ASTM E1839-20

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Designation: E1839 − 13 E1839 − 20
Standard Test Method Practice for
Efficacy of Slimicides for the Paper Industry—Bacterial and
Fungal Slime
This standard is issued under the fixed designation E1839; 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 presents a procedure to evaluate the efficacy of slimicides for the control of bacterial and fungal slimes
in paper mill systems and their counterparts.
1.2 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow
them where appropriate (40 CFR 160).
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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:
D1193 Specification for Reagent Water
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 TAPPI Standard:
T 205 Forming Handsheets for Physical Tests of Pulp
2.3 CFR Standard:
40 CFR Part 160 Good Laboratory Practice Standards
This test method practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agentsand is the direct responsibility
of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2013Jan. 1, 2020. Published May 2013March 2020. Originally approved in 1996. Last previous edition approved in 20072013 as
E1839 – 07.E1839 – 13. DOI: 10.1520/E1839-13.10.1520/E1839-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from Technical Association of the Pulp and Paper Industry (TAPPI), 15 Technology Parkway South, Norcross, GA 30092, http://www.tappi.org.
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3. Terminology
3.1 For definitions of terms related to this practice, see Terminology E2756.
3.2 Definitions:
3.2.1 furnish, n—pulp slurry fed to a paper machine. The type of pulp (sulfite, Kraft, mechanical), the source of fiber (virgin,
recycled including pre- or post-consumer waste paper), and the pH are used to designate a specific type of furnish.
3.2.2 microbicide, n—a physical or chemical agent that kills microorganisms.
3.2.3 pulp, n—wood separated by chemical or mechanical means into their fibrous components. The pulp is used to make paper,
paper board, or pulp sheets after specific treatments. Hardwood pulp is made from trees, such as maples or oaks, and softwood
pulp is produced from trees, such as pines.
3.2.4 pulp slurry, n—an aqueous combination of cellulosic fibers, fillers, and other additives used for specific grades of paper.
3.2.5 slime, n—biofilm.
3.2.6 slimicides, n—chemical agent added during pulp and paper processing to reduce the growth of slime-forming
microorganisms.
4. Summary of Test Method
4.1 Bacterial cells or fungal spores are added to acid or alkaline pulp slurries, or both, treated with slimicides to achieve final
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concentrations of 2 × 10 to 1 × 10 bacteria/mL and/or 10 to 10 fungal spores/mL, and incubated at appropriate temperature for
determined time periods. Aliquots of the test suspension are then neutralized, plated onto bacterial or fungal medium, and observed
for growth. Results with biocide are compared to results without biocide (control).
4.2 As a performance standard, an effective slimicide is one that shows a continued reduction in bacterial and fungal counts
relative to the control over the duration of the test.
5. Significance and Use
5.1 This test method is to be used to determine if a slime control agent has application in the paper industry for control of
bacterial or fungal slime/biofilm.
5.2 This test method is run in acid, alkaline, or acid and alkaline conditions to determine the efficacy of the slime control agent.
5.3 The test conditions may be modified to reflect intended use patterns in typical paper mill systems, including use of actual
paper mill furnish.
6. Apparatus
6.1 Balance:
6.1.1 Plant Balance, one sensitive to 0.1 g at load of 200 g, with platform to weigh furnish and bottles used in the test.
6.1.2 Analytical Balance, one sensitive to 0.1 mg used for weighing the candidate slime control agent.
6.2 Sample Containers (Sterile), 120-mL plastic specimen containers with screw-cap lids are ideal for holding test materials.
Other suitable containers include 150/160-mL milk dilution bottles or WHIRL-PAKS.
6.3 Culture Containers, Petri plates, tissue culture bottles, or glass tubes (15 × 125 mm or 18 × 150 mm without lip, preferably
of borosilicate glass).
6.4 Closures, for tubes and containers.
6.5 Disintegrators, Appendix A (Specifications and Care of Apparatus (Disintegrator)) of T 205.
6.6 Flaming Equipment—Depending upon circumstances, either an alcohol lamp, a bunsen burner, or electric incinerator may
be used to flame inoculating needles and other equipment.
6.7 Incubators Capable of maintaining temperatures of 28 to 70 6 2°C to provide proper incubation temperatures. Temperature
should be consistent with the temperature of the product to be preserved.
6.8 pH Meter—Any suitable for standardizing the pH of the culture medium.
6.9 Pipets—1.1-mL milk dilution type, 1.0 mL graduated in 0.01 mL, and 10 mL graduated in 0.1 mL. Pipetters may be used,
but not for highly viscous materials.
6.9.1 Pipetting Aid—Rubber bulb or other device to accomplish the transfer of liquid.
6.10 Sterilizers, pressurized steam sterilizer or hot-air oven capable of 180 6 2°C for 2 6 0.2 h.
6.11 Filter Apparatus for Filter Sterilizing, Disposable filter units, appropriate volume, 0.2-μm pore size.
6.12 Sterile Funnel, with sterile glass wool or sterile cotton gauze for filtration of spores.
6.13 Colony counter, manual, such as Quebec, Buck, and Wolfhuegel, or proven colony image analyzer (electronic/scanner
type) are suitable for counting plates after incubation.
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6.14 Swabs, sterile, for aiding in removal of fungal spores from agar surface.
6.15 Hemacytometer, for counting spore suspension.
6.16 Microscope, providing a magnification range of 400 to 1000× with a suitable light source. Phase contrast or dark field
capability may be necessary.
6.17 Constant Temperature Shaker—A reliable constant-temperature shaker (water bath or incubator type), shall be used to
provide mixing and aeration and to maintain a selected temperature (62°C) during the contact period.
6.18 Mechanical Stirrer—Magnetic or propeller-type stirrers or any other suitable device.
7. Reagents and Materials
7.1 Purity of Reagents—Reagent chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents
shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such
specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
7.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean distilled water or water of
equal purity (see Specification D1193, Type III).
7.3 Buffer for Suspending Spores and for Dilutions, sample containers having 100-mL phosphate buffer dilution water, sterile,
for spore suspension have solid, sterile glass beads in container.
7.3.1 0.25 M Phosphate Buffer Stock Solution—Dissolve 34 g of reagent grade KH PO in 500 mL of distilled water and mix.
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Adjust to pH 7.2 with 1 N NaOH and dilute to 1 l.
7.3.2 Phosphate Buffer Dilution Water—Add 1.25 mL of 0.25 M phosphate buffer stock solution to 1 L of distilled water and
mix. Dispense to sample container and sterilize.
7.4 Aluminum Sulfate (Alum) [Al (SO ) 18H O]—Prepare a 0.4 % solution of the hydrated aluminum in distilled water and
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sterilize in an autoclave. Any loss of water during sterilization is made up by adding sterile distilled water. Alternatively, the
solution may be filter sterilized.
7.5 Acid and Base for pH Adjustment to Make Acid and Alkaline Furnish:
7.5.1 Prepare a 2.0 N solution of sulfuric acid in water. Sterilize by filtration.
7.5.2 Prepare a 2.0 N solution of sodium hydroxide in water. Sterilize by filtration.
7.6 Pulp—Biocide-free pulp, consisting of two-third hardwood and one-third softwood, typical of current production
techniques, is recommended. Disintegrate the sheet in distilled water until free of fiber clots and undispersed fiber bundles (T 205).
Avoid methods which involve extensive cutting of fibers. The concentration of the pulp in water should be 1 %.
7.7 Bacterial and Fungal Culture Medium:
7.7.1 Bacteria—Standard dehydrated tryptone glucose extract agar or equivalent is recommended. Adjust pH of culture medium
to pH of the test system.
7.7.2 Fungi—Sabouraud Dextrose Agar or Potato Dextrose Agar are recommended for enumeration. Adjust pH of culture
medium to pH of the test system.
7.8 Stock Slimicide Solution—Weigh appropriate amount of slimicide and add to distilled water so that when 1 mL is added to
the furnish, it gives the desired final concentration.
8. Test Organisms
8.1 Bacteria: Enterobacter aerogenes (ATCC 13048) and Pseudomonas aeruginosa (ATCC 15442)—Maintain these organisms
on tryptone glucose extract agar at 32 6 2°C.
8.2 Fungi: Aspergillus niger (ATCC 6275)—Maintain and grow for sporulation on Sabouraud dextrose agar, grow at 25 6 2°C.
Chaetomium globosum (ATCC 6205)—Maintain and grow for sporulation on Mineral Salts Agar with sterile filter paper at 25 6
2°C. Sabouraud Dextrose agar may also be used to grow for sporulation.
9. Preparation of Inoculum
9.1 Bacteria:
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
The sole source of supply of the apparatus known to the committee at this time is Zellerbach, 808 Rhodes Ave., Columbus, OH 43205. If you are aware of alternative
suppliers, please provide this information to ASTM Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,
which you may attend.
American Type Culture Collection, ATCC Medium 329.
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9.1.1 Grow the bacterial cultures on tryptone glucose extract agar at 32 6 2°C for 24 h. Add 1 mL of sterile water to the surface
of the slant. Gently scrape and suspend the organisms in the liquid.
9.1.2 Transfer this volume to a tryptone glucose extract agar milk dilution bottle flat and incubate at 32 6 2°C for 24 h. The
organisms harvested from this bottle flat will serve as inoculum for the pulp cultures.
9.1.3 To obtain this inoculum, add 10 mL of sterile water to the bottle flat from a 99 6 2-mL sterile water blank. Disperse the
growth on the flat throughout the water by gently moving it over the surface of the agar medium. Transfer 5 mL of this suspension
to the remaining 89 mL of sterile water in the water blank and shake the contents of the water blank vigorously to break up any
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bacterial clumps. Let settle. Take 1 ml of from upper half of bottle, and add this inoculum (2 × 10 to 1 × 10 cells/mL) to each
bottle containing the pulp substrate in the test. This inoculum can also be cultured on tryptone glucose agar petri plates and
harvested in a similar fashion.
9.2 Fungal Spores:
9.2.1 Fungi are grown on appropriate agar slants at 25 6 2°C for 7 days. The fungal spores are harvested by adding 2 mL of
sterile, distilled water with a non-toxic wetting agent such as sodium dioctyl sulfosuccinate (0.05 g/L water) to the surface of the
slant. Gently scrape and suspend the spores in the liquid.
9.2.2 Inoculate these spores onto appropriate agar and incubate at 25 6 2°C for 7 days. Spores are harvested (as in 9.2.3) and
serve as the inoculum for the pulp cultures.
9.2.3 To obtain this inoculum, add 5 to 10 mL of sterile, phosphate buffer dilution water having a non-toxic wetting agent such
as sodium dioctyl sulfosuccinate (0.05 g/L water) to the surface of the agar. Disperse the fungal spores and mycelia in the bottle
by gently scraping the surface of the medium. Alternatively, remove the growth from the surface of the medium, add it to a
screw-cap container with buffer and glass beads, and shake vigorously. Set aside for 5 to 10 min to let the hyphae settle.
9.2.4 Carefully remove the supernate with a pipet and filter through cotton gauze or glass wool plugged sterile funnel.
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9.2.5 Count the conidial suspension with a hemacytometer and standardize to 10 to 10 /mL with phosphate dilution buffer.
10. Pulp Cultures
10.1 Weigh 50 g of pulp slurry into a sample container. To ensure a uniform pulp suspension in each container, constantly agitate
the stock solution with a mechanical stirrer. Use only pulp suspension prepared the same day. Stopper or cap and sterilize. After
sterilization, allow the bottles to cool to approximately 25 6 2°C or the temperature used for the test.
10.2 Make the following additions aseptically to each bottle in the order named and shake vigorously after each addition, using
20 complete cycles in a vertical motion.
10.3 Furnish:
10.3.1 For acid furnish, add a sufficient amount of 2.0 N sulfuric acid to adjust the pH to between pH 5.0 and 5.5. Add 0.1 mL
of 0.4 % solution of Alum.
10.3.2 For alkaline furnish, add a sufficient amount of 2.0 N sodium hydroxide to adjust the pH to between pH 8.0 and 8.5.
10.3.3 Add other furnishes, appropriate for the specific paper mill applications.
10.4 Add 1 mL of slimicide stock solution of sufficient concentration to each furnish to achieve the desired final concentration
in parts per million. To the negative controls, add 1 mL of sterile water. Include in each test, as a positive control, a reference
biocide that is known to have activity in this test. Include a minimum of five concentrations of the biocide under test and the
reference biocide in each experiment. Run each test and reference biocide in duplicate.
10.5 Add the amount of sterile distilled water that is necessary to bring the total weight of the contents of the bottle to 99 6
1 g after all other additions have been made.
10.6 Add 1 mL of an aqueous suspension of the test organism to each pulp slurry. Prepare this suspension just before it is to
be used. The time interval between the preparation of the suspension of the test organism for inoculating the bottles and the
addition of the suspension to each bottle may have some effect on the resistance of the organism. Final bacterial concentration
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should be between 2 × 10 to 1 × 10 bacterial/mL of pulp slurry. Final fungal spore concentration should be between 10 to 10
spores/mL of pulp slurry.
10.7 After the final addition of the test organism, place the inoculated bottles on an incubator shaker and maintain at a
temperature of 32 6 2°C for bacteria and 25 6 2°C for fungi. An alternate test temperature may be used reflecting appropriate
use conditions.
10.8 Bacterial Incubation and Enumeration:
10.8.1 Incubate for at least 3 h. If desired, additional contact times may be included in the test. However, the 3-h contact period
shall always be included in the test. A control flask with bacteria but without biocide must be run, and bacterial numbers determined
at T = 0 and at least 3 h after bacterial addition.
10.8.2 After incubation, vigorously shake the pulp cultures and immediately plate it in tryptone glucose extract agar in
accordance with standard dilution plating techniques. Make standard dilutions in phosphate buffer. Neutralization of biocide at the
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end of the contact period should be demonstrated (see Test Methods E1054). It is important that the pulp cultures be plated in the
same order as they were inoculated, thus ensuring a uniform contact time. After the medium has solidified in the petri dishes,
incubate them at 32 6 2°C for 48 6 2 h.
10.8.3 At the completion of the incubation period, count the number of bacterial colonies, following standard microbiological
protocols.
10.9 Fungal Incubation and Enumeration—Incubate for up to 24 h. Remove aliquots for fungal enumeration after T = 0 and
at least 2 additional contact times, including the 24 h time. (See Note 1). Serially dilute on the appropriate agar. Initial serial
dilutions should be done in neutralizer, followed by dilutions in phosphate buffer dilution water. Neutralization process should be
shown to be effective against the slimicide being tested (see Test Methods E1054). Incubate at 25 6 2°C or appropriate temperature
of test for up to 14 days. After completion of the incubation period, count the number of fungal colonies following standard
microbiological protocols.
NOTE 1—Suggested contact times are any two of the following 3, 6, and 24 h.
11. Calculation
11.1 Bacteria:
11.1.1 Report the results as the percent kill using the following equation:
~A 2 B!
Kill, %5 3100 (1)
A
~ !
where:
A = count of initial control, and
B = count after 3-h incubation or other selected contact time interval.
11.1.2 Plot the data and determine the percent kill for the desired slimicide concentration. If more than one contact time is used,
a reasonable progression in percent kill should be found. The control should either remain unchanged or increase over time. If it
does not, then the test must be repeated.
NOTE 2—It has been observed that an effective slimicide gives a reduction in bacterial counts (kills) of 99 % relative to the control. Data generated
in the laboratory, however, is only indicative of biocide performance in the field. Effective bacterial control may require adjustments in biocide dosages
due to specific environmental conditions and application procedures in the paper systems.
11.2 Fungi:
11.2.1 An effective slimicide is one that shows a continued reduction in fungal counts relative to the control over the duration
of the test.
11.2.2 If there is any question regarding the growth of the test organism in the test series, repeat the test.
NOTE 3—An effective agent should give a reduction in fungal counts of 90 % relative to the control. Data generated in the laboratory, however, is only
indicative of biocide performance in the field. Biocide dosages and feed points need to be determined in the actual paper system.
12. Precision and Bias
12.1 A precision and bias statement cannot be made for this test method at this time.
13. Keywords
13.1 acid furnish; alkaline furnish; Aspergillus niger, ATCC 6275; bacteria; Chaetomium globosum, ATCC 6205; Enterobacter
aerogenes, ATCC 13048; fungi; paper pulp; Pseudomonas aeruginosa, ATCC 15442; slimicide
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