ASTM D7818-12(2021)

Standard Test Method for Enumeration of Proteolytic Bacteria in Fresh (Uncured) Hides and Skins

SIGNIFICANCE AND USE
4.1 This test method enumerates proteolytic bacteria. Proteolytic bacteria have been known to cause damage to hides and skins.
SCOPE
1.1 This test method covers the enumeration of bacteria that can hydrolyze protein/collagen in 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, 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: Historical
• Publication Date: 31-Aug-2021
• 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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: D7818 − 12 (Reapproved 2021)
Standard Test Method for
Enumeration of Proteolytic Bacteria in Fresh (Uncured)
Hides and Skins
This standard is issued under the fixed designation D7818; 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 plates are incubated under aerobic conditions at 35 °C for 48 h.
After incubation, to determine bacteria that can hydrolyze
1.1 This test method covers the enumeration of bacteria that
protein (proteolytic), the plates are flooded with dilute acid and
can hydrolyze protein/collagen in fresh (uncured) hides and
the colonies showing a halo are counted.
skins. This test method is applicable to uncured hides and
skins.
4. Significance and Use
1.2 The values stated in SI units are to be regarded as
4.1 This test method enumerates proteolytic bacteria. Pro-
standard. No other units of measurement are included in this
teolytic bacteria have been known to cause damage to hides
standard.
and skins.
1.3 This standard does not purport to address all of the
5. Apparatus
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
5.1 Incubator, 35 6 1 °C.
priate safety, health, and environmental practices and deter-
5.2 Colony counter—(not mandatory, but highly recom-
mine the applicability of regulatory limitations prior to use.
mended).
1.4 This international standard was developed in accor-
5.3 Sterile pipets.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
5.4 Bent glass rods, sterile.
Development of International Standards, Guides and Recom-
5.5 Stomacher, for mixing initial dilution. (If stomacher is
mendations issued by the World Trade Organization Technical
unavailable, hand-mix.)
Barriers to Trade (TBT) Committee.
5.6 Balance.
2. Referenced Documents
5.7 Sterile petri dishes.
2.1 ASTM Standards:
5.8 Autoclave (sterilizer). (Check the effectiveness of ster-
D6715 Practice for Sampling and Preparation of Fresh or
ilization weekly. For example, place spore suspensions or strips
Salt-Preserved (Cured) Hides and Skins for Chemical and
of Bacillus stearothermophilus (commercially available) inside
Physical Tests
glassware for a full autoclave cycle. Follow manufacturer’s
E691 Practice for Conducting an Interlaboratory Study to
directions for sterilization of specific media.)
Determine the Precision of a Test Method
5.9 Stomacher bags, or sterile, sealable quart plastic bag
E177 Practice for Use of the Terms Precision and Bias in
(for example, food storage type, sterile bag).
ASTM Test Methods
5.10 Cutting tool, sterile (for example, scalpel blade and
3. Summary of Test Method
forcep, as needed for cutting fresh hides and skins).
3.1 Samples of uncured hides and skins are serially diluted
5.11 Vortex mixer, for mixing dilution tubes (optional).
and plated on agar containing casein from skim milk. The
5.12 pH meter.
5.13 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.
5.14 Autoclave thermometer.
Current edition approved Sept. 1, 2021. Published October 2021. Originally
approved in 2012. Last previous edition approved in 2016 as D7818 – 12 (2016).
6. Reagents and Materials
DOI: 10.1520/D7818-12R21.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or 6.1 5 % acetic acid.
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
6.2 Butterfield’s Phosphate Stock Solution: Dissolve 34 g
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. KH PO (Potassium Phosphate monobasic) in 500 mL DI
2 4
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7818 − 12 (2021)
water. Adjust the pH to 7.2 6 0.1 with 1N – 6N NaOH. Bring 9.2 Autoclave the prepared agar for 15 min at 121 °C. (See
volume to 1 L with DI water. Sterilize for 15 min at 121 °C. Note 1.)
NOTE 1—Typical autoclave setting is 120 – 124 °C. (See 5.8.)
9.3 Prepare a 10 % powdered skim milk mixture by adding
6.3 Butterfield’s Phosphate Diluent (BPD): Take 1.25 mL of
10 g powdered skim milk to 100 mL DI water, then stirring the
Butterfield’s Phosphate Stock solution (6.2) and bring to 1 L
mixture to dissolve it. Autoclave the mixture for 15 min at 121
with DI water. Dispense into 1-L bottles and 9-mL dilution
°C. (See Note 1.)
tubes. Sterilize for 15 min at 121 °C. (See Note 1.)
9.4 Cool the agar (9.2) to 45 6 1 °C, then add 100 mL of the
6.4 Standard plate count agar containing 100 mL of 10 %
sterile 10 % powdered skim milk mixture (9.3) per litre of agar.
powdered skim milk solution per litre of agar.
NOTE 2—Do not allow agar to solidify prior to pouring (9.5).
6.5 Alcohol (for flame sterilizing), for example, 70 %
9.5 Pour the sterile agar into petri dishes. Replace the cover
Isopropyl alcohol.
and swirl to evenly distribute the agar. Allow to solidify at
6.6 Bent glass rod (“hockey-stick”).
room temperature on a flat surface. When solid, invert the petri
6.7 Powdered skim milk.
dishes, with the cover on the bottom, leaving a slight opening
to allow the plates to dry for ⁄2 h.
6.8 Distilled or deionized water.
6.9 Bacillus stearothermophilus spore suspensions or strips
10. Procedure
(commercially available), or equivalent.
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g
6.10 1N – 6N NaOH.
specimen in a sterile bag. Include both flesh and hair side.
7. Hazards
10.2 Add 180 g of BPD (6.3) diluent into the same sterile
7.1 All reagents and chemicals should be handled with care.
bag (10.1). Stomach or hand-massage for 1 min. This provides
Before using any chemical, read and follow all safety precau-
a 1:10 dilution.
tions and instructions on the manufacturer’s label or MSDS
10.3 Prepare the following sample dilutions using 9-mL
(Material Safety Data Sheet).
-2 -3 -4 -5 -6 -7
dilution tubes (BPD): 10 , 10 , 10 , 10 , 10 , and 10 (see
8. Sampling
Fig. 1).
10.3.1 Control Blank—In 10.9, incubate one of the petri
8.1 The specimen shall be sampled in accordance with
dishes prepared in 9.5 as-is, with the sample plates.
Practice D6715, and placed in sterile containers.
-2 -1
Example: To obtain a 10 dilution, mix the 10 dilution and
9. Preparation of Standard Plate Count Agar
-1
pipet 1 mL of that 10 dilution into a 9-mL dilution tube.
9.1 Prepare the standard plate count agar per manufacturer
NOTE 3—When transferring the aliquots between the tubes, the analyst
label directions. must use a different pipet or pipet tip for each transfer.
FIG. 1 Plating
D7818 − 12 (2021)
estimated count for that dilution is 560 × 10 × 10,000 = 56,000,000.
10.4 Pipet an appropriate portion (0.1 mL or 0.2 mL), of the
-2
Estimated counts can also be made on plates with <25 colonies: report as
10 dilution and place the liquid in the middle of a dried, skim
estimated counts.
milk agar plate.
10.11 Record each plate’s dilution and count on the work-
10.5 Flame sterilize a bent glass rod, or obtain a sterile,
sheet. This initial count is the aerobic plate count (A).
autoclaved bent glass rod.
10.6 Using the glass rod, spread the liquid evenly on the 10.12 For the same plate(s) counted in 10.10, flood the
plate(s) with 5 % acetic acid.
agar surface.
NOTE 6—Use enough of the 5 % acetic acid to fully cover the surface
10.7 Replace the cover and allow the plate to dry at room
of the plate.
temperature.
10.13 Pour off the acetic acid and immediately count only
10.8 Repeat steps 10.4 – 10.7 for each dilution.
those colonies completely surrounded by a semi-clear zone.
10.9 Invert all plates and incubate at 35 6 1 °C for 48 6 3
h. NOTE 7—Proteolytic Colonies—Do not count all the colonies inside the
zone; only count the colonies that cause/affect the shape of the zone. When
10.10 Following incubation, count only those plates that
identifying the semi-clear zone(s), it is sometimes helpful to pick up the
have 25 – 250 colonies.
plate and change the angle of view slightly to determine if it is a true circle
NOTE 4—If a plate shows confluent growth (i.e. bacterial growth covers
or if it has another protruberance off its edge. (For weak semi-clear zones,
the entire plate, making it impossible to determine the existence of
it is helpful to rotate the plate in the light to be sure about the clearing).
discrete colonies), record that plate’s count as TNTC – “Too Numerous To
Look for a circle of clearing around a centrally locate
...

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: D7818 − 12 (Reapproved 2016) D7818 − 12 (Reapproved 2021)
Standard Test Method for
Enumeration of Proteolytic Bacteria in Fresh (Uncured)
Hides and Skins
This standard is issued under the fixed designation D7818; 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 hydrolyze protein/collagen in 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 safety, health, and healthenvironmental 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
2.1 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 casein from skim milk. The plates are
incubated under aerobic conditions at 35°C 35 °C for 48 h. After incubation, to determine bacteria that can hydrolyze protein
(proteolytic), the plates are flooded with dilute acid and the colonies showing a halo are counted.
4. Significance and Use
4.1 This test method enumerates proteolytic bacteria. Proteolytic bacteria have been known to cause damage to hides and skins.
5. Apparatus
5.1 Incubator, 35 6 1°C.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, 2016Sept. 1, 2021. Published October 2016October 2021. Originally approved in 2012. Last previous edition approved in 20122016 as
D7818 – 12.D7818 – 12 (2016). DOI: 10.1520/D7818-12R16.10.1520/D7818-12R21.
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
D7818 − 12 (2021)
5.2 Colony counter—(not mandatory, but highly recommended).
5.3 Sterile pipets.
5.4 Bent glass rods, sterile.
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 fresh hides and skins).
5.11 Vortex mixer, for mixing dilution tubes (optional).
5.12 pH meter.
5.13 Waterbath, 45 6 1°C.1 °C.
5.14 Autoclave thermometer.
6. Reagents and Materials
6.1 5 % acetic acid.
6.2 Butterfield’s Phosphate Stock Solution: Dissolve 34 g KH PO (Potassium Phosphate monobasic) in 500 mL DI water. Adjust
2 4
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.121 °C.
NOTE 1—Typical autoclave setting is 120 – 124°C. 120 – 124 °C. (See 5.8.)
6.3 Butterfield’s Phosphate Diluent (BPD): Take 1.25 mL of Butterfield’s Phosphate Stock solution (6.2) and bring to 1 L with
DI water. Dispense into 1-L bottles and 9-mL dilution tubes. Sterilize for 15 min at 121°C. 121 °C. (See Note 1.)
6.4 Standard plate count agar containing 100 mL of 10 % powdered skim milk solution per litre of agar.
6.5 Alcohol (for flame sterilizing), e.g. for example, 70 % Isopropyl alcohol.
6.6 Bent glass rod (“hockey-stick”).
6.7 Powdered skim milk.
6.8 Distilled or deionized water.
D7818 − 12 (2021)
6.9 Bacillus stearothermophilus spore suspensions or strips (commercially available), or equivalent.
6.10 1N – 6N NaOH.
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 Standard Plate Count Agar
9.1 Prepare the standard plate count agar per manufacturer label directions.
9.2 Autoclave the prepared agar for 15 min at 121°C. 121 °C. (See Note 1.)
9.3 Prepare a 10 % powdered skim milk mixture by adding 10 g powdered skim milk to 100 mL DI water, then stirring the mixture
to dissolve it. Autoclave the mixture for 15 min at 121°C. 121 °C. (See Note 1.)
9.4 Cool the agar (9.2) to 45 6 1°C, 1 °C, then add 100 mL of the sterile 10 % powdered skim milk mixture (9.3) per litre of
agar.
NOTE 2—Do not allow agar to solidify prior to pouring (9.5).
9.5 Pour the sterile agar into petri dishes. Replace the cover and swirl to evenly distribute the agar. Allow to solidify at room
temperature on a flat surface. When solid, invert the petri dishes, with the cover on the bottom, leaving a slight opening to allow
the plates to dry for ⁄2 h.
10. Procedure
10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 g specimen in a sterile bag. Include both flesh and hair side.
10.2 Add 180 g of BPD (6.3) 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 using 9-mL dilution tubes (BPD): 10 , 10 , 10 , 10 , 10 , and 10 (see Fig. 1).
10.3.1 Control Blank—In 10.9, incubate one of the petri dishes prepared in 9.5 as-is, 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 9-mL dilution tube.
NOTE 3—When transferring the aliquots between the tubes, the analyst must use a different pipet or pipet tip for each transfer.
-2
10.4 Pipet an appropriate portion (0.1 mL or 0.2 mL), of the 10 dilution and place the liquid in the middle of a dried, skim milk
agar plate.
10.5 Flame sterilize a bent glass rod, or obtain a sterile, autoclaved bent glass rod.
10.6 Using the glass rod, spread the liquid evenly on the agar surface.
10.7 Replace the cover and allow the plate to dry at room temperature.
D7818 − 12 (2021)
FIG. 1 Plating
10.8 Repeat steps 10.4 – 10.7 for each dilution.
10.9 Invert all plates and incubate at 35 6 1°C 1 °C for 48 6 3 h.
10.10 Following incubation, count only those plates that have 25 – 250 colonies.
NOTE 4—If a plate shows confluent growth (i.e. bacterial growth covers the entire plate, making it impossible to determine the existence of discrete
colonies), record that plate’s count as TNTC – “Too Numerous To Count”). See Figs. 2 and 3 for diagrams of a countable plate and a TNTC plate,
respectively.
FIG. 2 Diagram of a Countable Plate
D7818 − 12 (2021)
FIG. 3 Diagram of Confluent Growth (TNTC)
NOTE 5—Count all the distinct colonies on the plate. If there are similar-appearing colonies growing in close proximity but not touching, count them as
individual colonies, provided the distance between them is at least equal to the diameter of the smallest colony. Colonies that are impinging, and that differ
in appearance, such as morphology or color, are counted as individual colonies. Colonies that are a cluster, and are similar in appearance, such as
morphology or color, are counted as one colony (see arrow Fig. 4 – “7:00” position). There may also be “spreaders”: a chain of colonies n
...