ASTM D5246-92(1998)
(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
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 problems, 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.
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An American National Standard
Designation: D 5246 – 92 (Reapproved 1998)
Standard Test Method for
Isolation and Enumeration of Pseudomonas aeruginosa from
Water
This standard is issued under the fixed designation D 5246; 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 3.2.2 refrigeration—storage at 2 to 8°C.
1.1 This test method covers the isolation and enumeration of
4. Summary of Test Method
Pseudomonas aeruginosa (P. aeruginosa) from surface waters;
4.1 A water sample is passed through a 0.45 mm or
recreational waters; ground water, water supplies; especially
equivalent membrane filter. The filter carrying the retained
rural nonchlorinated sources; waste water; and saline waters.
organisms is placed on a selective medium (M-PA-C) and is
The detection limit of this test method is one microorganism
incubated at 41.5 6 0.5°C for 48 to 72 h. The resulting
per 100 mL.
pink-brown to black colonies of Pseudomonas aeruginosa are
1.2 This test method was used successfully with reagent
counted and reported per 100 mL of the sample. Colonies may
water and it is the user’s responsibility to ensure the validity of
be verified on skim milk agar.
this test method for waters of untested matrices.
1.3 The values stated in SI units are to be regarded as the
5. Significance and Use
standard.
5.1 Pseudomonas aeruginosa is an opportunistic pathogen,
1.4 This standard does not purport to address all of the
and has been linked as the causative agent of numerous
safety concerns, if any, associated with its use. It is the
infections that may be transmitted through a contaminated
responsibility of the user of this standard to establish appro-
water supply to a susceptible host. In addition to its direct
priate safety and health practices and determine the applica-
pathogenicity, the association of P. aeruginosa with human
bility of regulatory limitations prior to use. Specific hazard
fecal waste indicates that where elevated levels of P. aerugi-
statements are given in Section 10.
nosa are found, a serious health hazard may exist due to the
presence of other pathogens.
2. Referenced Documents
5.2 The membrane filtration procedure described is a rapid
2.1 ASTM Standards:
1 and reliable test method of detecting P. aeruginosa in water.
D 1129 Terminology Relating to Water
D 1193 Specification for Reagent Water
6. Interferences
D 2777 Practice for Determination of Precision and Bias of
1 6.1 For certain samples, bacterial cells may have been
Applicable Methods of Committee D-19 on Water
exposed to adverse environmental factors that lower their
D 3370 Practices for Sampling Water from Closed Con-
1 probability for survival and growth on a membrane filter
duits
medium. This effect may be pronounced in this test method due
3. Terminology to the presence of antibiotics and the elevated incubation
temperature.
3.1 Definitions:
6.2 The selection of an appropriate dilution volume is
3.1.1 For definitions of terms used in this test method, refer
essential. Too small a dilution volume may fail to detect any P.
to Terminology D 1129.
aeruginosa organisms, while too large a volume may cause an
3.2 Definitions of Terms Specific to This Standard:
overabundance of colonies that would interfere with an accu-
3.2.1 Pseudomonas aeruginosa—an aerobic, motile, gram
rate count.
negative rod that produces fluorescent pigments and pyocya-
6.3 Chemicals or a combination of chemicals in certain
nin. It is oxidase and caseinase positive, is able to grow at
samples can have a toxic effect upon P. aeruginosa when
42°C, is relatively resistant to many antibiotics, and may utilize
concentrated.
acetamide.
Available from BBL Microbiological Systems, Division of Becton Dickinson
Annual Book of ASTM Standards, Vol 11.01. 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.
D 5246 – 92 (1998)
6.4 Turbidity in samples may clog filter or effect color 9. Media Preparation
detection of organisms that develop on the filter. 2
9.1 M-PA-C Medium —Formula per litre of water:
6.5 Water samples containing residual chlorine can be
L-lysine 5.0 g
detrimental to P. aeruginosa. Utilize the procedure defined in
Sodium chloride 5.0 g
Yeast extract 2.0 g
Practices D 3370 to address chlorinated water samples.
Xylose 1.25 g
Sucrose 1.25 g
7. Apparatus Lactose 1.25 g
Phenol red 0.08 g
7.1 Top-Loading Balance, sensitive to 0.1 g.
Ferric ammonium citrate, anhydrous 0.80 g
Sodium thiosulfate, anhydrous 5.0 g
7.2 pH Meter and Surface pH Electrode.
Agar 12.0 g
7.3 Incubator, capable of maintaining temperature of 41.5
Magnesium sulfate, anhydrous 1.5 g
Kanamycin 0.008 g
6 0.5°C and 35 6 0.5°C.
Nalidixic acid 0.037 g
7.4 Stereoscopic Microscope, with a cool white fluorescent
9.1.1 M-PA-C Medium or Equivalent—Dissolve mixture of
light.
above items into 1 L of water, boiling for 1 min to solubilize
7.5 Colony Counter.
the chemicals. Cool to 45 to 50°C before dispensing. Pour one
7.6 Containers, with lids (for incubating test petri dishes
plate of medium and measure the pH of the surface with a
containing membrane filters under high humidity).
suitable pH electrode. The surface pH of the solidified medium
7.7 Long-Wave Ultraviolet Light.
should be 7.2 6 0.1. If it is not, adjust pH of the remaining
7.8 Autoclave, or other sterilizing equipment.
solution accordingly with potassium hydroxide solution.
7.9 Petri Dishes, sterile, 50 by 9 or 60 by 15 mm and 100 by
9.1.2 Aseptically dispense 5 to 6 mL of media into each
15 mm.
sterile 50 or 60 mm petri dish. This medium should be stored
7.10 Pipets, sterile, 1 and 10 mL, with 0.1-mL graduations
under refrigeration and used within one week after preparation.
and an accuracy of 65%.
9.2 Skim Milk Agar— Skim milk powder is high grade skim
milk reduced to powder by a spraying process. Slowly add 100
8. Reagents and Materials
g of skim milk powder to 500 mL of water and stir without heat
for approximately 30 min. Prepare an agar solution by adding
8.1 Purity of Reagents—Reagent grade chemicals shall be
15.0 g of agar to 500 mL of water and heat at 90°C for 10 to
used in all tests. Unless otherwise indicated, it is intended that
12 min. Autoclave the solutions separately at 121°C for 12
all reagents shall conform to the specifications of the Commit-
min. Cool, with stirring, until temperature reaches 50 to 55°C.
tee on Analytical Reagents of the American Chemical Society,
Add the skim milk solution to the agar solution, thoroughly
where such specifications are available. Other grades may be
mix, and dispense aseptically into sterile petri plates. The
used, provided it is first ascertained that the reagent is of
plates may be stored in sealed containers in the refrigerator for
sufficiently high purity to permit its use without lessening the
up to two weeks.
accuracy of the determination.
9.3 Soybean Casein Digest Agar —Formula per litre of
8.2 Purity of Water— Unless otherwise indicated, refer-
water:
ences to water shall be understood to mean reagent water
conforming to Type II of Specification 1193. Pancreatic digest of casein 15.0 g
Papaic digest of soybean meal 5.0 g
8.3 Buffered Water— Dispense 1.25 mL of buffered water
Sodium chloride 5.0 g
stock solution and 5.0 mL magnesium chloride solution (see Agar 15.0 g
8.5) and dilute to 1 L with water. Dispense in amount to
9.3.1 Soybean Casein Digest Agar—Prepare the media
provide 99 mL after sterilization.
according to manufacturer’s instructions and dispense it asep-
8.4 Buffered Water Stock—Dissolve 34.0 g potassium dihy-
tically into sterile petri dishes.
drogen phosphate (KH PO ) in 500 mL water, adjust to pH 7.2
2 4
with KOH solution (5.6 g/L) and dilute to 1 L with water.
10. Hazards
8.5 Magnesium Chloride Solution (81.1 g/L)—Dissolve
10.1 P. aeruginosa is an opportunistic human pathogen;
81.1 g magnesium chloride (MgCl 6H O) in water and dilute
2 2
thus handle all culture material (plates, pipets, dilution tubes)
to 1 L with water.
using accepted microbiological techniques, including steriliza-
8.6 Potassium Hydroxide Solution (5.6 g/L)—Dissolve 5.6
tion of all discarded material.
g of potassium hydroxide (KOH) in water and dilute to 1
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