ASTM D7819-12(2016)

Standard Test Method for Enumeration of Yeast and Mold on Fresh (Uncured) Hides and Skins

SIGNIFICANCE AND USE
4.1 This test method enumerates yeast and mold. Yeast and mold have been known to cause damage to hides and skins.
SCOPE
1.1 This test method covers the enumeration of yeast and mold on fresh (uncured) hides and skins. This test method is applicable to uncured hides and skins.  
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: D7819 − 12 (Reapproved 2016)
Standard Test Method for
Enumeration of Yeast and Mold on Fresh (Uncured) Hides
and Skins
This standard is issued under the fixed designation D7819; 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 5. Apparatus
1.1 This test method covers the enumeration of yeast and 5.1 Incubator, 20–25°C.
mold on fresh (uncured) hides and skins. This test method is
5.2 Colony counter (not mandatory, but highly recom-
applicable to uncured hides and skins.
mended).
1.2 The values stated in SI units are to be regarded as
5.3 Sterile pipets.
standard. No other units of measurement are included in this
5.4 Stomacher, for mixing initial dilution. (If stomacher is
standard.
unavailable, hand-mix.)
1.3 This standard does not purport to address all of the
5.5 Balance.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
5.6 Sterile petri dishes.
priate safety and health practices and determine the applica-
5.7 Autoclave (sterilizer). (Check the effectiveness of ster-
bility of regulatory limitations prior to use.
ilizationweekly.Forexample,placesporesuspensionsorstrips
of Bacillus stearothermophilus(commerciallyavailable)inside
2. Referenced Documents
glassware for a full autoclave cycle. Follow manufacturer’s
2.1 ASTM Standards:
directions for sterilization of specific media.)
D6715Practice for Sampling and Preparation of Fresh or
5.8 pH meter.
Salt-Preserved (Cured) Hides and Skins for Chemical and
Physical Tests
5.9 Waterbath, 45 6 1°C.
E691Practice for Conducting an Interlaboratory Study to
5.10 Stomacher bags, or sterile, sealable quart plastic bag
Determine the Precision of a Test Method
(e.g. Food storage type, sterile bag).
E177Practice for Use of the Terms Precision and Bias in
5.11 Cutting tool, sterile (e.g. scalpel blade and forcep, as
ASTM Test Methods
needed for cutting cured hides and skins).
3. Summary of Test Method
5.12 Vortex mixer, for mixing dilution tubes (optional).
3.1 Samples of uncured hides and skins are serially diluted
5.13 Autoclave thermometer, or equivalent for monitoring
andplatedonagarcontaininganantibioticsolution.Theplates
autoclave temperature.
are incubated at 20–25°C for 5 days.
6. Reagents and Materials
4. Significance and Use
6.1 Butterfield’s Phosphate Stock Solution: Dissolve 34 g
4.1 This test method enumerates yeast and mold. Yeast and
KH PO (Potassium Phosphate monobasic) in 500 mL DI
mold have been known to cause damage to hides and skins.
2 4
water.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.
ThistestmethodisunderthejurisdictionofASTMCommitteeD31onLeather
NOTE 1—Typical autoclave setting is 120–124°C at 15 psi. (See 5.7.)
and is the direct responsibility of Subcommittee D31.02 on Wet Blue.
Current edition approved Sept. 1, 2016. Published October 2016. Originally
6.2 Butterfield’s Phosphate Diluent (BPD):Take1.25mLof
approved in 2012. Last previous edition approved in 2012 as D7819–12. DOI:
Butterfield’s Phosphate Stock solution (6.1) and bring to 1 L
10.1520/D7819-12R16.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or with DI water. Dispense into 1-litre bottles and 9-mL dilution
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
tubes. Sterilize for 15 min at 121°C. (See Note 1.)
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 6.3 Potato Dextrose Agar (PDA).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7819 − 12 (2016)
6.4 Antibiotic solution (Chloramphenicol) – (needed to 9.3 Add 10 mL of chloramphenicol stock solution per litre
inhibit bacterial growth on agar). of agar to give a concentration of 100 ppm. Sterilize in the
autoclavefor15minat121°C.(SeeNote1.)Coolto45 61°C
6.5 Distilled or deionized water.
inawaterbath.Oncemediumhasbeentempered,itcanbeheld
6.6 NaOH, 1N – 6N.
for2–3hbeforeuse,providedthewaterlevelinthewaterbath
6.7 Bacillus stearothermophilus spore suspensions or strips is 2–3 cm above the surface of the agar. Final pH of the agar:
5.6 6 0.2.
(commercially available), or equivalent.
10. Procedure
7. Hazards
10.1 Using a sterile scalpel, aseptically obtain a 20 6 0.1 g
7.1 Allreagentsandchemicalsshouldbehandledwithcare.
specimen that includes both the flesh side and the hair side.
Before using any chemical, read and follow all safety precau-
Weighitintoasterilebag.Add180gofBPD(6.2)diluentinto
tions and instructions on the manufacturer’s label or MSDS
thesamesterilebag.Stomachorhand-massagefor1min.This
(Material Safety Data Sheet).
provides a 1:10 dilution.
8. Sampling
-2 -3 -4
10.2 Preparethefollowingsampledilutions:10 ,10 ,10 ,
-5 -6 -7
8.1 The specimen shall be sampled in accordance with
10 ,10 , and 10 (see Fig. 1).
-2 -1
Practice D6715, and placed in sterile containers.
Example:Toobtaina10 dilution,mixthe10 dilutionand
-1
pipet 1 mL of that 10 dilution into a 9-mL dilution tube.
9. Preparation of Potato Dextrose Agar and Antibiotic
NOTE 2—When transferring the aliquots between the tubes, the analyst
Solution
must use a different pipet or pipet tip for each transfer.
9.1 Prepare the antibiotic stock (10 000 ppm) solution by
10.3 Pipet 1 mL of each dilution into the appropriate,
dissolving1gof chloramphenicol in 100 mLsterile deionized
separate petri dishes.
or distilled water. Store this stock solution in a dark location at
10.4 Pour prepared agar (9.3) that has been previously
≤5°C for up to two months.
tempered to 45 6 1°C into the dish.
9.2 Suspend 39 g of Potato Dextrose Agar in 1 litre of NOTE 3—Add agar within 1–2 min after adding dilution to avoid
adherence of sample to bottom of dish. Do not pour agar directly on the
deionized or distilled water and heat to boiling to dissolve
sample. Replace the cover.
completely.
10.5 Swirltheplategentlyinafigure-eightmotiontoevenly
distribute the sample.
The sole source of supply known to the committee at this time is Sigma-
10.6 Allow agar to solidify.
Aldrich, Cat. # C0378 (25 g). If you are aware of alternative suppliers, please
provide this information toASTM International Headquarters.Your comments will
10.7 Incubate at 20–25°C for 5 days (a cabinet at room
receive careful consideration at a meeting of the responsible technical committee,
temperature is acceptable for use).
which you may attend.
FIG. 1 Plating
D7819 − 12 (2016)
NOTE4—Donotstackplateshigherthan3,anddonotinverttheplates.
laboratories participated in this study. Each of the two labora-
Let plates remain undisturbed until time for counting. Moving the plates
tories reported seven replicate test result
...

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: D7819 − 12 D7819 − 12 (Reapproved 2016)
Standard Test Method for
Enumeration of Yeast and Mold on Fresh (Uncured) Hides
and Skins
This standard is issued under the fixed designation D7819; 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 on fresh (uncured) hides and skins. This test method is applicable
to uncured hides and skins.
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 uncured hides and skins are serially diluted and plated on agar containing 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 yeast and mold. Yeast and mold have been known to cause damage to hides and skins.
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(If stomacher is unavailable, hand-mix.]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.
5.9 Waterbath, 45 6 1°C.
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
D7819 – 12. DOI: 10.1520/D7819-1210.1520/D7819-12R16.
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
D7819 − 12 (2016)
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, or equivalent for monitoring autoclave temperature.
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°C120 – 124°C at 15 psi. (See 5.7.)
6.2 Butterfield’s Phosphate Diluent (BPD): Take 1.25 mL of Butterfield’s Phosphate Stock solution (6.1) and bring to 1 L with
DI water. Dispense into 1 litre 1-litre 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 NaOH, 1N – 6N.
6.7 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 (10,000 (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 litre of deionized or distilled water and heat to boiling to dissolve completely.
9.3 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 obtain a 20 6 0.1 g specimen that includes both the flesh side and the hair side. Weigh
it into a sterile bag. Add 180 g of BPD (6.2) diluent into the same sterile bag. Stomach or hand-massage for 1 min. This provides
a 1:10 dilution.
-2 -3 -4 -5 -6 -7
10.2 Prepare the following sample dilutions: 10 , 10 , 10 , 10 , 10 , and 10 (see Fig. 1).
-2 -1 -1
Example: To obtain a 10 dilution, mix the 10 dilution and pipet 1 mL of that 10 dilution into a 9 mL 9-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.
10.3 Pipet 1 mL of each dilution into the appropriate, separate petri dishes.
10.4 Pour prepared agar (9.3) that has been previously tempered to 45 6 1°C into the dish.
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.
10.5 Swirl the plate gently in a figure-eight motion to evenly distribute the sample.
10.6 Allow agar to solidify.
10.7 Incubate at 20–25°C20 – 25°C for 5 days (a cabinet at room temperature is acceptable for use).
NOTE 4—Do not stack plates higher than 3, and do not invert the plates. Let plates remain undisturbed until time for counting. Moving the plates could
dislodge spores, thus creating extraneous growths that are not part of the original colony.
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 consid
...