ASTM D7816-12(2016)

Standard Test Method for Enumeration of Halophilic and Proteolytic Bacteria in Raceway Brine, Brine-Cured Hides and Skins

SIGNIFICANCE AND USE
4.1 This test method enumerates salt tolerant (halophilic) bacteria, and proteolytic bacteria that are also salt tolerant. Under the conditions of this test method those bacteria are equated as halophilic organisms. Salt tolerant proteolytic bacteria have been known to cause damage to hides and skins in raceway brine.
SCOPE
1.1 This test method covers the enumeration of bacteria that can tolerate high salt concentrations or can hydrolyze protein/collagen, or both. 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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: D7816 − 12 (Reapproved 2016)
Standard Test Method for
Enumeration of Halophilic and Proteolytic Bacteria in
1
Raceway Brine, Brine-Cured Hides and Skins
This standard is issued under the fixed designation D7816; 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 can hydrolyze protein (proteolytic), the plates are flooded with
dilute acid and the colonies showing a “halo” (semi-clear
1.1 Thistestmethodcoverstheenumerationofbacteriathat
zones) are counted.
can tolerate high salt concentrations or can hydrolyze protein/
collagen, or both. This test method is applicable to raceway
4. Significance and Use
brine, brine-cured hides and skins, and pre-charge raceway
liquor.
4.1 This test method enumerates salt tolerant (halophilic)
bacteria, and proteolytic bacteria that are also salt tolerant.
1.2 The values stated in SI units are to be regarded as
Under the conditions of this test method those bacteria are
standard. No other units of measurement are included in this
equated as halophilic organisms. Salt tolerant proteolytic
standard.
bacteria have been known to cause damage to hides and skins
1.3 This standard does not purport to address all of the
in 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 and health practices and determine the applica-
5.1 Incubator, 35 6 1°C.
bility of regulatory limitations prior to use.
5.2 Colony counter (not mandatory, but highly recom-
2. Referenced Documents
mended).
2
2.1 ASTM Standards:
5.3 Sterile pipets.
D6715Practice for Sampling and Preparation of Fresh or
5.4 Bent glass rods (“hockey-stick”), sterile. (If non-sterile,
Salt-Preserved (Cured) Hides and Skins for Chemical and
will need to flame sterilize.)
Physical Tests
E177Practice for Use of the Terms Precision and Bias in
5.5 Stomacher, for mixing initial dilution. (If stomacher is
ASTM Test Methods
unavailable, hand-mix.)
E691Practice for Conducting an Interlaboratory Study to
5.6 Balance.
Determine the Precision of a Test Method
5.7 Sterile petri dishes.
3. Summary of Test Method
5.8 Autoclave (sterilizer). (Check the effectiveness of ster-
3.1 Samples of brine-cured hides and skins, raceway brine,
ilizationweekly.Forexample,placesporesuspensionsorstrips
or pre-charge raceway liquor are serially diluted and plated on of Bacillus stearothermophilus(commerciallyavailable)inside
agar containing 7 % NaCl and casein from skim milk. The glassware for a full autoclave cycle. Follow manufacturer’s
plates are incubated under aerobic conditions at 35°C for 48 h. directions for sterilization of specific media.)
After incubation, to determine salt tolerant (halophilic)
5.9 Stomacher bags, or sterile, sealable quart plastic bag
bacteria, all colonies are counted. To determine bacteria that
(e.g. food storage type, sterile bag).
5.10 Cutting tool, sterile (e.g. scalpel blade and forcep, as
needed for cutting cured hides and skins).
1
ThistestmethodisunderthejurisdictionofASTMCommitteeD31onLeather
and is the direct responsibility of Subcommittee D31.02 on Wet Blue.
5.11 Vortex mixer, for mixing dilution tubes (optional).
Current edition approved Sept. 1, 2016. Published October 2016. Originally
approved in 2012. Last previous edition approved in 2012 as D7816–12. DOI:
5.12 pH meter.
10.1520/D7816-12R16.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or 5.13 Waterbath, 45 6 1°C.
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
5.14 Autoclave thermometer, or equivalent for monitoring
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. autoclave temperature.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D7816 − 12 (2016)
NOTE 3—Do not allow agar to solidify prior to pouring (9.5).
6. Reagents and Materials
9.5 Pour the sterile agar into petri dishes. Replace the cover
6.1 5 % acetic acid.
and swirl to evenly distribute the agar. Allow to solidify at
6.2 1N – 6N NaOH.
roomtemperatureonaflatsurface.Whensolid,invertthepetri
6.3 Standard plate count agar containing 100 mL of 10 %
dishes, with the cover on the bottom, leaving a slight opening
1
powdered skim milk solution and 77 g of salt per litre of agar.
to allow the plates to dry for ⁄2 h.
3
6.4 Ready-To-Use Plate, (optional) : Plate CountAgar + 10
10. Procedure
% skim milk + 7.7 % NaCl.
10.1 Using a sterile scalpel, aseptically wei
...

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: D7816 − 12 D7816 − 12 (Reapproved 2016)
Standard Test Method for
Enumeration of Halophilic and Proteolytic Bacteria in
1
Raceway Brine, Brine-Cured Hides and Skins
This standard is issued under the fixed designation D7816; 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 bacteria that can tolerate high salt concentrations or can hydrolyze
protein/collagen, or both. 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
2.1 ASTM Standards:
D6715 Practice for Sampling and Preparation of Fresh or Salt-Preserved (Cured) Hides and Skins for Chemical and Physical
Tests
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
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 casein from skim milk. The plates are incubated under aerobic conditions at 35°C for 48 h. After
incubation, to determine salt tolerant (halophilic) bacteria, all colonies are counted. To determine bacteria that can hydrolyze
protein (proteolytic), the plates are flooded with dilute acid and the colonies showing a “halo” (semi-clear zones) are counted.
4. Significance and Use
4.1 This test method enumerates salt tolerant (halophilic) bacteria, and proteolytic bacteria that are also salt tolerant. Under the
conditions of this test method those bacteria are equated as halophilic organisms. Salt tolerant proteolytic bacteria have been known
to cause damage to hides and skins in raceway brine.
5. Apparatus
5.1 Incubator, 35 6 1°C.
5.2 Colony counter (not mandatory, but highly recommended).
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 is unavailable, hand-mix.)
5.6 Balance.
5.7 Sterile petri dishes.
1
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
D7816 – 12. DOI: 10.1520/D7816-1210.1520/D7816-12R16.
2
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
1

---------------------- Page: 1 ----------------------
D7816 − 12 (2016)
5.8 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.)
5.9 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, as needed for cutting cured hides and skins).
5.11 Vortex mixer, for mixing dilution tubes (optional).
5.12 pH meter.
5.13 Waterbath, 45 6 1°C.
5.14 Autoclave thermometer, or equivalent for monitoring autoclave temperature.
6. Reagents and Materials
6.1 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.
3
6.4 Ready-To-Use Plate, (optional) : Plate Count Agar + 10 % skim milk + 7.7 % NaCl.
6.5 Butterfield’s Phosphate Stock Soluti
...