ASTM F838-20

Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration

SIGNIFICANCE AND USE
5.1 This test method is designed to assess the retentivity of a sterilizing filter under standard challenge conditions.  
5.1.1 A challenge of 107 bacteria per cm2 of effective filtration area is selected to provide a high degree of assurance that the method has sufficient sensitivity to detect oversized pores and that the filter will quantitatively retain large numbers of organisms. The model challenge organism, B. diminuta, is widely considered to be a small bacterium and is recognized as an industry standard for qualifying sterilizing filters. Other species may represent a worst-case test in terms of ability to penetrate a filter. This test does not provide assurance that filters can completely retain such bacteria.  
5.1.2 The analytical procedure utilized in this test method provides a method to assign a numerical value to the filtration efficiency of the filter being evaluated under standard filtration conditions. For the purpose of product sterility assurance, additional process-specific studies should be performed.
SCOPE
1.1 This test method determines the bacterial retention characteristics of membrane filters for liquid filtration using Brevundimonas diminuta as the challenge organism. This test method can be used to evaluate any membrane filter system used for liquid sterilization.  
1.2 This test method is not intended to be used in performance of product- and process-specific validation of the bacterial retention characteristics of membrane filters to be used in pharmaceutical or biopharmaceutical sterilizing filtration, or both. Process- and product-specific bacterial retention validation should be carried out using the intended product manufacturing process parameters and the product solution or surrogate as the carrier fluid.  
1.3 The values stated in SI units are to be regarded as standard.  
1.3.1 Exception—The inch-pound values given for units of pressure are to be regarded as standard; SI unit conversions are shown in parentheses.  
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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 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: 30-Sep-2020
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
• Drafting Committee: E55.14 - Measurement Systems and Analysis

Relations

• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999

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: F838 − 20
Standard Test Method for
Determining Bacterial Retention of Membrane Filters
Utilized for Liquid Filtration
This standard is issued under the fixed designation F838; 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 2. Referenced Documents
1.1 This test method determines the bacterial retention 2.1 ASTM Standards:
characteristics of membrane filters for liquid filtration using D1193 Specification for Reagent Water
Brevundimonas diminuta as the challenge organism. This test
3. Terminology
method can be used to evaluate any membrane filter system
used for liquid sterilization.
3.1 Definitions:
3.1.1 log reduction value, n—the logarithm to the base 10 of
1.2 This test method is not intended to be used in perfor-
the ratio of the number of microorganisms in the challenge to
mance of product- and process-specific validation of the
the number of organisms in the filtrate.
bacterial retention characteristics of membrane filters to be
used in pharmaceutical or biopharmaceutical sterilizing
4. Summary of Test Method
filtration, or both. Process- and product-specific bacterial
4.1 After sterilization, the test filter is challenged with a
retention validation should be carried out using the intended
3 7
suspension of B. diminuta (ATCC 19146 ) to provide 10
product manufacturing process parameters and the product
organisms per cm of effective filtration area (EFA) at a
solution or surrogate as the carrier fluid.
maximum differential pressure across the test filter of 30 psig
1.3 The values stated in SI units are to be regarded as –3
(206 kPa) and a flux of 2 to 4 × 10 L per min (3.3 to 6.7 ×
standard. –8 3 2
10 m /s) per cm of effective filtration area. The entire filtrate
1.3.1 Exception—The inch-pound values given for units of
is passed through an analytical membrane filter disc, which is
pressure are to be regarded as standard; SI unit conversions are
subsequently incubated on a solidified growth medium. Micro-
shown in parentheses.
organisms that are not retained by the filter being tested will
1.4 This standard does not purport to address all of the
develop into visible colonies on the analysis membrane and can
safety concerns, if any, associated with its use. It is the
then be enumerated.
responsibility of the user of this standard to establish appro-
5. Significance and Use
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
5.1 This test method is designed to assess the retentivity of
1.5 This international standard was developed in accor-
a sterilizing filter under standard challenge conditions.
7 2
dance with internationally recognized principles on standard-
5.1.1 A challenge of 10 bacteria per cm of effective
ization established in the Decision on Principles for the
filtration area is selected to provide a high degree of assurance
Development of International Standards, Guides and Recom-
that the method has sufficient sensitivity to detect oversized
mendations issued by the World Trade Organization Technical
pores and that the filter will quantitatively retain large numbers
Barriers to Trade (TBT) Committee.
of organisms. The model challenge organism, B. diminuta, is
1 2
This test method is under the jurisdiction of ASTM Committee E55 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Manufacture of Pharmaceutical and Biopharmaceutical Products and is the direct contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
responsibility of Subcommittee E55.14 on Measurement Systems and Analysis. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Oct. 1, 2020. Published October 2020. Originally the ASTM website.
ɛ1 3
approved in 1983. Last previous edition published in 2015 as F838 – 15a . DOI: Available from American Type Culture Collection (ATCC), 10801 University
10.1520/F0838-20. Boulevard, Manassas, VA 20110, http://www.atcc.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F838 − 20
widely considered to be a small bacterium and is recognized as the specifications of the American Chemical Society, where
an industry standard for qualifying sterilizing filters. Other such specifications are available.
species may represent a worst-case test in terms of ability to
7.2 Purity of Water—Unless otherwise indicated, references
penetrate a filter. This test does not provide assurance that
to water shall mean reagent water, Type IV as defined in
filters can completely retain such bacteria.
Specification D1193.
5.1.2 The analytical procedure utilized in this test method
7.2.1 Additionally, any water used in this test method must
provides a method to assign a numerical value to the filtration
conform to the requirements for non-bacteriostatic water speci-
efficiency of the filter being evaluated under standard filtration
fied in the current edition of Standard Methods for the
conditions. For the purpose of product sterility assurance, 5
Examination of Water and Wastewater.
additional process-specific studies should be performed.
8. Reagents and Materials
6. Apparatus
8.1 Saline Lactose Broth Medium:
6.1 Assemble the apparatus described below as in Fig. 1:
8.1.1 Lactose Broth—Dissolve 1.3 g of dehydrated lactose
6.1.1 Stainless Steel Pressure Vessel, 12-L capacity (or
broth medium in 100 mL of water.
larger), fitted with a 0 to 50-psi (0 to 350-kPa) pressure gauge.
8.1.2 Sodium Chloride Solution—Dissolve 7.6 g of sodium
6.1.2 Air Regulator.
chloride (NaCl) in 970 mL of water in a 2-L flask with an
6.1.3 47-mm or 142-mm Analysis Disc Filter Assemblies,
appropriate closure.
two or more, with hose or sanitary connections as applicable.
8.1.3 Add 30 mL of lactose broth (8.1.1) to 970 mL of
6.1.4 Diaphragm-Protected 0 to 50-psi (0 to 350-kPa)
sodium chloride solution. Autoclave at 121 °C for 15 min.
Pressure Gauge, for upstream pressure reading.
8.2 Frozen Cell Paste Method:
6.1.5 Manifold, with valves (autoclavable) and hose connec-
8.2.1 Growth Medium A—Dissolve in water and dilute to 1
tions.
L. Autoclave at 121 °C for 15 min (pH 6.8 to 7.0).
6.1.6 Autoclavable Tubing, (must be able to withstand a
pressure of 50 psi (350 kPa)).
6.1.7 Filter Housing, with hose connections.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
6.1.8 Hose Clamps.
Standard-Grade Reference Materials, American Chemical Society, Washington,
6.1.9 Test Filter.
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,
7. Purity of Reagents and Materials U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
copeial Convention, Inc. (USPC), Rockville, MD.
7.1 Purity of Reagents—Reagent grade chemicals shall be 5
Available from the American Public Health Association (APHA), 800 I Street,
used. Unless otherwise indicated, all reagents shall conform to NW, Washington, DC 20001-3710, http://www.apha.org.
FIG. 1 Test Set-Up for Bacterial Retention Testing
F838 − 20
9.3.2 Transfer 2 mL of agitated broth culture to 1 L of sterile
Tryptic Peptone (or Casitone) 7.5 g
Yeast Extract 2.5 g
saline lactose broth, swirl to mix inoculum and incubate at 30
Sodium Chloride (NaCl) 0.5 g
6 2 °C for 24 h with agitation. Check purity of seed broth.
Magnesium Sulfate (MgSO ·3H O) 0.35 g
4 2
NOTE 1—Saline lactose broth suspension may be stored at 4 °C for up
8.2.2 Harvesting Buffer—Dissolve 0.790 g of monobasic
to 8 h prior to use.
potassium phosphate (KH PO ) and 1.0 g of dibasic potassium
2 4
phosphate (K HPO ) in 100 mL of glycerol (C H O ). Adjust
9.3.3 Determine the concentration of viable cells in the
2 4 3 8 3
to pH 7.2 with 0.1 N potassium hydroxide solution. Dilute to 1
challenge suspension according to Section 11 (expected con-
7 8
L with water and sterilize at 121 °C for 15 min.
centration is 10 to 10 cells/mL).
8.2.3 Potassium Hydroxide Solution (0.1 N)—Dissolve 5.61
9.3.4 Identify the organisms as B. diminuta in accordance
g of potassium hydroxide (KOH) in water and dilute to 1 L in
with Section 10.
a volumetric flask.
9.4 Preparation of Frozen Cell Paste of B. diminuta:
8.2.4 Tryptic Soy Agar—Prepare according to manufactur-
9.4.1 Inoculate 10 mL of Sterile Growth Medium A (8.2.1)
er’s instructions.
with the stock culture (9.2.1) and incubate at 30 6 2 °C for 24
8.2.5 Tryptic Soy Broth—Prepare according to manufactur-
h.
er’s instructions.
9.4.2 Transfer 10 mL of the bacterial suspension from 9.4.1
8.3 Analytical Reagents and Materials:
into 500 mL of Sterile Growth Medium A and incubate at 30 6
8.3.1 M-Plate Count Agar—Prepare according to manufac-
2 °C for 24 h.
turer’s instructions.
9.4.3 Prepare 10 L of a seed culture by transferring 200 mL
8.3.2 Peptone Water (1 g/L)—Dissolve the peptone in water.
of the bacterial suspension from 9.4.2 into 10 L of Sterile
Dispense suitable volumes, for preparing decimal dilutions,
Growth Medium A. Incubate at 30 6 2 °C for 24 h.
into suitable containers. Autoclave at 121 °C for 15 min.
9.4.4 Inoculate the 10 L of the seed culture into 500 L of
8.4 B. diminuta (ATCC 19146). Growth Medium A. Grow aerobically at 30 6 2 °C. Monitor
growth spectrophotometrically at 500 nm, and plot growth
8.5 Analytical Membrane Filters, 47-mm or 142-mm
curve.
diameter, 0.45 μm nominal pore size, 130 to 160 μm thick.
9.4.5 When the culture reaches the stationary phase, harvest
8.6 Petri Dishes, 150-mm diameter.
the cells by continuous flow centrifugation.
8.7 Incubator, 30 6 2 °C.
9.4.6 Re-suspend cells in two to three volumes of cold
sterile harvesting buffer.
8.8 Unidirectional Airflow Bench.
9.4.7 Centrifuge suspension and re-suspend cells in an equal
volume of harvesting buffer. Determine the cell concentration
9. Methods for Preparation of Bacterial Challenge Stock
(expected concentration of viable cells is 1 × 10 cells/mL).
Suspension
9.4.8 Transfer aliquots (for example, 50 mL) of cell paste
9.1 General—The following two methods have been used
into sterile plastic centrifuge tubes, and freeze using dry
extensively for the preparation of B. diminuta challenge
ice-acetone batch or liquid nitrogen. Store frozen cell paste at
suspensions. The presentation of these methods is not meant to
−70 °C.
exclude other equally valid methods for the preparation of B.
9.5 Preparation of Challenge Stock Suspension from Frozen
diminuta. It is important, however, that any B. diminuta
Cell Paste:
challenge suspension used is monodispersed and meets the
9.5.1 Disinfect the tube containing the cell paste by dipping
criteria set forth in Section 10.
tube in 80 % ethyl alcohol and flaming just long enough to
9.2 Reconstitute the culture according to directions pro-
burn off most of the alcohol. Use sterile tongs to hold tube.
vided by the American Type Culture Collection (ATCC).
9.5.2 Aseptically remove the cap from the tube and drop the
Check the purity of the reconstituted culture by means of streak
tube into a sterile Erlenmeyer flask containing a sterile mag-
plates. Examine for uniform colony morphology and identify
netic stirring bar and 20 cell volumes of a sterile solution of
single-cell isolates as B. diminuta in accordance with Section
0.9 % NaCl which contains 0.001 to 0.002 M MgCl at room
10.
temperature (for example, transfer a 50-mL aliquot of frozen
9.2.1 Stock Cultures—Prepare stock cultures from single
cell paste into 1 L of sterile solution).
cell isolates of 9.2. Inoculate tryptic soy agar slants and
incubate at 30 6 2 °C for 24 h. Overlay slants with sterile
NOTE 2—MgCl must be in the solution prior to adding the frozen cell
paste to prevent clumping during thaw.
mineral oil and store at 4 °C. Check weekly for viability and
purity. Alternatively, tryptic soy semisolid agar stab cultures
9.5.3 Place the flask on a magnetic stirring unit, and mix
may be substituted for the slant cultures.
until the entire contents of the tube is suspended evenly (about
9.2.2 Long Term Storage of Cultures—Lyophilize or store in
40 min).
liquid nitrogen.
9.5.4 Determine the concentration of viable cells according
to Section 11 (expected concentration of the cell suspension is
9.3 Preparation of Challenge Stock Suspension in Saline
1 to 2 × 10 cells/mL).
Lactose Broth:
9.3.1 Inoculate 10-mL sterile tryptic soy broth with stock 9.5.5 Identify the organism as B. diminuta in accordance
culture (9.2.1) and incubate at 30 6 2 °C for 24 h. with Section 10.
F838 − 20
10. Identification of B. diminuta 11.5 Perform viable cell assay, in duplicate, using the
membrane filter assay or direct spread plate assay under
10.1 Colonial Morphology:
conditions that are similar to those specified for microbial
10.1.1 Colonies of B. diminuta are yellow-beige, slightly
enumeration testing in the current edition of the United States
convex, complete and shiny.
Pharmacopeia.
10.1.2 At 30 °C (optimum growth temperature) colonies are
11.5.1 For the membrane filter assay, use 1 mL from the
microscopic to pinpoint after 24 h and 1 to 2-mm diameter after
–4 –6
10 through the 10 dilutions. Place 50 mL of sterile 0.9 %
36 to 48 h.
NaCl solution into the funnel of the filter holder prior to adding
10.2 Microscopic Examination:
the 1.0 mL aliquots of the decimal dilutions. Filter and wash
10.2.1 Prepare a Gram stain.
the walls of the funnel with 50 mL of sterile 0.9 % NaCl
10.2.1.1 Examine the preparation with a compound light
solution. Remove assay membrane from funnel, and place on
microscope fitted with a calibrated ocular micrometer and an
agar medium.
oil immersion objective lens with good resolving power (for
11.5.2 For the direct spread plate assay, use 0.1 mL from
–3 –4 –5
example, a plan achromatic objective with a numerical aperture
10 , 10 , 10 dilutions.
of 1.2 or greater)
...

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.
´1
Designation: F838 − 15a F838 − 20
Standard Test Method for
Determining Bacterial Retention of Membrane Filters
Utilized for Liquid Filtration
This standard is issued under the fixed designation F838; 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.
NOTE—Fig. 1 was editorially updated and the year date changed on Sept. 30, 2015.
ε NOTE—9.1 was editorially corrected in August 2018.
1. Scope
1.1 This test method determines the bacterial retention characteristics of membrane filters for liquid filtration using Brevundi-
monas diminuta as the challenge organism. This test method maycan be employedused to evaluate any membrane filter system
used for liquid sterilization.
1.2 This test method is not intended to be used in performance of product- and process-specific validation of the bacterial retention
characteristics of membrane filters to be used in pharmaceutical or biopharmaceutical sterilizing filtration, or both. Process- and
product-specific bacterial retention validation should be carried out using the intended product manufacturing process parameters
and the product solution or surrogate as the carrier fluid.
1.3 The values stated in SI units are to be regarded as standard.
1.3.1 Exception—The inch-pound values given for units of pressure are to be regarded as standard; SI unit conversions are shown
in parentheses.
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, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.5 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:
D1193 Specification for Reagent Water
3. Terminology
3.1 Definitions:
This test method is under the jurisdiction of ASTM Committee E55 on Manufacture of Pharmaceutical and Biopharmaceutical Products and is the direct responsibility
of Subcommittee E55.03 on General Pharmaceutical Standards.
Current edition approved Sept. 30, 2015Oct. 1, 2020. Published October 2015October 2020. Originally approved in 1983. Last previous edition published in 2015 as
ɛ1
F838 – 15.F838 – 15a . DOI: 10.1520/F0838-15AE01.10.1520/F0838-20.
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’sstandard’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
F838 − 20
3.1.1 log reduction value—value, n—the logarithm to the base 10 of the ratio of the number of microorganisms in the challenge
to the number of organisms in the filtrate.
4. Summary of Test Method
4.1 After sterilization, the test filter is challenged with a suspension of B. diminuta (ATCC 19146 ) at a concentration of to provide
7 2
10 organisms per cm of effective filtration area (EFA) at a maximum differential pressure across the test filter of 30 psig (206
–3 –8 3 2
kPa) and a flow rate flux of 2 to 4 × 10 LPM per L per min (3.3 to 6.7 × 10 m /s) per cm of effective filtration area. The entire
filtrate is then filtered passed through an analytical membrane filerfilter disc, which is subsequently incubated on a solidified growth
medium. Microorganisms that are not retained by the filter being tested will develop into visible colonies on the analysis membrane
and can then be enumerated.
5. Significance and Use
5.1 This test method is designed to assess the retentivity of a sterilizing filter under standard challenge conditions.
7 2
5.1.1 A challenge of 10 bacteria per cm of effective filtration area is selected to provide a high degree of assurance that the filter
will be challenged uniformly across the membrane surface to assure itmethod has sufficient sensitivity to detect oversized pores
and that the filter will quantitatively retain large numbers of organisms. The model challenge organism, B. diminuta, is widely
considered to be a small bacterium and is recognized as an industry standard for qualifying sterilizing filters. Other species may
represent a worst-case test in terms of ability to penetrate a filter. This test does not provide assurance that filters can completely
retain such bacteria.
5.1.2 The analytical procedure utilized in this test method provides a method to assign a numerical value to the filtration efficiency
of the filter being evaluated under standard filtration conditions. For the purpose of product sterility assurance, additional
process-specific studies should be performed.
6. Apparatus
6.1 Assemble the apparatus described below as in Fig. 1:
6.1.1 Stainless Steel Pressure Vessel, 12-L capacity (or larger), fitted with a 0 to 50-psi (0 to 350-kPa) pressure gauge.
FIG. 1 Test Set-Up for BacteriaBacterial Retention Testing
Available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, http://www.atcc.org.
F838 − 20
6.1.2 Air Regulator.
6.1.3 47-mm–142-mm 47-mm or 142-mm Analysis Disc Filter Assemblies, two or more, with hose or sanitary connections as
applicable.
6.1.4 Diaphragm-Protected 0 to 50-psi (0 to 350-kPa) Pressure Gauge, for upstream pressure reading.
6.1.5 Manifold, with valves (autoclavable) and hose connections.
6.1.6 Autoclavable Tubing, (must be able to withstand a pressure of 50 psi (350 kPa)).
6.1.7 Filter Housing, with hose connections.
6.1.8 Hose Clamps.
6.1.9 Incubator, 30 6 2°C.
6.1.10 Laminar Flow Bench.
6.1.11 Smooth-Tip Forceps.
6.1.9 Test Filter.
7. Purity of Reagents and Materials
7.1 Purity of Reagents—Reagent grade chemicals shall be used. Unless otherwise indicated, all reagents shall conform to the
specifications of the American Chemical Society, where such specifications are available.
7.2 Purity of Water—Unless otherwise indicated, references to water shall mean reagent water, Type IV as defined in Specification
D1193.
7.2.1 Additionally, any water used in this test method must conform to the requirements for non-bacteriostatic water specified in
the current edition of Standard Methods for the Examination of Water and Wastewater.
8. Reagents and Materials
8.1 Saline Lactose Broth Medium:
8.1.1 Lactose Broth—Dissolve 1.3 g of dehydrated lactose broth medium in 100 mL of water.
8.1.2 Sodium Chloride Solution—Dissolve 7.6 g of sodium chloride (NaCl) in 970 mL of water in a 2-L flask with an appropriate
closure.
8.1.3 Add 30 mL of lactose broth (8.1.1) to 970 mL of sodium chloride solution. Autoclave at 121°C 121 °C for 15 min.
8.2 Frozen Cell Paste Method:
8.2.1 Growth Medium A—Dissolve in water and dilute to 1 L. Autoclave at 121°C 121 °C for 15 min (pH 6.8 to 7.0).
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC, www.chemistry.org. 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, http://www.usp.org.MD.
Available from the American Public Health Association (APHA), 800 I Street, NW, Washington, DC 20001-3710, http://www.apha.org.
F838 − 20
Tryptic Peptone (or Casitone) 7.5 g
Yeast Extract 2.5 g
Sodium Chloride (NaCl) 0.5 g
Magnesium Sulfate (MgSO ·3H O) 0.35 g
4 2
8.2.2 Harvesting Buffer—Dissolve 0.790 g of monobasic potassium phosphate (KH PO ) and 1.0 g of Kdibasic potassium
2 4
phosphate (K HPO ) in 100 mL of glycerol (C H O ). Adjust to pH 7.2 with 0.1 N potassium hydroxide solution. Dilute to 1 L
2 4 3 8 3
with water and sterilize at 121°C 121 °C for 15 min.
8.2.3 Potassium Hydroxide Solution (0.1 N)—Dissolve 5.61 g of potassium hydroxide (KOH) in water and dilute to 1 L in a
volumetric flask.
8.2.4 Tryptic Soy Agar—Prepare according to manufacturer’s instructions.
8.2.5 Tryptic Soy Broth—Prepare according to manufacturer’s instructions.
8.3 Analytical Reagents and Materials:
8.3.1 M-Plate Count Agar—Prepare according to manufacturer’smanufacturer’s instructions.
8.3.2 Peptone Water (1 g/L)—Dissolve the peptone in water. Dispense suitable volumes, for preparing decimal dilutions, into
screw-capsuitable containers. Autoclave at 121°C 121 °C for 15 min.
8.4 B. diminuta (ATCC 19146).
8.5 Analytical Membrane Filters, 47-mm or 142-mm diameter, 0.45 μm nominal pore size, 130 to 160 μm thick.
8.6 Petri Dishes, 150-mm diameter.
8.7 Incubator, 30 6 2 °C.
8.8 Unidirectional Airflow Bench.
9. Methods for Preparation of Bacterial Challenge Stock Suspension
9.1 General—The following two methods have been used extensively for the preparation of B. diminuta challenge suspensions.
The presentation of these methods is not meant to exclude other equally valid methods for the preparation of B. diminuta. It is
important, however, that any B. diminuta challenge suspension used is monodispersemonodispersed and meets the criteria set forth
in Section 10.
9.2 Reconstitute the culture according to directions provided by the American Type Culture Collection (ATCC). Check the purity
of the reconstituted culture by means of streak plates. Examine for uniform colony morphology,morphology and identify
single-cell isolates as B. diminuta in accordance with Section 10.
9.2.1 Stock Cultures—Prepare stock cultures from single cell isolates of 9.2. Inoculate tryptic soy agar slants and incubate at 30
6 2°C 2 °C for 24 h. Overlay slants with sterile mineral oil and store at 4°C. 4 °C. Check weekly for viability and purity.
Alternatively, tryptic soy semisolid agar stab cultures may be substituted for the slant cultures.
9.2.2 Long Term Storage of Cultures—Lyophilize or store in liquid nitrogen.
9.3 Preparation of Challenge Stock Suspension in Saline Lactose Broth:
9.3.1 Inoculate 10-mL sterile tryptic soy broth with stock culture (9.2.1) and incubate at 30 6 2°C 2 °C for 24 h.
9.3.2 Transfer 2 mL of agitated broth culture to 1 L of sterile saline lactose broth, swirl to mix inoculum and incubate at 30 6
2°C 2 °C for 24 h. h with agitation. Check purity of seed broth.
F838 − 20
NOTE 1—Saline lactose broth suspension may be stored at 4°C 4 °C for up to 8 h prior to use.
9.3.3 Determine the concentration of viable cells in the challenge suspension according to Section 11 (expected concentration is
7 8
10 to 10 cells/mL).
9.3.4 Identify the organisms as B. diminuta in accordance with Section 10.
9.4 Preparation of Frozen Cell Paste of B. diminuta:
9.4.1 Inoculate 10 mL of Sterile Growth Medium A (8.2.1) with the stock culture (9.2.1) and incubate at 30 6 2°C 2 °C for 24
h.
9.4.2 Transfer 10 mL of the bacterial suspension from 9.3.19.4.1 into 500 mL of Sterile Growth Medium A and incubate at 30 6
2°C 2 °C for 24 h.
9.4.3 Prepare 10 L of a seed culture by transferring 200 mL of the bacterial suspension from 9.4.2 into 10 L of Sterile Growth
Medium A. Incubate at 30 6 2°C 2 °C for 24 h.
9.4.4 Inoculate the 10 L of the seed culture into 500 L of Growth Medium A. Grow aerobically at 30 6 2°C. 2 °C. Monitor growth
spectrophotometrically at 500 nm, and plot growth curve.
9.4.5 When the culture reaches the stationary phase, harvest the cells by continuous flow centrifugation.
9.4.6 Re-suspend cells in two to three volumes of cold sterile harvesting buffer.
9.4.7 Centrifuge suspension and re-suspend cells in an equal volume of harvesting buffer. Determine the cell concentration
(expected concentration of viable cells is 1 × 10 cells/mL).
9.4.8 Transfer aliquots (for example, 50 mL) of cell paste into sterile plastic centrifuge tubes, and freeze using dry ice-acetone
batch or liquid nitrogen. Store frozen cell paste at −70°C.−70 °C.
9.5 Preparation of Challenge Stock Suspension from Frozen Cell Paste:
9.5.1 Disinfect the tube containing the cell paste by dipping tube in 80 % ethyl alcohol and flaming just long enough to burn off
most of the alcohol. Use sterile tongs to hold tube.
9.5.2 Aseptically remove the cap from the tube and drop the tube into a sterile Erlenmeyer flask containing a sterile magnetic
stirring bar and 20 cell volumes of a sterile solution of 0.9 % NaCl which contains 0.001 to 0.002 M MgCl at room temperature
(for example, transfer a 50-mL aliquot of frozen cell paste into 1 L of sterile solution).
NOTE 2—MgCl must be in the solution prior to adding the frozen cell paste to prevent dumpingclumping during thaw.
9.5.3 Place the flask on a magnetic stirring unit, and mix until the entire contents of the tube is suspended evenly (about 40 min).
9.5.4 Determine the concentration of viable cells according to Section 11 (expected concentration of the cell suspension is 1 to
2 × 10 cells/mL).
9.5.5 Identify the organism as B. diminuta in accordance with Section 10.
10. Identification of B. diminuta
10.1 Colonial Morphology:
10.1.1 Colonies of B. diminuta are yellow-beige, slightly convex, complete and shiny.
F838 − 20
10.1.2 At 30°C 30 °C (optimum growth temperature) colonies are microscopic to pinpoint after 24 h and 1 to 2-mm diameter after
36 to 48 h.
10.2 Microscopic Examination:
10.2.1 Prepare a Gram stain.
10.2.1.1 Examine the preparation with a compound light microscope fitted with a calibrated ocular micrometer and an oil
immersion objective lens with good resolving power (for example, a planachromatic plan achromatic objective with a numerical
aperture of 1.2 or greater). Observe several microscopic fields for organisms’ size and arrangement of cells.
10.2.1.2 Stained preparations should reveal a Gram-negative, small, rod-shaped organism about 0.3 to 0.4 μm by 0.6 to 1.0 μm
in size, occurring primarily as single cells.
10.2.2 Prepare a flagella stain (optional). B. diminuta is characterized by a single, polar flagellum.
10.3 Biochemical Characterization:
10.3.1 Perform a number of the following biochemical characterization tests. B. diminuta gi
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