ASTM D7816-23

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, 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

• Status: Published
• Publication Date: 31-Aug-2023
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: D31 - Leather
• Drafting Committee: D31.02 - Wet Blue

Relations

• Refers: ASTM D6715-13(2020) - Standard Practice for Sampling and Preparation of Fresh or Salt-Preserved (Cured) Hides and Skins for Chemical and Physical Tests
• Effective Date: 01-Apr-2020

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: D7816 − 23
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 agar containing 7 % NaCl and casein from skim milk. The
plates are incubated under aerobic conditions at 35 °C for 48 h.
1.1 This test method covers the enumeration of bacteria that
After incubation, to determine salt tolerant (halophilic)
can tolerate high salt concentrations or can hydrolyze protein/
bacteria, all colonies are counted. To determine bacteria that
collagen, or both. This test method is applicable to raceway
can hydrolyze protein (proteolytic), the plates are flooded with
brine, brine-cured hides and skins, and pre-charge raceway
dilute acid and the colonies showing a “halo” (semi-clear
liquor.
zones) are counted.
1.2 The values stated in SI units are to be regarded as
4. Significance and Use
standard. No other units of measurement are included in this
standard.
4.1 This test method enumerates salt tolerant (halophilic)
bacteria, and proteolytic bacteria that are also salt tolerant.
1.3 This standard does not purport to address all of the
Under the conditions of this test method those bacteria are
safety concerns, if any, associated with its use. It is the
equated as halophilic organisms. Salt tolerant proteolytic
responsibility of the user of this standard to establish appro-
bacteria have been known to cause damage to hides and skins
priate safety, health, and environmental practices and deter-
in raceway brine.
mine the applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accor-
5. Apparatus
dance with internationally recognized principles on standard-
5.1 Incubator, 35 °C 6 1 °C.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
5.2 Colony counter (not mandatory, but highly recom-
mendations issued by the World Trade Organization Technical
mended).
Barriers to Trade (TBT) Committee.
5.3 Sterile pipets.
2. Referenced Documents
5.4 Bent glass rods (“hockey-stick”), sterile. (If non-sterile,
2
2.1 ASTM Standards: will need to flame sterilize.)
D6715 Practice for Sampling and Preparation of Fresh or
5.5 Stomacher, for mixing initial dilution. (If stomacher is
Salt-Preserved (Cured) Hides and Skins for Chemical and
unavailable, hand-mix.)
Physical Tests
5.6 Balance.
E177 Practice for Use of the Terms Precision and Bias in
ASTM Test Methods
5.7 Sterile petri dishes.
E691 Practice for Conducting an Interlaboratory Study to
5.8 Autoclave (sterilizer). (Check the effectiveness of ster-
Determine the Precision of a Test Method
ilization weekly. For example, place spore suspensions or strips
of Bacillus stearothermophilus (commercially available) inside
3. Summary of Test Method
glassware for a full autoclave cycle. Follow manufacturer’s
3.1 Samples of brine-cured hides and skins, raceway brine,
directions for sterilization of specific media.)
or pre-charge raceway liquor are serially diluted and plated on
5.9 Stomacher bags, or sterile, sealable quart plastic bag
1
(for example, food storage type, sterile bag).
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.
5.10 Cutting tool, sterile (for example, scalpel blade and
Current edition approved Sept. 1, 2023. Published September 2023. Originally
forcep, as needed for cutting cured hides and skins).
approved in 2012. Last previous edition approved in 2021 as D7816 – 12 (2021).
DOI: 10.1520/D7816-23.
5.11 Vortex mixer, for mixing dilution tubes (optional).
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
5.12 pH meter.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 5.13 Waterbath, 45 °C 6 1 °C.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D7816 − 23
5.14 Autoclave thermometer, or equivalent for monitoring 9.4 Cool the agar (9.2) to 45 °C 6 1 °C, then add 100 mL
autoclave temperature. of the sterile 10 % powdered skim milk mixture (9.3) per litre
of ag
...

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 (Reapproved 2021) D7816 − 23
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, 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.
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
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, 2021Sept. 1, 2023. Published October 2021September 2023. Originally approved in 2012. Last previous edition approved in 20162021
as D7816 – 12 (2016).(2021). DOI: 10.1520/D7816-12R21.10.1520/D7816-23.
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 − 23
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.35 °C 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.
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. (for example, food storage type, sterile bag).
5.10 Cutting tool, sterile (e.g. (for example, 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
...