ASTM E1427-00
(Guide)Standard Guide for Selecting Test Methods to Determine the Effectiveness of Antimicrobial Agents and Other Chemicals for the Prevention, Inactivation and Removal of Biofilm
Standard Guide for Selecting Test Methods to Determine the Effectiveness of Antimicrobial Agents and Other Chemicals for the Prevention, Inactivation and Removal of Biofilm
SCOPE
1.1 Microorganisms attach to surfaces and grow, forming communities that are called biofilms. In addition to microorganisms, biofilms may contain the by-products of microbial growth ( that is, polysaccharides, enzymes, etc.), inorganic ions (that is, Mg, Ca, Fe, etc.) and organic materials (that is, oil, exudates from plants or animals, etc.). Biofilms may be found in many places, including on cooling system equipment ( that is, cooling towers, heat exchangers, etc.), water and oil pipelines, food and pharmaceutical processing surfaces and lines, dental water unit lines and medical prosthetic devices.
1.2 Biofilm formation may lead to reduced heat transfer in cooling towers, decreased fluid flow in pipelines, corrosion of metal surfaces, spoilage of food and pharmaceutical products, and infection in humans. The adverse impact of biofilm growth has led to the need for chemical or physical treatments for controlling them. This may involve preventing biofilm formation, inactivating microbes in biofilms and removing biofilms.
1.3 Since biofilms may form in many different types of systems, no one method can be presented that evaluates all the factors affecting biofilm control; therefore, many methods are presented for forming biofilms. Detecting and measuring biofilms and microorganisms within biofilms are important in evaluating control procedures. Many procedures are listed and referenced for measurement of microorganisms in biofilms and biofilm mass and activity.
1.4 The purpose of this guide is to inform the investigator of methods that can be used for biofilm formation and measurement, allowing development of test procedures for determining the effectiveness of chemical treatments for prevention, inactivation, and removal of unwanted biofilm. This guide is a teaching tool that will help the researcher in planning studies for controlling biofilms. This guide is not an exhaustive survey of biofilm methods. It is recommended that the researcher consult the latest information on biofilm methods from the published scientific literature and from appropriate internet sites, using biofilm as the keyword.
1.5 Discussions of various methods for evaluating efficacy of potential control materials against microorganisms in solution are available.
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Designation: E 1427 – 00
Standard Guide for
Selecting Test Methods to Determine the Effectiveness of
Antimicrobial Agents and Other Chemicals for the
Prevention, Inactivation and Removal of Biofilm
This standard is issued under the fixed designation E 1427; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope biofilm as the keyword.
1.5 Discussions of various methods for evaluating efficacy
1.1 Microorganisms attach to surfaces and grow, forming
of potential control materials against microorganisms in solu-
communities that are called biofilms. In addition to microor-
tion are available.
ganisms, biofilms may contain the by-products of microbial
growth (polysaccharides, enzymes, etc.), inorganic ions (Mg,
2. Referenced Documents
Ca, Fe, etc.) and organic materials (oil, exudates from plants or
2.1 This guide lists methods that can be used in forming and
animals, etc.). Biofilms may be found in many places, includ-
measuring biofilms, which allows development of test methods
ing on cooling system equipment (cooling towers, heat ex-
for determining the effectiveness of chemical and physical
changers, etc.), water and oil pipelines, food and pharmaceu-
treatments for prevention, inactivation, and removal of un-
tical processing surfaces and lines, dental water unit lines and
wanted biofilm. Published procedures for biofilm formation
medical prosthetic devices.
and measurement (Sections 4 and 5) are referenced.
1.2 Biofilm formation may lead to reduced heat transfer in
cooling towers, decreased fluid flow in pipelines, corrosion of
3. Significance and Use
metal surfaces, spoilage of food and pharmaceutical products,
3.1 This guide should be used by individuals responsible for
and infection in humans. The adverse impact of biofilm growth
the following:
has led to the need for chemical or physical treatments for
3.1.1 The maintenance of systems in which fluids come in
controlling them. This may involve preventing biofilm forma-
contact with surfaces, which adversely could be effected by the
tion, inactivating microbes in biofilms and removing biofilms.
presence of biofilm.
1.3 Since biofilms may form in many different types of
3.1.2 The development of methods, that is, chemicals, to
systems, no one method can be presented that evaluates all the
prevent, inactivate, or remove biofilm from various systems.
factors affecting biofilm control; therefore, many methods are
3.1.3 The verification of specific claims for chemicals to
presented for forming biofilms. Detecting and measuring
prevent, inactivate, or remove biofilm from specific systems.
biofilms and microorganisms within biofilms are important in
3.2 The systems considered include, but are not limited to,
evaluating control procedures. Many procedures are listed and
those designed for drinking water distribution, food processing,
referenced for measurement of microorganisms in biofilms and
industrial process fluids, and treated or untreated body fluids.
biofilm mass and activity.
3.2.1 The adverse effects of biofilm in these systems include
1.4 The purpose of this guide is to inform the investigator of
product spoilage, loss of production, corrosion, reduced heat
methods that can be used for biofilm formation and measure-
transfer, increased morbidity and mortality of the general
ment, allowing development of test procedures for determining
population, and outbreaks of hospital-acquired infections.
the effectiveness of chemical treatments for prevention, inac-
Since many different published methods, which have not
tivation, and removal of unwanted biofilm. This guide is a
undergone the rigors of ASTM Interlaboratory Testing, are
teaching tool that will help the researcher in planning studies
referenced, it is the responsibility of the investigator to verify
for controlling biofilms. This guide is not an exhaustive survey
the validity of the methods selected or developed for the
of biofilm methods. It is recommended that the researcher
intended application.
consult the latest on biofilm methods from the published
3.3 The information presented in Section 4 is a limited
scientific literature and from appropriate internet sites, using
Suggested internet sites are PubMed at the National Center for Biotechnology
Information (www.ncbi.nlm.nih.gov) and the American Society for Microbiology
This guide is under the jurisdiction of ASTM Committee E35 on Pesticides and
(www.journals.asm.org). Utilizing this technology the researcher may obtain the
is the direct responsibility of Subcommittee E35.15 on Antimicrobial Agents.
latest information on biofilms, and tailor their search for the specific information
Current edition approved Oct. 10, 2000. Published January 2001. Originally
they need.
published as E 1427 – 91. Last previous edition E 1427 – 91.
ASTM Standards on Materials and Environmental Microbiology, 2nd Edition,
1993.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1427
listing of test procedures, with references, for biofilm forma- 5.1.2.3 Rhodamine 123 (59).
tion. These procedures are a guide to the many ways that are 5.1.2.4 Tetrazolium salts (60-65).
used to form biofilms. Selection of specific test parameters 5.1.3 Molecular Probes:
enables simulation of applicable field conditions. Among the 5.1.3.1 r-RNA (66).
parameters that should be considered are nutrients, miscella- 5.1.3.2 Immunologic probes (66, 67).
neous nonnutrients organics, inorganic salts and ions, corrosion 5.1.4 Colony forming units or most probable number meth-
products, temperature, pH, redox potential, aerobic conditions, ods:
flow rate, shear, time, substratum (type and texture), and 5.1.4.1 Scraping and plating (68, 69).
microorganism types and their interactions (1,2). Methods that 5.1.4.2 Swabbing and plating (70).
can be used to measure biofilm formation are outlined in 5.1.4.3 Sonicating and plating (71).
Section 5. These are a limited number of referenced methods 5.1.4.4 Agar contact method (72, 73).
and are intended only as a guide. Methods selected by 5.1.4.5 Squeegee and rinse (74).
investigators depend on which criteria are most important in 5.1.4.6 Alginate or hydrogel/dissolve/plate (15, 75).
the system, that is, microbial population densities, biomasses 5.1.4.7 Biofilm growth in microtiter plates (31).
accumulation, or metabolic activities, or a combination thereof. 5.1.5 Radiolabelling to determine population density:
In any case, these methods should be used by individuals 5.1.5.1 Microautoradiography (76, 77).
familiar with microbiological techniques. 5.1.5.2 Radiolabelled cells (78).
5.2 Metabolic Activity—Gross activity of biofilm:
4. Substratum and Laboratory Methods for Biofilm
5.2.1 Bioluminance (76),
Formation, Either Static or Dynamic Models
5.2.1.1 ATP (77)
(Continuous or Batch) (1-9)
5.2.1.2 Lux gene (78, 79)
5.2.1.3 Tryptophan (80)
4.1 Coupons overlayed with microbial suspension (10, 11).
4.2 Coupons (metals, plastic, glass, etc.) in beakers or 5.2.2 Radiolabelled substrate uptake or metabolism of sub-
strate with release of radioactive compound (54, 84-86).
fouling loops 12-14).
4.3 Coupons overlayed with hydrogel (15). 5.2.3 Enzymatic (80, 87-90).
5.2.4 Impedance (91-93).
4.4 Polycarbonate membranes overlain with microbial sus-
pension (16). 5.2.5 Respirometry (94–95).
5.2.6 Microcalorimetry (96).
4.5 Plexiglass, reactor (17).
4.6 Glass beakers (18). 5.2.7 Nuclear magnetic Resonance (97).
5.2.8 Attenuated-total-reflection (ATR) Fourier-transform-
4.7 Powders or small beads in column or beaker (19-22).
4.8 Hydroxyapatite beads or discs (23, 24). infrared-spectroscopy (FTIR) (98).
5.3 Biomass—Total Viable and nonviable cells with associ-
4.9 Alginate beads (25, 26).
4.10 Tubing or pipe sections filled with or immersed in ated biofilm material:
5.3.1 Microscopy (99):
microorganism suspension (27).
4.11 Tubing/ or pipes in lab biofouling loop (28, 29). 5.3.1.1 Brightfield.
5.3.1.2 Phase contrast.
4.12 Prescored sample sections (30).
4.13 Stainless steel rings (14). 5.3.1.3 Epifluorescence (100-104).
5.3.1.4 Scanning Electron Microscope (105–106).
4.14 Microtiter plates (31-33).
4.15 Plugs (Robbin’s device), discs in rubber strips (5, 34). 5.3.1.5 Interference reflection and light section (107).
4.16 Rototorque (annular reactor) (35-37). 5.3.1.6 Differential interference contrast microscopy (108).
5.3.1.7 Electron microscope (109, 110).
4.17 Constant depth film fermentor (38).
4.18 Rotating Biological Reactor (39). 5.3.1.8 Confocal microscope (111).
5.3.2 Spectroscopic:
4.19 Rotating Disc Reactor Method (29, 40).
4.20 Model cooling tower (41, 42). 5.3.2.1 Bacteria on translucent surface (13, 112).
5.3.2.2 DNA absorption (260 nm/280 nm).
4.21 Parallel plate flow chamber or cell (43-46).
4.22 Capillary tubes (flowcells) (9, 47, 48). 5.3.3 Components of microorganisms (organic nitrogen,
carbon, chlorophyll, lipopolysaccharide lipid, protein, carbo-
5. Measurements of Biofilm
hydrate, fatty acid analysis, glycocalyx (4, 20, 88, 101,
113-116).
5.1 Population Viable Cell Density:
5.3.4 Weight (dry at 103°C, volatile 550°C) (117, 118).
5.1.1 Microscopic methods (Brightfield, Epifluorescence,
5.3.5 Thickness of biofilm (119).
Scanning Confocal).
5.3.6 Biofilm mass (120).
5.1.1.1 Growth response to nalidixic acid (52-54).
5.3.7 Heat transfer resistance (17).
5.1.2 Vital Dyes:
5.3.8 Pressure gradients/friction resistance (15, 120, 121).
5.1.2.1 Viablue 2 (59).
5.1.2.2 Fluorescein diacetate (56-58).
6. Keywords
6.1 biofilm; biomass; formation; inactivation; microbial ac-
tivity; population density; prevention; removal; sessile popu-
The boldface numbers in parentheses refers to the list of references at the end
of this standard. lation
E 1427
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