ASTM D5246-92(2004)
(Test Method)Standard Test Method for Isolation and Enumeration of Pseudomonas aeruginosa from Water
Standard Test Method for Isolation and Enumeration of <i>Pseudomonas aeruginosa</i> from Water
SIGNIFICANCE AND USE
Pseudomonas aeruginosa is an opportunistic pathogen, and has been linked as the causative agent of numerous infections that may be transmitted through a contaminated water supply to a susceptible host. In addition to its direct pathogenicity, the association of P. aeruginosa with human fecal waste indicates that where elevated levels of P. aeruginosa are found, a serious health hazard may exist due to the presence of other pathogens.
The membrane filtration procedure described is a rapid and reliable test method of detecting P. aeruginosa in water.
SCOPE
1.1 This test method covers the isolation and enumeration of Pseudomonas aeruginosa (P. aeruginosa) from surface waters; recreational waters; ground water, water supplies; especially rural nonchlorinated sources; waste water; and saline waters. The detection limit of this test method is one microorganism per 100 mL.
1.2 This test method was used successfully with reagent water and it is the user's responsibility to ensure the validity of this test method for waters of untested matrices.
1.3 The values stated in SI units are to be regarded as the 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. Specific hazard statements are given in Section 10.
General Information
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Standards Content (Sample)
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Designation: D5246 − 92 (Reapproved2004)
Standard Test Method for
Isolation and Enumeration of Pseudomonas aeruginosa from
Water
This standard is issued under the fixed designation D5246; 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 3.2.1 Pseudomonas aeruginosa—an aerobic, motile, gram
negative rod that produces fluorescent pigments and pyocya-
1.1 Thistestmethodcoverstheisolationandenumerationof
nin. It is oxidase and caseinase positive, is able to grow at
Pseudomonas aeruginosa(P. aeruginosa)fromsurfacewaters;
42°C,isrelativelyresistanttomanyantibiotics,andmayutilize
recreational waters; ground water, water supplies; especially
acetamide.
rural nonchlorinated sources; waste water; and saline waters.
3.2.2 refrigeration—storage at 2 to 8°C.
The detection limit of this test method is one microorganism
per 100 mL.
4. Summary of Test Method
1.2 This test method was used successfully with reagent
4.1 A water sample is passed through a 0.45 mm or
wateranditistheuser’sresponsibilitytoensurethevalidityof
equivalent membrane filter. The filter carrying the retained
this test method for waters of untested matrices.
organisms is placed on a selective medium (M-PA-C) and is
1.3 The values stated in SI units are to be regarded as the
incubated at 41.5 6 0.5°C for 48 to 72 h. The resulting
standard.
pink-brown to black colonies of Pseudomonas aeruginosa are
1.4 This standard does not purport to address all of the
counted and reported per 100 mLof the sample. Colonies may
safety concerns, if any, associated with its use. It is the
be verified on skim milk agar.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
5. Significance and Use
bility of regulatory limitations prior to use. Specific hazard
5.1 Pseudomonas aeruginosa is an opportunistic pathogen,
statements are given in Section 10.
and has been linked as the causative agent of numerous
infections that may be transmitted through a contaminated
2. Referenced Documents
water supply to a susceptible host. In addition to its direct
2.1 ASTM Standards:
pathogenicity, the association of P. aeruginosa with human
D1129Terminology Relating to Water
fecal waste indicates that where elevated levels of P. aerugi-
D1193Specification for Reagent Water
nosa are found, a serious health hazard may exist due to the
D2777Practice for Determination of Precision and Bias of
presence of other pathogens.
Applicable Test Methods of Committee D19 on Water
5.2 The membrane filtration procedure described is a rapid
D3370Practices for Sampling Water from Closed Conduits
and reliable test method of detecting P. aeruginosa in water.
3. Terminology
6. Interferences
3.1 Definitions:
6.1 For certain samples, bacterial cells may have been
3.1.1 For definitions of terms used in this test method, refer
exposed to adverse environmental factors that lower their
to Terminology D1129.
probability for survival and growth on a membrane filter
3.2 Definitions of Terms Specific to This Standard:
medium.Thiseffectmaybepronouncedinthistestmethoddue
to the presence of antibiotics and the elevated incubation
temperature.
This test method is under the jurisdiction of ASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
6.2 The selection of an appropriate dilution volume is
Current edition approved June 1, 2004. Published June 2004. Originally
essential.Too small a dilution volume may fail to detect any P.
approved in 1992. Last previous edition approved in 1998 as D5246–92 (1998).
DOI: 10.1520/D5246-92R04.
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 Available from BBL Microbiological Systems, Division of Becton Dickinson
the ASTM website. and Co., Cockeysville, MD 21030. Other suppliers may be utilized if equivalent.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5246 − 92 (2004)
aeruginosa organisms, while too large a volume may cause an 8.5 Magnesium Chloride Solution (81.1 g/L)—Dissolve
overabundance of colonies that would interfere with an accu- 81.1 g magnesium chloride (MgCl 6H O) in water and dilute
2 2
rate count. to 1 L with water.
6.3 Chemicals or a combination of chemicals in certain
8.6 Potassium Hydroxide Solution(5.6g/L)—Dissolve5.6g
samples can have a toxic effect upon P. aeruginosa when
of potassium hydroxide (KOH) in water and dilute to 1 Lwith
concentrated.
water.
6.4 Turbidity in samples may clog filter or effect color
8.7 Membrane Filters, sterile, 47 mm with grid (0.45 µm
detection of organisms that develop on the filter.
pore size) or equivalent.
6.5 Water samples containing residual chlorine can be
9. Media Preparation
detrimental to P. aeruginosa. Utilize the procedure defined in
Practices D3370 to address chlorinated water samples.
9.1 M-PA-C Medium —Formula per litre of water:
L-lysine 5.0 g
7. Apparatus
Sodium chloride 5.0 g
Yeast extract 2.0 g
7.1 Top-Loading Balance, sensitive to 0.1 g.
Xylose 1.25 g
7.2 pH Meter and Surface pH Electrode.
Sucrose 1.25 g
Lactose 1.25 g
7.3 Incubator,capableofmaintainingtemperatureof41.5 6
Phenol red 0.08 g
0.5°C and 35 6 0.5°C. Ferric ammonium citrate, anhydrous 0.80 g
Sodium thiosulfate, anhydrous 5.0 g
7.4 Stereoscopic Microscope, with a cool white fluorescent
Agar 12.0 g
Magnesium sulfate, anhydrous 1.5 g
light.
Kanamycin 0.008 g
7.5 Colony Counter. Nalidixic acid 0.037 g
7.6 Containers, with lids (for incubating test petri dishes 9.1.1 M-PA-C Medium orEquivalent—Dissolvemixtureof
containing membrane filters under high humidity). above items into 1 L of water, boiling for 1 min to solubilize
the chemicals. Cool to 45 to 50°C before dispensing. Pour one
7.7 Long-Wave Ultraviolet Light.
plate of medium and measure the pH of the surface with a
7.8 Autoclave, or other sterilizing equipment.
suitablepHelectrode.ThesurfacepHofthesolidifiedmedium
7.9 Petri Dishes,sterile,50by9or60by15mmand100by should be 7.2 6 0.1. If it is not, adjust pH of the remaining
15 mm. solution accordingly with potassium hydroxide solution.
9.1.2 Aseptically dispense 5 to 6 mL of media into each
7.10 Pipets, sterile, 1 and 10 mL, with 0.1-mL graduations
sterile 50 or 60 mm petri dish. This medium should be stored
and an accuracy of 65%.
underrefrigerationandusedwithinoneweekafterpreparation.
8. Reagents and Materials
9.2 Skim Milk Agar—Skimmilkpowderishighgradeskim
8.1 Purity of Reagents—Reagent grade chemicals shall be
milkreducedtopowderbyasprayingprocess.Slowlyadd100
used in all tests. Unless otherwise indicated, it is intended that gofskimmilkpowderto500mLofwaterandstirwithoutheat
all reagents shall conform to the specifications of the Commit-
for approximately 30 min. Prepare an agar solution by adding
tee onAnalytical Reagents of theAmerican Chemical Society, 15.0 g of agar to 500 mL of water and heat at 90°C for 10 to
where such specifications are available. Other grades may be
12 min. Autoclave the solutions separately at 121°C for 12
used, provided it is first ascertained that the reagent is of min. Cool, with stirring, until temperature reaches 50 to 55°C.
sufficiently high purity to permit its use without lessening the
Add the skim milk solution to the agar solution, thoroughly
accuracy of the determination. mix, and dispense aseptically into sterile petri plates. The
plates may be stored in sealed containers in the refrigerator for
8.2 Purity of Water—Unless otherwise indicated, references
up to two weeks.
towatershallbeunderstoodtomeanreagentwaterconforming
to Type II of Specification D1193.
9.3 Soybean Casein Digest Agar —Formula per litre of
water:
8.3 Buffered Water—Dispense 1.25 mL of buffered water
stock solution and 5.0 mL magnesium chloride solution (see Pancreatic digest of casein 15.0 g
Papaic digest of soybean meal 5.0 g
8.5) and dilute to 1 L with water. Dispense in amount to
Sodium chloride 5.0 g
provide 99 mL after sterilization.
Agar 15.0 g
8.4 Buffered Water Stock—Dissolve 34.0 g potassium dihy-
9.3.1 Soybean Casein Digest Agar—Prepare the media ac-
drogenphosphate(KH PO )in500mLwater,adjusttopH7.2
2 4
cording to manufacturer’s instructions and dispense it asepti-
with KOH solution (5.6 g/L)
...
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