ASTM E2800-11(2017)

Standard Practice for Characterization of <emph type="ital">Bacillus</emph> Spore Suspensions for Reference Materials

SIGNIFICANCE AND USE
5.1 Standard practices for the characterization of spores used as reference materials are important to ensure a uniform basis for testing the performance of detection devices and laboratory instruments. Bacillus spore suspensions can be used for a large variety of purposes including testing environmental sampling techniques, inactivation methods, decontamination methods and basic research.  
5.2 The practice is intended for both manufacturers and end users of Bacillus spore suspensions. The results of the characterization measurements are presented in a report of analysis (ROA). The ROA should provide sufficient detail about the measurement technique to enable the customers to replicate the measurements, allowing them to determine if the properties of the spore suspension changed during shipping and storage.  
5.3 The enumeration of the viable spores and determination of homogeneity by microscopic analysis are two basic measurements required for the minimal characterization of reference materials. Phase contrast microscopy does not require staining to distinguish the “phase bright” dormant spores from phase dark spores, dark vegetative cells and clumps. When spores germinate they appear phase dark under phase contrast imaging (5). Germinated spores in a reference sample will soon die due to lack of nutrients. It is important in storing samples to prevent the premature germination of the spores. This standard practice includes the important steps for these measurements and includes guidance for advanced measurements. Additional guidance is given for advanced techniques to characterize spore suspensions that may be used to provide a higher level of characterized Bacillus  spore reference samples.  
5.4 The specific properties of the spores used for their intended application, such as susceptibility to disinfectant processes, should be determined in addition to the basic measurements covered in this practice. Additional information on the measurement of spore proper...
SCOPE
1.1 This practice is focused on two basic measurements to characterize Bacillus reference materials, the enumeration of spores using growth of colonies on nutrient media and using phase contrast microscopy to determine spore quality and homogeneity. Additional information on advanced methods for characterization is provided in Appendix X1.  
1.2 This document will provide the user with recommendations for measurement methods, and the details and conditions that should be employed to ensure reliable and high-quality data are obtained. The practice will help ensure that results obtained from the characterization are reported in a uniform manner. This will allow others to replicate the measurements and facilitate the comparison of different lots of Bacillus spore suspensions used as reference materials. It is important to note that the Bacillus species are a heterogeneous group and their specific requirements for growth and sporulation may vary. Users of this practice are encouraged to consult the literature for specific information on the species of Bacillus bacteria they are using (1).2  
1.3 This standard practice does not provide guidance for the identification of unknown species of bacteria. The identification of Bacillus species has been traditionally based on colony morphology, growth on selective media, and biochemical tests, but more recently nucleic acid technologies have enabled the phylogenetic analysis of this group based on 16S DNA sequence similarities (1).  
1.4 Some Bacillus spp. are pathogenic to humans and animals and the user is advised to adhere to safe laboratory procedures and practices for handling spores from these species (2). 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...

General Information

• Status: Published
• Publication Date: 31-Aug-2017
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: E54 - Homeland Security Applications
• Drafting Committee: E54.01 - CBRNE Detection and CBRN Protection

Relations

• Refers: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels&#x2014;Filtration and Culture Procedures
• Effective Date: 01-May-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM E2197-17 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E2197-17e1 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E2458-17 - Standard Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents and Toxins from Nonporous Surfaces
• Effective Date: 15-May-2017
• Refers: ASTM D4455-85(2014) - Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure (Withdrawn 2019)
• Effective Date: 01-Jan-2014
• Refers: ASTM D6974-09(2013)e1 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels&mdash;Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM D6974-09(2013)e2 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels-Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM D6974-09(2013) - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels&mdash;Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM E2197-11 - Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
• Effective Date: 01-Jan-2011
• Refers: ASTM E2458-10 - Standard Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents from Nonporous Surfaces
• Effective Date: 01-Oct-2010
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D6974-09 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels&#8212;Filtration and Culture Procedures
• Effective Date: 01-Jun-2009
• Refers: ASTM D4455-85(2009) - Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure
• Effective Date: 01-May-2009
• Refers: ASTM E1873-06 - Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique (Withdrawn 2014)
• Effective Date: 01-Nov-2006

Overview

ASTM E2800-11(2017), Standard Practice for Characterization of Bacillus Spore Suspensions for Reference Materials, provides a uniform basis for assessing Bacillus spore suspensions used as reference materials. Developed by ASTM, this international standard enables both manufacturers and end users to reliably characterize and compare Bacillus spore samples. Accurate characterization is crucial for applications such as testing detection devices, validating laboratory instruments, evaluating decontamination methods, and conducting scientific research.

By detailing the minimal and advanced requirements for spore enumeration and analysis, ASTM E2800 ensures data consistency and traceability throughout production, testing, and storage of Bacillus spore suspensions. The standard aims to foster reproducibility and reliability across laboratories and industries that require precise microbial reference materials.

Key Topics

• Spore Enumeration: The standard outlines practices for quantifying viable Bacillus spores by plating on nutrient agar to count colony forming units (CFU/mL). Serial dilution and aseptic techniques are emphasized to ensure robust measurements.

• Phase Contrast Microscopy: Methods are presented for evaluating spore suspension quality and homogeneity. Phase contrast microscopy is used to distinguish between phase bright (dormant, viable) spores and phase dark vegetative cells or debris, allowing users to assess sample purity and stability.

• Reporting and Replicability: Users are required to document the characterization process via a report of analysis (ROA), ensuring all relevant details about measurement techniques, results, and sample handling are reported for reproducibility.

• Advanced Characterization Guidance: Appendices guide users through advanced analytical techniques that can further enhance the level of sample characterization, such as measurement of spore resistance, density, DNA content, and surface properties.

• Storage and Safety: Recommendations are included for the proper storage of Bacillus spore suspensions to prevent premature germination, maintain viability, and avoid contamination. Safe handling practices and laboratory biosafety measures are emphasized due to the pathogenic potential of some Bacillus species.

Applications

ASTM E2800 is widely applicable in settings that depend on standardized, high-quality microbial reference materials:

• Calibration and Verification: Used in the calibration and verification of biological detection systems and environmental sampling devices.
• Decontamination and Inactivation Studies: Supports assessment of decontamination protocols and inactivation methods for Bacillus spores.
• Basic and Applied Research: Facilitates reproducible results in microbiological and biochemical research involving Bacillus species.
• Manufacturing Quality Control: Guides quality assurance in the production and certification of Bacillus spore reference materials.
• Regulatory and Compliance Testing: Supports laboratories in meeting international standards for test method validation, sterility testing, and compliance with health and safety regulations.

Related Standards

For comprehensive reference material characterization and laboratory best practices, users should consult these related standards and documents:

• ASTM D6974 – Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels
• ISO 4833 – Microbiology of Food and Animal Feeding Stuffs: Enumeration of Microorganisms (Colony-Count Technique)
• ISO 21528-1 – Microbiology of Food: Detection and Enumeration of Enterobacteriaceae
• USP Microbiological Best Laboratory Practices (USP 29 Suppl 2)
• Standard Methods for Examination of Water and Wastewater (9215 & 9218)
• EPA Standard Procedures for Enumeration of Bacterial Inocula

Practical Value

Implementing ASTM E2800 ensures Bacillus spore suspensions used in laboratory testing and validation processes are characterized reliably and consistently. This standard delivers:

• Improved comparability of analytical results across laboratories
• Consistent reporting and traceability for regulatory compliance
• Increased confidence in the quality and stability of reference materials
• Enhanced biosafety through recommended handling and storage protocols

By following ASTM E2800, organizations can achieve high-quality Bacillus spore reference materials essential for testing, research, and product validation in both public health and industrial settings.

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: E2800 − 11 (Reapproved 2017)
Standard Practice for
Characterization of Bacillus Spore Suspensions for
Reference Materials
This standard is issued under the fixed designation E2800; 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.
INTRODUCTION
Bacillus spp. are aerobic, rod-shaped, Gram positive bacteria that produce endospores under
nutrient limiting conditions. The endospores are designed to persist in extreme environments and
consequently are highly resistant to inactivation by heat, chemicals and irradiation. A few species of
Bacillus are medically important because of their impact on human and animal health while others
have important agricultural and industrial applications. Measurement of viable Bacillus spores present
in a suspension can be performed using classical microbiology techniques, such as growth on nutrient
medium. The spore suspension is diluted, an aliquot spread on solid nutrient medium, incubated at an
appropriate temperature, and the resulting colonies counted. The selection of the type of growth
medium and incubation temperature for the optimal growth of a particular Bacillus species should be
determined by consultation of relevant literature or by comparison of different growth media and
incubation temperatures.
Bacillus spore reference materials have many important applications in agriculture, basic research,
medical diagnosis, detector validation, and sterility testing. Uniform methods for the characterization
of spores will improve the comparison of different lots of materials and results between different
laboratories.
1. Scope Users of this practice are encouraged to consult the literature
for specific information on the species of Bacillus bacteria they
1.1 This practice is focused on two basic measurements to
are using (1).
characterize Bacillus reference materials, the enumeration of
spores using growth of colonies on nutrient media and using 1.3 This standard practice does not provide guidance for the
phase contrast microscopy to determine spore quality and identification of unknown species of bacteria. The identifica-
homogeneity. Additional information on advanced methods for tion of Bacillus species has been traditionally based on colony
characterization is provided in Appendix X1. morphology, growth on selective media, and biochemical tests,
but more recently nucleic acid technologies have enabled the
1.2 This document will provide the user with recommenda-
phylogenetic analysis of this group based on 16S DNA
tions for measurement methods, and the details and conditions
sequence similarities (1).
that should be employed to ensure reliable and high-quality
data are obtained. The practice will help ensure that results 1.4 Some Bacillus spp. are pathogenic to humans and
obtained from the characterization are reported in a uniform animals and the user is advised to adhere to safe laboratory
manner. This will allow others to replicate the measurements procedures and practices for handling spores from these
and facilitate the comparison of different lots of Bacillus spore species (2). This standard does not purport to address all of the
suspensions used as reference materials. It is important to note safety concerns, if any, associated with its use. It is the
that the Bacillus species are a heterogeneous group and their responsibility of the user of this standard to establish appro-
specific requirements for growth and sporulation may vary. priate safety and health practices and determine the applicabil-
ity of regulatory limitations prior to use (2).
1.5 This practice assumes a basic knowledge of microbiol-
This practice is under the jurisdiction of ASTM Committee E54 on Homeland ogy and molecular biology and access to the cited references.
Security Applications and is the direct responsibility of Subcommittee E54.01 on
CBRNE Detection and CBRN Protection.
Current edition approved Sept. 1, 2017. Published October 2017. Originally
approved in 2011. Last previous edition approved in 2011 as E2800 – 11. DOI: The boldface numbers in parentheses refer to a list of references at the end of
10.1520/E2800-11R17. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2800 − 11 (2017)
1.6 The values stated in SI units are to be regarded as ISO 21528-1:2004 Microbiology of food and animal feeding
standard. No other units of measurement are included in this stuffs -- Horizontal methods for the detection and enu-
standard. meration of Enterobacteriaceae – Part 1: Detection and
enumeration by MPN technique with pre-enrichment
1.7 This international standard was developed in accor-
2.4 United States Pharmacopeia Standards:
dance with internationally recognized principles on standard-
USP. 2006 Microbiological Best Laboratory Practices. USP
ization established in the Decision on Principles for the
29 Suppl 2 pp. 3804-3807
Development of International Standards, Guides and Recom-
USP. 2003 Good Microbiological Laboratory Practices.
mendations issued by the World Trade Organization Technical
Pharmacopeial Forum 29(3):842-850.
Barriers to Trade (TBT) Committee.
USP. 2004 Microbiological Best Laboratory Practices. Phar-
2. Referenced Documents macopeial Forum 29(3):1713-1721
2.1 ASTM Standards:
3. Terminology
D1129 Terminology Relating to Water
3.1 Definitions:
D4455 Test Method for Enumeration of Aquatic Bacteria by
3.1.1 colony forming unit (CFU), n—units for the number of
Epifluorescence Microscopy Counting Procedure (With-
viable particles present in a solution. A CFU can result from a
drawn 2019)
single viable bacterial cell or from a clump of cells. (D1129)
D6974 Practice for Enumeration of Viable Bacteria and
Fungi in Liquid Fuels—Filtration and Culture Procedures 3.1.2 vortex mixing, v—applying a tube containing a liquid
E1873 Guide for Detection of Nucleic Acid Sequences by sample to a special laboratory mixer that establishes a vigorous
the Polymerase Chain Reaction Technique (Withdrawn circular motion in the bottom of the tube.
2014) 3.1.2.1 Discussion—The circular mixing motion results in a
E2197 Quantitative Disk Carrier Test Method for Determin- vortex in the tube that ensures the complete suspension of the
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal, entire tube contents.
and Sporicidal Activities of Chemicals
4. Summary of Practice
E2414 Test Method for Quantitative Sporicidal Three-Step
Method (TSM) to Determine Sporicidal Efficacy of
4.1 Viable Spore Concentration by Plating on Nutrient
Liquids, Liquid Sprays, and Vapor or Gases on Contami-
Agar—Plating bacteria on nutrient media is a well-established
nated Carrier Surface (Withdrawn 2014)
method for detection of bacteria in water (3). Suspensions of
E2458 Practices for Bulk Sample Collection and Swab
spores are first mixed well by vortex mixing or pipetting up and
Sample Collection of Visible Powders Suspected of Being
down vigorously to insure homogeneity of the spore suspen-
Biological Agents and Toxins from Nonporous Surfaces
sion and then serially diluted using an appropriate buffer. Three
2.2 Standard Methods for the Examination of Water and
serial dilutions are prepared from a spore reference sample. An
Wastewater: aliquot of the diluted spore suspension is placed on a nutrient
Method 9218 Aerobic Endospores (2007)
agar plate and spread using aseptic techniques. After
Method 9215 Heterotrophic Plate Count (2004) incubation, the colonies on the plates are counted and the
Environmental Protection Agency Standard Procedure for
numbers of viable spores are referred to as colony forming
Enumeration of Bacterial Inocula on Carriers (Carrier units (CFU). The average number of colonies obtained from
Counts) for the Germicidal Spray Products as Disinfec-
the diluted suspension are used to calculate the concentration
tants Test, Disinfectant Towelette Test, and the Tubercu- of the original stock solution and reported as CFU/mL. In order
locidal Activity of Disinfectants Test SOP Number: MD-
to obtain consistent results, careful attention must be paid to
04-05 Date Revised: 01-13-09 detail to ensure adequate dispersion of spores and avoiding
2.3 ISO Standards:
losses during the process.
ISO 4833:2003 Microbiology of food and animal feeding
4.2 Spore Quality and Homogeneity Determined by Phase
stuffs -- Horizontal method for the enumeration of micro-
Contrast Microscopy—An inexpensive, rapid and effective
organisms – Colony-count technique at 30 degrees C
method to determine quality and homogeneity of spore prepa-
rations. A drop of the spore sample is placed on a microscope
slide and covered with a coverslip. The spore preparation is
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
examined using a high power objective (typically 100×) with a
Standards volume information, refer to the standard’s Document Summary page on
phase contrast microscope. Phase bright spores, phase dark
the ASTM website.
spores, phase dark vegetative cells, and spore clumps are
The last approved version of this historical standard is referenced on
www.astm.org. counted either manually, using automated counting devices or
Available from American Public Health Association, Standard Methods for the
by digital imaging and computer software techniques (4). The
Examination of Water and Wastewater, Washington, DC 20001, http://
percentage of phase bright spores in the sample is calculated
www.standardmethods.org/.
and reported.
Available from United States Environmental Protection Agency (EPA), Ariel
Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http://
www.epa.gov. 5. Significance and Use
Available from International Organization for Standardization (ISO), 1, ch. de
5.1 Standard practices for the characterization of spores
la Voie-Creuse, Case postale 56, CH-1211, Geneva 20, Switzerland, http://
www.iso.ch. used as reference materials are important to ensure a uniform
E2800 − 11 (2017)
basis for testing the performance of detection devices and 6.12 Glass Microscope Slides, precleaned, typically 25 by
laboratory instruments. Bacillus spore suspensions can be used 75 mm.
for a large variety of purposes including testing environmental
6.13 Glass Coverslips.
sampling techniques, inactivation methods, decontamination
6.14 Immersion Oil, as recommended by microscope manu-
methods and basic research.
facturer suitable for objective used and coverslips.
5.2 The practice is intended for both manufacturers and end
users of Bacillus spore suspensions. The results of the charac-
7. Reagents
terization measurements are presented in a report of analysis
7.1 Purity of Water—Water used for preparation of solutions
(ROA). The ROA should provide sufficient detail about the
should be sterile and high purity; either reverse osmosis,
measurement technique to enable the customers to replicate the
de-ionized or distilled.
measurements, allowing them to determine if the properties of
the spore suspension changed during shipping and storage. 7.2 Phosphate Buffered Saline (PBS)—A typical composi-
tion is composed of 0.137 M NaCl, 0.0027 M KCl, 0.01 M
5.3 The enumeration of the viable spores and determination
sodium phosphate, pH 7.4. Other similar formulations may be
of homogeneity by microscopic analysis are two basic mea-
used. The solution should be sterilized by autoclaving or
surements required for the minimal characterization of refer-
filtration.
ence materials. Phase contrast microscopy does not require
staining to distinguish the “phase bright” dormant spores from 7.3 Triton X100™ Stock Solution (10 % vol./vol.), prepared
phase dark spores, dark vegetative cells and clumps. When
in sterile water in a sterile container.
spores germinate they appear phase dark under phase contrast
7.4 Nutrient Agar Plates—May be prepared in the labora-
imaging (5). Germinated spores in a reference sample will soon
tory or purchased. Plates should be stored and used within
die due to lack of nutrients. It is important in storing samples
expiration date. Typically, laboratory prepared plates are stored
to prevent the premature germination of the spores. This
at 4°C and used within 2 weeks. Specific media such as 5 %
standard practice includes the important steps for these mea-
(vol./vol.) sheep blood agar plates may provide important
surements and includes guidance for advanced measurements.
colony morphology that can assist in the conformation of
Additional guidance is given for advanced techniques to
bacteria.
characterize spore suspensions that may be used to provide a
7.5 Bleach, freshly diluted, 10 % (vol./vol.). Confirm that
higher level of characterized Bacillus spore reference samples.
the stock solution (commercial bleach, sodium hypochlorite)
5.4 The specific properties of the spores used for their
has not expired.
intended application, such as susceptibility to disinfectant
7.6 Ethanol, 70 % (vol./vol.), prepared in sterile water.
processes, should be determined in addition to the basic
measurements covered in this practice. Additional information
8. Hazards
on the measurement of spore properties is located in the
appendix.
8.1 Considerations for safe handling of spore suspensions.
Some Bacillus spp. cause disease in human and animals. Prior
6. Apparatus
to using these materials, the user must fully investigate the
safety hazards associated with the particular Bacillus spp. and
6.1 Pipettes, fixed volume or adjustable. The performance
follow the appropriate safety guidelines. The correct training of
of the pipettes should be checked to determine correct dispens-
personnel and the proper use of personal protective equipment
ing volume. (pipettors should be tested for proper performance
(PPE) is essential. A good source for the information on
frequently).
laboratory safety is the publication “Biosafety in Microbiologi-
6.2 Sterile Pipette Tips.
cal and Biomedical laboratories” (2). Route of infection and
6.3 Vortex Mixer.
infectious dose and toxin production impact potential for
infection/toxicity.
6.4 Incubator, capable of maintaining 30 to 70 6 2°C.
8.2 Use of good microbiology practices is important for
6.5 Autoclave, for preparing sterile media and sterilizing
safely working with the pathogenic Bacillus species. Interna-
waste.
tional guidelines for microbiological safety should be followed
6.6 Plate Spinner (optional).
when appropriate (6).
6.7 Bunsen Burner or Alcohol Lamp (optional).
9. Sample Storage
6.8 Sterile Glass or Sterile Plastic Disposable Spreader
Rods.
9.1 Appropriate storage conditions are essential to preserve
the properties of the spores. Preservation of spore viability
6.9 Phase Contrast Microscope—Low power (10 to 20×)
(prevention of germination), sterility, and lack of clumping are
and high power phase (40, 60, or 100×) objectives are
the goals. Traditionally, spores have been stored in solutions of
preferred.
sterile water, 20 to 70 % (vol./vol.) ethanol or 1 % (wt./vol.)
6.10 Disposable Plastic (or Glass) Petri Dishes, typically
phenol in water at 4°C to prevent bacterial growth. Spores can
100 mm in diameter and 15 mm deep, sterile.
be frozen, but the effect of freezing and thawing on the
6.11 Dilution Tubes, sterile. properties of the spores has to be determined.
E2800 − 11 (2017)
9.2 It is essential to prevent premature germination of used. The conditions for pre-drying the plates in an incubator
spores, clumping, and loss of spores during storage. One study overnight or under a sterile hood with the lid ajar should be
used borosilicate glass vials with PTFE lined caps to store determined for each type of agar media (11).
spores in a number of solutions including sterile deionized
10.1.5 After spreading, the covers are replaced and the
water, 20 % ethanol and 1 % phenol for periods up to several
plates are placed in an incubator at the desired temperature
years at 4°C and found the spore viability for B. anthracis
(typically 30 to 37 6 2°C). The temperature should be
Sterne to be stable for over several years (7).
measured with a NIST-traceable thermometer.
10.1.6 A good source of the composition and performance
9.3 It is important that the storage condition for each
TM TM
of growth media is the Difco & BBL (12), Manual of
particular source of spores should be investigated to determine
TM
Microbiological Culture Media, Remel catalog or the ASM
the effect of storage on specific properties and viability.
Media Manual. Quality control of the growth media should be
Traditionally spore preparations are considered to be high
performed to ensure reliable results (13). The storage condition
quality if they contain greater than 90 % phase-bright viable
and shelf life of growth media, as recommended by the
spores as determined in the following sections.
manufacturer, should be followed.
10.1.7 The plates are removed from the incubator, typically
10. Procedure
after 18 h, but the optimal time should be established depend-
10.1 Spore Viability and Concentration by Growth on Agar-
ing upon the species, temperature and media used. The
Containing Media:
bacterial colonies are counted either manually, with an elec-
10.1.1 Several important factors should be considered when
tronic counter or imaged with a camera. Only those plates
using Bacillus spore suspensions, including the selection of the
having between 30 and 300 colonies should be used for
solution used for dilution, the containers used for dilution, and
determining the concentration of the spores.
any treatment to reduce potential aggregates (clumps) of spores
10.1.8 The colony forming units (CFUs) in the sample are
present in the samples. The choice of dilution media and
calculated by dividing the actual colonies on a plate by the
container should be selected to reduce losses of the spores due
volume applied to the plate. This result is divided by the
to adhesion to the surfaces of the container (and pipette
dilution factor to calculate CFU/mL. The average CFU values
surfaces) during dilution. A suitable solution for dilution of
and the standard deviations of the measurement should be
TM
spore suspensions is PBS containing Triton X-100 (0.05%
reported. An example of the calculation is shown in the
vol./vol) (8). Other diluents and treatments can be used (7-10),
appendix.
but their suitability for the spores from a particular species of
10.1.9 Low Concentrations of Spores—It is anticipated that
Bacillus bacteria should be determined.
spore suspensions used as reference materials will be suffi-
10.1.2 Spore suspensions have to be adequately mixed
ciently concentrated for plate count determinations or visual-
immediately before dilution and sampling because the spores
ization by phase contrast microscopy. However, with low
will settle in the bottom of the tubes. Vortex mixing the tubes
concentrations of spores, samples can be concentrated by either
for 30 s before removing a sample is an effective step to
centrifugation or filtration as used for detection of spores in
suspend the spores.
water systems (14).
10.1.3 At least three replicates of the serial dilutions should
10.1.9.1 A short centrifugation, for example 12 000 × g for
be done on the spore samples. A typical dilution would be to
5 min can be used. The spores can then be suspended in a
vortex mix the stock suspension (30 s), immediately remove
smaller volume. Care must be taken to ensure this step has not
0.1 mL and add this to 0.9 mL of the diluents in a new tube,
-1 -1
resulted in losses or in the formation of spore clumps. Avoid
resulting in a 10 dilution. Repeating this process from the 10
-2
excessive centrifugation or heating of sampl
...