ASTM D4454-85(2009)
(Test Method)Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015)
Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015)
SIGNIFICANCE AND USE
Measurement of bacterial densities is generally the first step in establishing a relationship between bacteria and other biochemical processes. It is known that the classical plate count procedure underestimates bacterial densities while the epifluorescence direct microscopic procedure more accurately depicts the total numbers of nonviable or dormant and viable cells in a water sample. The acridine-orange INT-formazan reduction technique provides information on the total concentrations of bacteria as well as that proportion which are actively respiring and thus involved in degradative processes.
The acridine-orange INT-formazan reduction technique is both quantitative and precise.
This procedure is ideal for enumerating both pelagic and epibenthic bacteria in all fresh water and marine environments.
The process can be employed in survey studies to characterize the bacteriological densities and activities of environmental waters.
SCOPE
1.1 This test method covers the detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure. This test method is applicable to environmental waters and potable waters.
1.2 Certain types of debris and other microorganisms may fluoresce in acridine-orange stained smears.
1.3 The procedure described requires a trained microbiologist or technician who is capable of distinguishing bacteria from other fluorescing bodies on the basis of morphology when viewed at higher magnifications.
1.4 Use of bright light permits differentiation of single bacteria where reduced formazan is deposited at the polar ends.
1.5 Approximately 104 cells/mL are required for detection by this test method.
1.6 Minimal cell size which allows the detection of formazan deposits is represented by bacteria of 0.4 μm.2
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 This standard does not purport to address 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.
WITHDRAWN RATIONALE
This test method covers the detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure.
Formerly under the jurisdiction of Committee D19 on Water, this test method was withdrawn in December 2015 in accordance with section 10.6.3 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D4454 − 85 (Reapproved 2009)
StandardTest Method for
Simultaneous Enumeration of Total and Respiring Bacteria
in Aquatic Systems by Microscopy
This standard is issued under the fixed designation D4454; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope D1193Specification for Reagent Water
1.1 This test method covers the detection and enumeration
3. Terminology
of aquatic bacteria by the use of an acridine-orange epifluo-
rescence direct-microscopic counting procedure. This test
3.1 Definitions—For definitions of terms used in this test
method is applicable to environmental waters and potable
method, refer to Terminology D1129.
waters.
4. Summary of Test Method
1.2 Certain types of debris and other microorganisms may
fluoresce in acridine-orange stained smears.
4.1 A water sample is treated with an aqueous solution of
INT-dye(2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazo-
1.3 The procedure described requires a trained microbiolo-
lium chloride) for 20 min. The reaction then is stopped by
gist or technician who is capable of distinguishing bacteria
adding a 37% solution of formaldehyde. Sample is filtered
fromotherfluorescingbodiesonthebasisofmorphologywhen
through a 0.1-µm pore size polycarbonate membrane filter
viewed at higher magnifications.
(presoaked in sudan black solution or equivalent), and stained
1.4 Use of bright light permits differentiation of single
with acridine orange for 3 min.
bacteriawherereducedformazanisdepositedatthepolarends.
4.2 The filter is then air-dried and examined under oil
1.5 Approximately 10 cells/mL are required for detection
immersionfortotalbacteriaunderepifluorescenceillumination
by this test method.
and for respiring bacteria under transmitted bright light illu-
1.6 Minimal cell size which allows the detection of forma-
mination.
zan deposits is represented by bacteria of 0.4 µm.
5. Significance and Use
1.7 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
5.1 Measurement of bacterial densities is generally the first
standard.
step in establishing a relationship between bacteria and other
biochemical processes. It is known that the classical plate
1.8 This standard does not purport to address the safety
concerns, if any, associated with its use. It is the responsibility count procedure underestimates bacterial densities while the
epifluorescence direct microscopic procedure more accurately
of the user of this standard to establish appropriate safety and
health practices and determine the applicability of regulatory depicts the total numbers of nonviable or dormant and viable
cells in a water sample. The acridine-orange INT-formazan
limitations prior to use.
reduction technique provides information on the total concen-
2. Referenced Documents
trationsofbacteriaaswellasthatproportionwhichareactively
respiring and thus involved in degradative processes.
2.1 ASTM Standards:
D1129Terminology Relating to Water
5.2 The acridine-orange INT-formazan reduction technique
is both quantitative and precise.
This test method is under the jurisdiction ofASTM Committee D19 on Water
5.3 Thisprocedureisidealforenumeratingbothpelagicand
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
epibenthicbacteriainallfreshwaterandmarineenvironments.
Current edition approved May 1, 2009. Published June 2009. Originally
approved in 1985. Last previous edition approved in 2002 as D4454–85 (2002).
DOI: 10.1520/D4454-85R09.
DIFCOTechnicalInformation—BactoAcridineOrangeStain,isavailablefrom
Difco Laboratories, P.O. Box 1058, Detroit, MI 48201. Zimmerman, et al, “Simultaneous Determination of Total Number of Aquatic
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Bacteria and the Number Thereof Involved in Respiration ,” Applied and Environ-
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM mental Microbiology , Vol 36, 1978, pp. 926–935
Standards volume information, refer to the standard’s Document Summary page on Cherry, et al, “Temperature Influence on Bacterial Populations in Aquatic
the ASTM website. Systems,” Water Res., Vol 8, 1974, pp. 149–155.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4454 − 85 (Reapproved 2009)
5.4 The process can be employed in survey studies to 7.6 Sudan Dye Solution—Dissolve 100 mg of Sudan Black
characterize the bacteriological densities and activities of B or equivalent in 75 mL of absolute ethanol then add 75 mL
environmental waters. of water and mix.
7.7 Immersion Oil, very low fluorescing (equivalent to
6. Apparatus
Cargille Type A).
6.1 Fluorescence Microscope, with an oil immersion objec-
7.8 Formaldehyde, 37% solution.
tive lens (100×).
8. Procedure
6.2 Eye Pieces, 12.5×, equipped with a net micrometer (10
by 10 mm) (25×2-mm squares).
8.1 Sample Processing:
8.1.1 Place 10 mL of the sample into a clean, sterile test
6.3 Condenser, 1.25×, suitable for the microscope.
tube.Add 1 mL of 0.2% aqueous INT-dye 2-( p-iodophenyl)-
6.4 High-Pressure Mercury Lamp , 200-W, on a UV light
3-(p-nitrophenyl)-5-phenyl tetrazolium chloride.
sourcegivingverticalillumination,andafilterunitH2(Leitz)
8.1.2 Mixcarefullyandholdthesampleinthedarkat in situ
with BG12 and BG38 transmission filters or equivalents.
temperature for approximately 20 min.
6.5 Stage Micrometer, 2 by 200 parts.
8.1.3 Stop the reaction by adding 0.1 mL of 37% formal-
dehyde that also acts as preservative (at this stage the sample
6.6 Membrane Filter Support, sterile, particle-free, fritted-
can be stored at 4°C up to one month).
glass, 25 mm.
8.2 Membrane Filtration and Microscopic Examination:
6.7 Funnel, 15-mL capacity or equivalent.
8.2.1 Filter 1 mL of the (INT) treated/preserved sample
6.8 Membrane Filter, sterile plain regular polycarbonate,
through 0.1-µm polycarbonate membrane which has been
25-mm (0.1-µm pore size).
presoaked for 24 h in a solution of sudan black B (BDH) in
6.9 Filter Apparatus, that should contain vacuum source,
50% ethanol.
filtering flask, and a filtering flask as a water trap.
8.2.2 Stainthefilterwith3mLofacridineorangefor3min.
8.2.3 Filter the acridine orange.
6.10 Forceps(flattip),Alcohol,BunsenBurner,CleanGlass
8.2.4 Remove the filter, and air-dry for 1
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