ASTM D7817-12(2021)
(Test Method)Standard Test Method for Enumeration of Yeast and Mold in Raceway Brine, Brine-Cured Hides and Skins
Standard Test Method for Enumeration of Yeast and Mold in Raceway Brine, Brine-Cured Hides and Skins
SIGNIFICANCE AND USE
4.1 This test method enumerates salt tolerant yeast and mold, and under the conditions of this test method those are equated as halophilic organisms. Salt tolerant yeast and mold have been known to cause damage to hides and skins in raceway brine.
SCOPE
1.1 This test method covers the enumeration of yeast and mold. This test method is applicable to raceway brine, brine-cured hides and skins, and pre-charge raceway liquor.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D7817 − 12 (Reapproved 2021)
Standard Test Method for
Enumeration of Yeast and Mold in Raceway Brine, Brine-
Cured Hides and Skins
This standard is issued under the fixed designation D7817; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope agarcontaining7%NaClandanantibioticsolution.Theplates
are incubated at 20–25 °C for 5 days.
1.1 This test method covers the enumeration of yeast and
mold. This test method is applicable to raceway brine, brine-
4. Significance and Use
cured hides and skins, and pre-charge raceway liquor.
4.1 This test method enumerates salt tolerant yeast and
1.2 The values stated in SI units are to be regarded as
mold, and under the conditions of this test method those are
standard. No other units of measurement are included in this
equated as halophilic organisms. Salt tolerant yeast and mold
standard.
have been known to cause damage to hides and skins in
1.3 This standard does not purport to address all of the
raceway brine.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
5. Apparatus
priate safety, health, and environmental practices and deter-
5.1 Incubator, 20–25 °C.
mine the applicability of regulatory limitations prior to use.
5.2 Colony counter—(not mandatory, but highly recom-
1.4 This international standard was developed in accor-
mended).
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
5.3 Sterile pipets.
Development of International Standards, Guides and Recom-
5.4 Stomacher, for mixing initial dilution. (If stomacher is
mendations issued by the World Trade Organization Technical
unavailable, hand-mix.)
Barriers to Trade (TBT) Committee.
5.5 Balance.
2. Referenced Documents
5.6 Sterile petri dishes.
2.1 ASTM Standards:
5.7 Autoclave (sterilizer)—(Check the effectiveness of ster-
D6715Practice for Sampling and Preparation of Fresh or
ilizationweekly.Forexample,placesporesuspensionsorstrips
Salt-Preserved (Cured) Hides and Skins for Chemical and
of Bacillus stearothermophilus(commerciallyavailable)inside
Physical Tests
glassware for a full autoclave cycle. Follow manufacturer’s
E691Practice for Conducting an Interlaboratory Study to
directions for sterilization of specific media.)
Determine the Precision of a Test Method
5.8 pH meter.
E177Practice for Use of the Terms Precision and Bias in
ASTM Test Methods
5.9 Waterbath, 45 6 1 °C.
5.10 Stomacher bags, or sterile, sealable quart plastic bag
3. Summary of Test Method
(for example, food storage type, sterile bag).
3.1 Samples of brine-cured hides and skins, raceway brine,
5.11 Cutting tool, sterile (for example, scalpel blade and
or pre-charge raceway liquor are serially diluted and plated on
forcep, as needed for cutting cured hides and skins).
5.12 Vortex mixer, for mixing dilution tubes (optional).
ThistestmethodisunderthejurisdictionofASTMCommitteeD31onLeather 5.13 Autoclave thermometer.
and is the direct responsibility of Subcommittee D31.02 on Wet Blue.
Current edition approved Sept. 1, 2021. Published October 2021. Originally
6. Reagents and Materials
approved in 2012. Last previous edition approved in 2016 as D7817–12 (2016).
DOI: 10.1520/D7817-12R21.
6.1 Butterfield’s Phosphate Stock Solution—Dissolve 34 g
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
KH PO (Potassium Phosphate monobasic) in 500 mL DI
2 4
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
water.Adjust the pH to 7.2 6 0.1 with 1N – 6N NaOH. Bring
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. volume to 1 L with DI water. Sterilize for 15 min at 121 °C.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7817 − 12 (2021)
NOTE 1—Typical autoclave setting is 120–124 °C. (See 5.7.)
9. Preparation of Potato Dextrose Agar and Antibiotic
Solution
6.2 Butterfield’s Phosphate Diluent with salt (BPD w/salt)—
Take 1.25 mL of Butterfield’s Phosphate Stock solution (6.1)
9.1 Prepare the antibiotic stock (10 000 ppm) solution by
andbringto1LwithDIwater,thenadd77gofsalt(NaCl)per
dissolving1gof chloramphenicol in 100 mLsterile deionized
litre prior to autoclaving. Dispense into 1-L bottles and 9-mL
or distilled water. Store this stock solution in a dark location at
dilution tubes. Sterilize for 15 min at 121 °C. (See Note 1.)
≤5 °C for up to two months.
6.3 Potato Dextrose Agar (PDA).
9.2 Suspend 39 g of Potato Dextrose Agar in 1 L of
deionized or distilled water and heat to boiling to dissolve
6.4 Antibiotic solution—(Chloramphenicol) – (needed to
inhibit bacterial growth on agar). completely.
6.5 Distilled or deionized water. 9.3 Add 77 g of NaCl per litre of agar. Add 10 mL of
chloramphenicol stock solution per litre of agar to give a
6.6 Salt (NaCl), Sodium chloride – reagent grade.
concentrationof100ppm.Sterilizeintheautoclavefor15min
6.7 1N – 6N NaOH.
at 121 °C. (See Note 1.) Cool to 45 6 1 °C in a waterbath.
6.8 Bacillus stearothermophilus spore suspensions or strips Once medium has been tempered, it can be held for 2–3 h
(commercially available), or equivalent. before use, provided the water level in the waterbath is 2–3
cm above the surface of the agar. Final pH of the agar: 5.6 6
7. Hazards
0.2.
7.1 Allreagentsandchemicalsshouldbehandledwithcare.
10. Procedure
Before using any chemical, read and follow all safety precau-
tions and instructions on the manufacturer’s label or MSDS
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g
(Material Safety Data Sheet).
specimen in a sterile bag. For brine-cured hides and skins,
include both flesh and hair side.
8. Sampling
10.2 Add 180 g of BPD w/salt (6.2) diluent into the same
8.1 The specimen shall be sampled in accordance with
sterile bag (10.1). Stomach or hand-massage for 1 min. This
Practice D6715, and placed in sterile containers.
provides a 1:10 dilution.
-2 -3 -4
10.3 Preparethefollowingsampledilutions:10 ,10 ,10 ,
-5 -6 -7
10 ,10 , and 10 (see Fig. 1).
The sole source of supply known to the committee at this time is Sigma-
10.3.1 Control Blank—In 10.5, pour melted media that has
Aldrich, Cat. # C0378 (25 g). If you are aware of alternative suppliers, please
been previously tempered to 45 6 1 °C into a dish, then
provide this information toASTM International Headquarters.Your comments will
continue with 10.6 as with the sample plates.
receive careful consideration at a meeting of the responsible technical committee,
which you may attend.
FIG. 1 Plating
D7817 − 12 (2021)
-2 -1
Example:Toobtaina10 dilution,mixthe10 dilutionand where:
-1
pipet 1 mL of that 10 dilution into a 9-mL dilution tube.
A = number of colonies counted in step 10.10, and
NOTE 2—When transferring the aliquots between the tubes, the analyst
C = dilution factor (see Table 1).
must use a different pipet or pipet tip for each transfer.
Mold 5 B 3 C (2)
10.4 Pipet 1 mL of each dilution into the appropriate,
separate petri dishes.
where:
B = number of colonies counted in step 10.10, and
10.5 Pour prepared agar (9.3) that has been previously
C = dilution factor (see Table 1).
tempered to 4
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