ASTM D7817-12(2016)

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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 31-Aug-2016
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: D31 - Leather
• Drafting Committee: D31.02 - Wet Blue

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D7817 − 12 (Reapproved 2016)
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 equated as halophilic organisms. Salt tolerant yeast and mold
have been known to cause damage to hides and skins in
1.1 This test method covers the enumeration of yeast and
raceway brine.
mold. This test method is applicable to raceway brine, brine-
cured hides and skins, and pre-charge raceway liquor.
5. Apparatus
1.2 The values stated in SI units are to be regarded as
5.1 Incubator, 20–25°C.
standard. No other units of measurement are included in this
5.2 Colony counter—(not mandatory, but highly recom-
standard.
mended).
1.3 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 5.3 Sterile pipets.
responsibility of the user of this standard to establish appro-
5.4 Stomacher, for mixing initial dilution. (If stomacher is
priate safety and health practices and determine the applica-
unavailable, hand-mix.)
bility of regulatory limitations prior to use.
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
5.9 Waterbath, 45 6 1°C.
ASTM Test Methods
5.10 Stomacher bags, or sterile, sealable quart plastic bag
3. Summary of Test Method
(e.g. food storage type, sterile bag).
3.1 Samples of brine-cured hides and skins, raceway brine,
5.11 Cutting tool, sterile (e.g. scalpel blade and forcep, as
or pre-charge raceway liquor are serially diluted and plated on
needed for cutting cured hides and skins).
agarcontaining7%NaClandanantibioticsolution.Theplates
5.12 Vortex mixer, for mixing dilution tubes (optional).
are incubated at 20–25°C for 5 days.
5.13 Autoclave thermometer.
4. Significance and Use
6. Reagents and Materials
4.1 This test method enumerates salt tolerant yeast and
mold, and under the conditions of this test method those are
6.1 Butterfield’s Phosphate Stock Solution—Dissolve 34 g
KH PO (Potassium Phosphate monobasic) in 500 mL DI
2 4
water.Adjust the pH to 7.2 6 0.1 with 1N – 6N NaOH. Bring
ThistestmethodisunderthejurisdictionofASTMCommitteeD31onLeather
volume to 1 L with DI water. Sterilize for 15 min at 121°C.
and is the direct responsibility of Subcommittee D31.02 on Wet Blue.
NOTE 1—Typical autoclave setting is 120–124°C. (See 5.7.)
Current edition approved Sept. 1, 2016. Published October 2016. Originally
6.2 Butterfield’s Phosphate Diluent with salt (BPD w/salt)—
approved in 2012. Last previous edition approved in 2012 as D7817–12. DOI:
10.1520/D7817-12R16.
Take 1.25 mL of Butterfield’s Phosphate Stock solution (6.1)
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
andbringto1LwithDIwater,thenadd77gofsalt(NaCl)per
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
litre prior to autoclaving. Dispense into 1-L bottles and 9-mL
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. dilution tubes. Sterilize for 15 min at 121°C. (See Note 1.)
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7817 − 12 (2016)
6.3 Potato Dextrose Agar (PDA). 9.2 Suspend 39 g of Potato Dextrose Agar in 1 L of
3 deionized or distilled water and heat to boiling to dissolve
6.4 Antibiotic solution—(Chloramphenicol) – (needed to
completely.
inhibit bacterial growth on agar).
9.3 Add 77 g of NaCl per litre of agar. Add 10 mL of
6.5 Distilled or deionized water.
chloramphenicol stock solution per litre of agar to give a
6.6 Salt (NaCl), Sodium chloride – reagent grade.
concentrationof100ppm.Sterilizeintheautoclavefor15min
at121°C.(SeeNote1.)Coolto45 61°Cinawaterbath.Once
6.7 1N – 6N NaOH.
medium has been tempered, it can be held for 2–3 h before
6.8 Bacillus stearothermophilus spore suspensions or strips
use,providedthewaterlevelinthewaterbathis2–3cmabove
(commercially available), or equivalent.
the surface of the agar. Final pH of the agar: 5.6 6 0.2.
7. Hazards
10. Procedure
7.1 Allreagentsandchemicalsshouldbehandledwithcare.
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g
Before using any chemical, read and follow all safety precau-
specimen in a sterile bag. For brine-cured hides and skins,
tions and instructions on the manufacturer’s label or MSDS
include both flesh and hair side.
(Material Safety Data Sheet).
10.2 Add 180 g of BPD w/salt (6.2) diluent into the same
8. Sampling
sterile bag (10.1). Stomach or hand-massage for 1 min. This
8.1 The specimen shall be sampled in accordance with
provides a 1:10 dilution.
Practice D6715, and placed in sterile containers.
-2 -3 -4
10.3 Preparethefollowingsampledilutions:10 ,10 ,10 ,
-5 -6 -7
10 ,10 , and 10 (see Fig. 1).
9. Preparation of Potato Dextrose Agar and Antibiotic
10.3.1 Control Blank—In 10.5, pour melted media that has
Solution
been previously tempered to 45 6 1°C into a dish, then
9.1 Prepare the antibiotic stock (10 000 ppm) solution by
continue with 10.6 as with the sample plates.
dissolving1gof chloramphenicol in 100 mLsterile deionized -2 -1
Example:Toobtaina10 dilution,mixthe10 dilutionand
or distilled water. Store this stock solution in a dark location at -1
pipet 1 mL of that 10 dilution into a 9-mL dilution tube.
≤5°C for up to two months.
NOTE 2—When transferring the aliquots between the tubes, the analyst
must use a different pipet or pipet tip for each transfer.
10.4 Pipet 1 mL of each dilution into the appropriate,
The sole source of supply known to the committee at this time is Sigma-
separate petri dishes.
Aldrich, Cat. # C0378 (25 g). If you are aware of alternative suppliers, please
provide this information toASTM International Headquarters.Your comments will 10.5 Pour prepared agar (9.3) that has been previously
receive careful consideration at a meeting of the responsible technical committee,
tempered to 45 6 1°C into the dish.
which you may attend.
FIG. 1 Plating
D7817 − 12 (2016)
NOTE 3—Add agar within 1–2 min after adding dilution to avoid
12. Report
adherence of sample to bottom of dish. Do not pour agar directly on the
12.1 Report the results from 11.1 asYeast (salt tolerant) cfu
sample. Replace the cover.
pergof sample, Mold (salt tolerant) cfu per g of sample
10.6 Swirltheplategentlyinafigure-eightmotiontoevenly
respectively.
distribute the sample.
NOTE 9—When requested, the counts for yeast and mold may be
combined as one
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D7817 − 12 D7817 − 12 (Reapproved 2016)
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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 and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2.1 ASTM Standards:
D6715 Practice for Sampling and Preparation of Fresh or Salt-Preserved (Cured) Hides and Skins for Chemical and Physical
Tests
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
3. Summary of Test Method
3.1 Samples of brine-cured hides and skins, raceway brine, or pre-charge raceway liquor are serially diluted and plated on agar
containing 7 % NaCl and an antibiotic solution. The plates are incubated at 20–25°C20 – 25°C for 5 days.
4. 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.
5. Apparatus
5.1 Incubator, 20–25°C.20 – 25°C.
5.2 Colony counter—(not mandatory, but highly recommended).
5.3 Sterile pipets.
5.4 Stomacher, for mixing initial dilution. (If stomacher is unavailable, hand-mix.)
5.5 Balance.
5.6 Sterile petri dishes.
5.7 Autoclave (sterilizer)—(Check the effectiveness of sterilization weekly. For example, place spore suspensions or strips of
Bacillus stearothermophilus (commercially available) inside glassware for a full autoclave cycle. Follow manufacturer’s directions
for sterilization of specific media.)
5.8 pH meter.
This test method is under the jurisdiction of ASTM Committee D31 on Leather and is the direct responsibility of Subcommittee D31.02 on Wet Blue.
Current edition approved Sept. 1, 2012Sept. 1, 2016. Published October 2012October 2016. Originally approved in 2012. Last previous edition approved in 2012 as
D7817 – 12. DOI: 10.1520/D7817-1210.1520/D7817-12R16.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7817 − 12 (2016)
5.9 Waterbath, 45 6 1°C.
5.10 Stomacher bags, or sterile, sealable quart plastic bag (e.g. food storage type, sterile bag).
5.11 Cutting tool, sterile (e.g. scalpel blade and forcep, as needed for cutting cured hides and skins).
5.12 Vortex mixer, for mixing dilution tubes (optional).
5.13 Autoclave thermometer.
6. Reagents and Materials
6.1 Butterfield’s Phosphate Stock Solution—Dissolve 34 g KH PO (Potassium Phosphate monobasic) in 500 mL DI water.
2 4
Adjust the pH to 7.2 6 0.1 with 1N – 6N NaOH. Bring volume to 1 L with DI water. Sterilize for 15 min at 121°C.
NOTE 1—Typical autoclave setting is 120–124°C.120 – 124°C. (See 5.7.)
6.2 Butterfield’s Phosphate Diluent with salt (BPD w/salt)—Take 1.25 mL of Butterfield’s Phosphate Stock solution (6.1) and
bring to 1 L with DI water, then add 77 g of salt (NaCl) per litre prior to autoclaving. Dispense into 1 L 1-L bottles and 9 mL 9-mL
dilution tubes. Sterilize for 15 min at 121°C. (See Note 1.)
6.3 Potato Dextrose Agar (PDA).
6.4 Antibiotic solution—(Chloramphenicol(Chloramphenicol) ) – – (needed to inhibit bacterial growth on agar).
6.5 Distilled or deionized water.
6.6 Salt (NaCl), Sodium chloride – reagent grade.
6.7 1N – 6N NaOH.
6.8 Bacillus stearothermophilus spore suspensions or strips (commercially available), or equivalent.
7. Hazards
7.1 All reagents and chemicals should be handled with care. Before using any chemical, read and follow all safety precautions
and instructions on the manufacturer’s label or MSDS (Material Safety Data Sheet).
8. Sampling
8.1 The specimen shall be sampled in accordance with Practice D6715, and placed in sterile containers.
9. Preparation of Potato Dextrose Agar and Antibiotic Solution
9.1 Prepare the Antibioticantibiotic stock (10000 (10 000 ppm) solution by dissolving 1 g of chloramphenicol in 100 mL sterile
deionized or distilled water. Store this stock solution in a dark location at ≤5°C for up to two months.
9.2 Suspend 39 g of Potato Dextrose Agar in 1 L of deionized or distilled water and heat to boiling to dissolve completely.
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 concentration
of 100 ppm. Sterilize in the autoclave for 15 min at 121°C. (See Note 1.) Cool to 45 6 1°C in a waterbath. Once medium has been
tempered, it can be held for 2–32 – 3 h before use, provided the water level in the waterbath is 2–32 – 3 cm above the surface of
the agar. Final pH of the agar: 5.6 6 0.2.
10. Procedure
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g specimen in a sterile bag. For brine-cured hides and skins, include
both flesh and hair side.
10.2 Add 180 g of BPD w/salt (6.2) diluent into the same sterile bag (10.1). Stomach or hand-massage for 1 min. This provides
a 1:10 dilution.
-2 -3 -4 -5 -6 -7
10.3 Prepare the following sample dilutions: 10 , 10 , 10 , 10 , 10 , and 10 (see Fig. 1).
10.3.1 Control Blank—In 10.5, pour melted media that has been previously tempered to 45 6 1°C into a dish, then continue
with 10.6 as with the sample plates.
-2 -1 -1
Example: To obtain a 10 dilution, mix the 10 dilution and pipet 1 mL of that 10 dilution into a 9mL9-mL dilution tube.
NOTE 2—When transferring the aliquots between the tubes, the analyst must use a different pipet or pipet tip for each transfer.
10.4 Pipet 1 mL of each dilution into the appropriate, separate petri dishes.
10.5 Pour prepared agar (9.3) that has been previously tempered to 45 6 1°C into the dish.
The sole source of supply known to the committee at this time is Sigma-Aldrich, Cat. # C0378 (25 g). If you are aware of alternative suppliers, please provide this
information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may
attend.
D7817 − 12 (2016)
FIG. 1 Plating
NOTE 3—Add agar within 1–21 – 2 min after adding dilution to avoid adherence of sample to bottom of dish. Do not pour agar directly on the sample.
Replace the cover.
10.6 Swirl the plate gently in a figure-eight motion to evenly distribute the sample.
10.7 Allow agar to solidify.
10.8 Incubate at 20–25
...