6715 Practice for Sampling and Preparation of Salt Pre-served (Cured) Hides and Skins for Chemical and Physi-cal TestsE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Su
6、mmary of Test Method3.1 Samples of brine-cured hides and skins, raceway brineor pre-charge raceway liquor are serially diluted and plated onagar containing 7 % NaCl and casein from skim milk. Theplates are incubated under aerobic conditions at 35C for 48 h.After incubation, to determine salt toleran
7、t (halophilic)bacteria, all colonies are counted. To determine bacteria thatcan hydrolyze protein (proteolytic), the plates are flooded withdilute acid and the colonies showing a “halo” (semi-clearzones) are counted.4. Significance and Use4.1 This test method enumerates salt tolerant (halophilic)bac
8、teria, and proteolytic bacteria that are also salt tolerant.Under the conditions of this test method those bacteria areequated as halophilic organisms. Salt tolerant proteolyticbacteria have been known to cause damage to hides and skinsin raceway brine.5. Apparatus5.1 Incubator, 35 6 1C.5.2 Colony c
9、ounter (not mandatory, but highly recom-mended).5.3 Sterile pipets.5.4 Bent glass rods (“hockey-stick”), sterile. (If non-sterile,will need to flame sterilize.)5.5 Stomacher, for mixing initial dilution. (If stomacher isunavailable, hand-mix.)5.6 Balance.5.7 Sterile petri dishes.5.8 Autoclave (steri
10、lizer). (Check the effectiveness of ster-ilization weekly. For example, place spore suspensions or stripsof Bacillus stearothermophilus (commercially available) insideglassware for a full autoclave cycle. Follow manufacturersdirections for sterilization of specific media.)5.9 Stomacher bags, or ster
11、ile, 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 cured hides and skins).5.11 Vortex mixer, for mixing dilution tubes (optional).5.12 pH meter.5.13 Waterbath, 45 6 1C.5.14 Autoclave thermometer, or equ
12、ivalent for monitoringautoclave temperature.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 approved Sept. 1, 2012. Published October 2012. DOI: 10.1520/D7816-122For referenced ASTM standar
13、ds, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards 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 1
14、9428-2959. United States16. Reagents and Materials6.1 5 % acetic acid.6.2 1N 6N NaOH.6.3 Standard plate count agar containing 100 mL of 10 %powdered skim milk solution and 77 g of salt per litre of agar.6.4 Ready-To-Use Plate, (optional)3: Plate CountAgar + 10% skim milk + 7.7 % NaCl.6.5 Butterfield
15、s Phosphate Stock SolutionDissolve 34 gKH2PO4(Potassium Phosphate monobasic) in 500 mL DIwater. Adjust the pH to 7.2 6 0.1 with 1N 6N NaOH. Bringvolume to 1 L with DI water. Sterilize for 15 min at 121C.NOTE 1Typical autoclave setting is 120124C at 15 psi. (See 5.8.)6.6 Butterfields Phosphate Diluen
16、t with salt (BPD w/salt)Take 1.25 mL of Butterfields Phosphate Stock solution (6.5)and bring to 1 Lwith DI water, then add 77 g of salt (NaCl) perlitre prior to autoclaving. Dispense into 1 L bottles and 9 mLdilution tubes. Sterilize for 15 min at 121C. (See Note 1.)6.7 Alcohol (for flame sterilizin
17、g), e.g. 70 % Isopropylalcohol.6.8 Powdered skim milk.6.9 Distilled or deionized water.6.10 Salt (NaCl), Sodium chloride reagent grade.6.11 Bacillus stearothermophilus spore suspensions orstrips (commercially available), or equivalent.7. Hazards7.1 All reagents and chemicals should be handled with c
18、are.Before using any chemical, read and 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 Coun
19、t AgarNOTE 2Omit steps 9.1-9.5 if using Reagent 6.4.9.1 Prepare the standard plate count agar per manufacturerlabel directions.9.2 Add 77 g of salt per litre of agar and autoclave for 15min at 121C.9.3 Prepare a 10 % powdered skim milk mixture by adding10 g powdered skim milk to 100 mL DI water, the
20、n stirring themixture to dissolve it. Autoclave the mixture for 15 min at121C.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.Rotate bottle gently to mix.NOTE 3Do not allow agar to solidify prior to pouring (9.5).9.5 Pour the ste
21、rile agar into petri dishes. Replace the coverand swirl to evenly distribute the agar. Allow to solidify 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 steril
22、e scalpel, aseptically weigh a 20 6 0.1gspecimen in a sterile bag. For brine-cured hides and skinsinclude both flesh and hair side.10.2 Add 180 g of BPD w/salt (6.6) diluent into the samesterile bag (10.1). Stomach or hand-massage for 1 min. Thisprovides a 1:10 dilution.10.3 Prepare the following sa
23、mple dilutions using 9mLdilution tubes (BPD w/salt): 10-2,10-3,10-4,10-5,10-6, and 10-7(see Fig. 1).10.3.1 Control BlankIn 10.9, incubate one of the petridishes prepared in 9.5 as-is, with the sample plates.Example: To obtain a 10-2dilution, mix the 10-1dilution andpipet 1mL of that 10-1dilution int
24、oa9mLdilution tube.NOTE 4When transferring the aliquots between the tubes, the analystmust use a different pipet or pipet tip for each transfer.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. (If you expect
25、a high bacteria load use 0.1 mLaliquot, otherwise use 0.2 mL aliquot. If approximate range ofbacteria load is unknown then use both 0.1 and 0.2 aliquots.Refer to Table 1 for Plating Factor.)10.5 Flame sterilize a bent glass rod, or obtain a sterile,autoclaved bent glass rod.10.6 Using the glass rod,
26、 spread the liquid evenly 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 25250 colonies.NOTE
27、5If a plate shows confluent 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 6Ha
28、lophilic ColoniesCount all the distinct colonies on theplate. If there are similar-appearing colonies growing in close proximitybut not touching, count them as individual colonies, provided the distancebetween them is at least equal to the diameter of the smallest colony.Colonies that are impinging,
29、 and that differ in appearance, such asmorphology or color, are counted as individual colonies. Colonies that area cluster, and are similar in appearance, such as morphology or color, arecounted as one colony (see arrow Fig. 4 “7:00” position). There mayalso be “spreaders”: a chain of colonies not d
30、istinctly separated. Count asone colony if a chain of colonies appears to be caused by disintegration ofa 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 ofpetri dish. Count as one colony if a colony forms in a
31、 film of water at theedge or over the agar surface.3The sole source of supply of the reagent known to the committee at this timeis Edge Biologicals, Inc., Memphis, TN, Cat.# 4P-468. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comm
32、ents will receive careful consideration at a meeting of theresponsible technical committee, which you may attend.D7816 122Estimated 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 abou
33、t 56 cm2, therefore, use a factorof 56 when estimating the count. Example: 0.1mL of a 10-4dilution wasplated 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.
34、Estimated counts can also be made on plates with 25 colonies: report asestimated counts.10.11 Record each plates dilution and count on the work-sheet. This initial count is the halophilic count (A).10.12 For the same plate(s) counted in 10.10, flood theplate(s) with 5 % acetic acid.NOTE 7Use enough
35、of the 5 % acetic acid to fully cover the surfaceof the plate.10.13 Pour off the acetic acid and immediately count onlythose colonies surrounded by a semi-clear zone.NOTE 8Proteolytic ColoniesDo not count all the colonies inside thezone; only count the colonies that cause/affect the shape of the zon
36、e.Whenidentifying the semi-clear zone(s) it is sometimes helpful to pick up theplate and change 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 th
37、e clearing.)Look for a circle of clearing around a centrally located colony. If theclearing is 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
38、in thecenters of each of the circles. When there are multiple colonies in the sameclear zone, 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 separa
39、te colony.NOTE 9Colonies on the edge of the zone that are not fully encom-passed or do not affect the shape of the zone should not be counted.10.14 Record each platess dilution and count on the work-sheet. This second count is the proteolytic count (B).11. Calculation of Results for Halophilic and P
40、roteolyticBacteria11.1 Calculate the result for halophilic bacteria by using thefollowing formula:Halophilic (salt tolerant) bacteria 5 A 3 D 3 E (1)where:A = number of colonies counted in step 10.10,D = plating factor (see Table 1), andE = dilution factor (see Table 2).11.2 Calculate the result for
41、 proteolytic bacteria by using thefollowing formula:Proteolytic bacteria 5 B 3 D 3 E (2)where:B = number of colonies counted in step 10.13,D = plating factor (see Table 1), andE = dilution factor (see Table 2).FIG. 1 PlatingTABLE 1 Plating FactormL Plated (10.4) Plating Factor0.1 mL 100.2 mL 5TABLE
42、2 Dilution FactorTest Tube Plated (10.4) Dilution Factor10-210010-31,00010-410,00010-5100,00010-61,000,00010-710,000,000D7816 12312. Report12.1 Report the result from 11.1 as Halophilic (salt tolerant)bacteria per gram of sample and the result from 11.2 asProteolytic bacteria per gram of sample.12.2
43、 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 Test Method D7816 conducted in2009. One laboratory participated in this study. The laboratoryreported five replicate test results for six dif
44、ferent specimentypes. Every “test result” reported represents an individualdetermination. Except for the use of only one laboratory,Practice E691 was followed for the design and analysis of thedata.413.1.1 Repeatability Limit (r)Two test results obtainedwithin one laboratory shall be judged not equi
45、valent if theydiffer by more than the “r” value for that material; “r”istheinterval 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
46、 in Tables 3 and 4.4Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D31-1015.FIG. 2 Diagram of a Countable PlateFIG. 3 Diagram of Confluent Growth (TNTC)D7816 12413.1.2 Reproducibility Limit (R)Two test results shall bejudged not
47、 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 cannot be
48、determined withdata from only one reporting laboratory.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 13.1.1 wouldnormally have an approximate 95 % probability of beingcorrect, however the precisio
49、n statistics obtained in this ILSmust not be treated as exact mathematical quantities which areapplicable to all circumstances and uses. The limited numberof locations tested and laboratories reporting results guaranteesthat there will be times when differences greater than predictedby the ILS results will arise, sometimes with considerablygreater or smaller frequency than the 95 % probability limitwould imply. Consider the repeatability limit as a generalguide, and the associated probability of 95 % as only a roughindicator
