RECREATIONAL WATERS (9213)/Membrane Filter Technique for Pseudomonas aeruginosa
5. CABELLI, V.J., H. KENNEDY & M.A. LEVIN. 1976. Pseudomonas
aeruginosa and fresh recreational waters. J. Water Pollut. Control
Fed. 48:367.
6. SHERRY, J.P., S.R. KUCHMA & B.J. DUTKA. 1979. The occurrence of
Candida albicans in Lake Ontario bathing beaches. Can. J. Micro-
biol. 25:1036.
7. STEVENS, A.R., R.L. TYNDALL, C.C. COUTANT & E. WILLAERT. 1977.
Isolation of the etiological agent of primary amoebic meningoen-
cephalitis from artificially heated waters. Appl. Environ. Microbiol.
34:701.
8. WELLINGS, F.M., P.T. AMUSO, S.L. CHANG & A.L. LEWIS. 1977.
Isolation and identification of pathogenic Naegleria from Florida
lakes. Appl. Environ. Microbiol. 34:661.
9. N’DIAYE, A., P. GEORGES, A. N’GO & B. FESTY. 1985. Soil amoebas
as biological markers to estimate the quality of swimming pool
waters. Appl. Environ. Microbiol. 49:1072.
9213 E. Membrane Filter Technique for Pseudomonas aeruginosa
1. Laboratory Apparatus
See Section 9222B.1.
2. Culture Media
a. M-PA agar: This agar may not be available in dehydrated
form and may require preparation from the basic ingredients.
L-Lysine HCl ......................................................................... 5.0 g
Sodium chloride (NaCl) ........................................................ 5.0 g
Yeast extract .......................................................................... 2.0 g
Xylose .....................................................................................2.5 g
Sucrose................................................................................... 1.25 g
Lactose ................................................................................... 1.25 g
Phenol red.............................................................................. 0.08 g
Ferric ammonium citrate ....................................................... 0.8 g
Sodium thiosulfate (Na2S2O3) .............................................. 6.8 g
Agar ..................................................................................... 15.0 g
Reagent-grade water.............................................................. 1 L
Adjust pH to 6.5 ⫾ 0.1 and sterilize via autoclaving. Cool to
55 to 60°C; readjust pH to 7.1 ⫾ 0.2 and add the following dry
antibiotics per liter of agar base: sulfapyridine,* 176 mg; kana-
mycin,* 8.5 mg; nalidixic acid,* 37.0 mg; and cycloheximide,*
150 mg. After mixing, dispense in 3-mL quantities in 50 ⫻ 12 mm
Petri plates. Store poured plates at 2 to 8°C. Discard unused
medium after 1 month.
b. Modified M-PA agar†
c. Milk agar (Brown and Scott Foster Modification):
1) Mixture A:
Instant nonfat milk‡ .......................................................... 100 g
Reagent-grade water ........................................................... 500 mL
2) Mixture B:
Nutrient broth .................................................................... 12.5 g
Sodium chloride (NaCl) ...................................................... 2.5 g
* Sigma Chemical Co., St. Louis, MO, or equivalent.
† Commercially available as M-PA-C agar. Contains magnesium, sulfate, kana-
mycin, and nalidixic acid.
‡ Carnation, or equivalent.
https://doi.org/10.2105/SMWW.2882.187
10. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1986. Ambient Water
Quality Criteria for Bacteria—1986; EPA-440/5-84-002. U.S. En-
vironmental Protection Agency, Washington, D.C.
11. CHAROENCA, N. & R. FUJIOKA. 1993. Assessment of Staphylococcus
bacteria in Hawaii’s marine recreational waters. Water Sci. Technol.
27:283.
10. Bibliography
OLIVIERI, V.P., C.W. DRUSE & K. KAWATA. 1977. Microorganisms in
Urban Stormwater; EPA-600/2-77-087. U.S. Environmental Pro-
tection Agency, Cincinnati, Ohio.
RICE, E.W., T.C. COVERT, D.K. WILD, D. BERMAN, S.A. JOHNSON & C.H.
JOHNSON. 1993. Comparative resistance of Escherichia coli and
Enterococci to chlorination. J. Environ. Health A28:89.
Agar .................................................................................... 15.0 g
Reagent-grade water......................................................... 500 mL
Separately prepare and sterilize Mixtures A and B; cool rap-
idly to 55°C; aseptically combine mixtures and dispense 20 to
25 mL per Petri dish.
3. Procedure
a. Presumptive tests: Filter 200 mL or less of natural waters or
up to 500 mL of swimming pool waters through sterile mem-
brane filters. Place each membrane on a poured plate of modified
M-PA agar so there is no air space between the membrane and
the agar surface.
Invert plates and incubate at 41.5 ⫾ 0.5°C for 72 h.
Typically, P. aeruginosa colonies are 0.8 to 2.2 mm in diam-
eter and appear flat with light outer rims and brownish to
greenish-black centers. Count typical colonies, preferably from
filters containing 20 to 80 colonies. Use a 10- to 15-power
magnifier as an aid in colony counting.
A two-plate method also may be used; this method will help
recover chlorine-stressed P. aeruginosa (e.g., from swimming
pools and whirlpools). Use membrane filter technique to prepare
samples. Place membrane filters on R2A agar (Section
9215A.6c) and incubate at 37 ⫾ 0.5°C for 24 h. Aseptically
transfer the membranes to M-PA agar and incubate at 35 ⫾
0.5°C for another 24 h. Random and atypical colonies can be
verified using confirmation tests.
b. Confirmation tests: Use milk agar to confirm a number of
typical and atypical colonies. Make a single streak (2 to 4 cm
long) from an isolated colony on a milk agar plate and incubate
at 35 ⫾ 1.0°C for 24 h. P. aeruginosa hydrolyzes casein and
produces a yellowish to green diffusible pigment.
4. Interpretation and Calculation of Density
Confirmation is not routinely required. In the absence of
confirmation, report results as the number of presumptive
P. aeruginosa/100 mL.
7
 RECREATIONAL WATERS (9213)/Multiple-Tube Technique for Pseudomonas aeruginosa
5. Bibliography
DRAKE, C.H. 1966. Evaluation of culture media for the isolation and
enumeration of Pseudomonas aeruginosa. Health Lab. Sci. 3:10.
BROWN, M.R.W. & J.H. SCOTT FOSTER. 1970. A simple diagnostic milk
medium for Pseudomonas aeruginosa. J. Clin. Pathol. 23:172.
LEVIN, M.A. & V.J. CABELLI. 1972. Membrane filter technique for
enumeration of Pseudomonas aeruginosa. Appl. Microbiol. 24:864.
9213 F. Multiple-Tube Technique for Pseudomonas aeruginosa
1. Laboratory Apparatus
See Section 9221.
2. Culture Media
a. Asparagine broth: This medium may not be available in
dehydrated form and may require preparation from the basic
ingredients.
Asparagine, DL .........................................................................
Anhydrous dipotassium hydrogen phosphate (K2HPO4) .......
Magnesium sulfate (MgSO4 䡠 7H2O) ......................................
Reagent-grade water ................................................................
Adjust pH to between 6.9 and 7.2 before sterilization.
b. Acetamide broth: This medium may not be available in
dehydrated form and may require preparation from the basic
ingredients.
Acetamide ............................................................................ 10.0
Sodium chloride (NaCl) ........................................................ 5.0
Anhydrous dipotassium hydrogen phosphate (K2HPO4) ..... 1.39
Anhydrous potassium dihydrogen phosphate (KH2PO4) ..... 0.73
Magnesium sulfate (MgSO4 䡠 7H2O).................................... 0.5
Dissolve 1.2 g phenol red in 100 mL 0.01N NaOH and add
1 mL/L of acetamide broth. Use phenol red stock solution
https://doi.org/10.2105/SMWW.2882.187
DUTKA, B.J. & K.K. KWAN. 1977. Confirmation of the single-
step membrane filter procedure for estimating Pseudomonas
aeruginosa densities in water. Appl. Environ. Microbiol.
33:240.
BRODSKY, M.H. & B.W. CIEBIN. 1978. Improved medium for recovery
and enumeration of Pseudomonas aeruginosa from water using
membrane filters. Appl. Environ. Microbiol. 36:26.
within 1 year. Adjust pH to between 7.1 and 7.3 before
sterilization. Final pH should be 7.0 ⫾ 0.2. Prepare acetamide
broth as described above. If agar slants are preferred, prepare
as described above but add 15 g agar/L, heat to dissolve agar,
and dispense 8-mL quantities in 16-mm tubes. After autoclav-
ing, incline tubes while cooling to provide a large slant
surface.
3. Procedure
3.0 g
1.0 g a. Presumptive test: Perform a five-tube test. Use 10 mL
0.5 g single-strength asparagine broth for inocula of 1 mL or less and
1 L 10 mL double-strength asparagine broth for 10-mL inocula.
Higher dilutions may be necessary for swimming pools. Incubate
inoculated tubes at 35 to 37°C. After 24 h and again after 48 h
of incubation, examine tubes under long-wave ultraviolet light
(black light) in a darkened room. Production of a green fluores-
cent pigment constitutes a positive presumptive test.
g b. Confirmed test: Confirm positive tubes by inoculating
g 0.1 mL of culture into acetamide broth or onto the surface of
g acetamide agar slants. Development of purple color (alkaline
g
pH) within 24 to 36 h of incubation at 35 to 37°C is a positive
g
confirmed test for Pseudomonas aeruginosa.
c. Computing and reporting results: Refer to Table 9221:V
and Section 9221C.
8
 9215 HETEROTROPHIC PLATE COUNT*
9215 A. Introduction
1. Applications
The heterotrophic plate count (HPC), formerly known as the
standard plate count, is a procedure for estimating the number of
live, culturable heterotrophic bacteria in water and for measuring
changes in swimming pools or during water treatment and dis-
tribution. Colonies may arise from pairs, chains, clusters, or
single cells—all of which are included in the term colony-
forming units (CFU). The final count also depends on interaction
among developing colonies. Choose the procedure and medium
that best suit how the resulting information will be used. Only
compare data generated using the same procedure and medium.
Four methods and five media are described.
2. Selection of Method
a. Pour plate method: The pour plate method (9215B) is
simple to perform and can accommodate sample or diluted-
sample volumes ranging from 0.1 to 2.0 mL. It produces sub-
surface colonies that are relatively small, compact, and less apt
to encroach on each other than surface colonies do. However,
submerged colonies often are slower growing and difficult to
transfer. A thermostatically controlled water bath is essential for
tempering the agar.
b. Spread plate method: The spread plate method (9215C)
causes no heat shock. All colonies are on the agar surface, where
analysts typically can easily distinguish them from particles and
bubbles, and discern different colony morphologies. If not, they
can transfer colonies quickly and easily discern colony morphol-
ogy via comparisons to published descriptions. However, the
agar in this method can only absorb a small volume of sample or
diluted sample (0.1 to 0.5 mL, depending on the degree to which
prepoured plates have been dried). Also, spreading colonies
could be interferences. To use this procedure, maintain a supply
of suitable pre-dried, absorbent agar plates (see 9215C.4).
c. Membrane filter method: The membrane filter method
(9215D) can be used to analyze large volumes of low-turbidity
water and is the method of choice for waters with low numbers
of heterotrophic organisms (⬍1 to 10 CFU/mL). It produces no
heat shock. However, there is the expense of the membrane filter,
the smaller area for colony development, possible damage to
cells by excessive filtration pressures, possible variations in
membrane-filter quality (see Section 9020B.5i), and the need to
detect colonies via reflected light against a white background if
colored filters or contrast stains are not used.
d. Enzyme substrate method: The enzyme substrate method,
SimPlate® (9215E), can be used to analyze drinking- and source-
water samples with a wide range of bacterial concentrations. It
* Approved by Standard Methods Committee, 2016.
Joint Task Group: Gil Dichter (co-chair), Mark W. LeChevallier (co-chair),
Jennifer Best, Eric Duderstadt, Dennis Hill, Kimberly Phillips.
https://doi.org/10.2105/SMWW.2882.188
produces no heat shock. Comparable to the pour plate method,
this method† uses a medium in which substrates are hydrolyzed
by multiple microbial enzymes, causing the release of 4-meth-
ylumbelliferone after 48 h of incubation at 35 ⫾ 5°C. The
4-methylumbelliferone fluoresces when exposed to long-wave-
length (365–366 nm) ultraviolet (UV) light, and the number of
blue fluorescing wells corresponds to a most probable number
(MPN) of bacteria in the sample. Unfortunately, individual col-
onies cannot be directly recovered for subsequent analysis
3. Work Area
Provide a level table or bench top with ample area in a clean,
draft-free, well-lighted room or within a horizontal-flow laminar
hood. Use tables or bench tops with nonporous surfaces, and
disinfect before any analysis is made (Sections 9020B.3c and e).
4. Samples
Collect water as directed in Section 9060A. Initiate analysis as
soon as possible after collection to minimize changes in bacterial
populations, and do not exceed a holding time of 24 h (or, if the
sample was collected for regulatory purposes, the maximum
holding time specified by the regulation). If the sample cannot be
tested within 30 min after collection, maintain it at ⬍10°C but do
not allow it to freeze during transit.
5. Sample Preparation
Mark each plate with sample number, dilution, date, and any
other necessary information before examination. For pour- or
spread-plate methods, use sterile glass (65 cm2) or presterilized
disposable plastic (57 cm2) Petri dishes. For pour-plate, spread-
plate, or membrane-filtration methods, prepare at least two rep-
licate plates for each volume of sample or dilution examined for
compliance testing. Duplicates also are recommended for non-
compliance testing.
Thoroughly mix all samples or dilutions by shaking vigor-
ously for 7 s by hand (approximately 25 times with a 1-ft
movement) or via a mechanical shaker for 15 s at low speed.
Analytical results depend on adequate sample mixing; if the
sample is not adequately shaken before aliquots are removed, the
actual bacterial density could be underestimated.
6. Media
Users should ensure that the formulations of purchased media
match those described below. Compare new lots of media with
current lot in use according to Sections 9020B.5j and 9b.
† SimPlate威 for HPC, Idexx Laboratories, Westbrook, ME.
1