MEMBRANE FILTER TECHNIQUE (g222yThermotolerant (Fecal) Coliform Membrane Fiiter Procedure
gins on transport medium when high temperatures are encoun-
Dissolve ingredients by rehydrating in water. Sterilize by
autoclaving ar 121-124"C for 15 min. Final pH should be 8.6 +
0.2. Dispense at least 2.0 to 3.0 mL (depending on pad manu-
facturer) to tight-lidded plastic culture dishes containing an
absorbent pad, and carefully remove excess liquid from pad by
decanting the plate. Store plates in the refrigerator for use within
96 h.
3. Procedure
a. Sunple presenation and shipnxent' Place absorbent pad in
bottom of sterile Petri dish and saturate with selected coliform
holding medium (see 9222C.2). Remove membrane fllter from
filtration unit with sterile forceps and roll it, grid side up, onto
surface of medium-saturated pad. Protect membrane from mois-
ture loss by tightly closing plastic Petri dish. Seal loose-fltting
dishes with an appropriate sealing tapet to prevent rnembrane
dehydration during transit. Place culture dish containing mem-
brane in an appropria.te shipping container and send to laboratory
for test completion. The sample can be held without visiblo
growth for a maximum of 72 h at ambient temperature on the
holding/preservative medium. Visible growth occasionally be-
t Parafilm, Sigma-Aldrich Co. LLC, or equivalent.
9222 D. Thermotolerant (Fecal) Coliform Membrane Filter Procedure
Thermotolerant (formerly fecal) coliform bacterial densities
may be deterrnined by either multiple-tube procedure or MF
technique. {See Sectisn 9225 for differen*iation af Escherichia
e.'/i.) Tte thenno*oieraat cr&ifsrm !# procedum uses am sa-
riched lactcre modium and hcuba$on terriperature of ,&{.5 -r
0.2'C for selectivity. Because incubation temperafls-e is critieal,
submerge waterpreoled (plastic bag enclosures) L{F culnr,-es in a
water bath for incubation at the elevated iemp€rature or usc an
approprriate -cr:)lid heat-sirrk or other incubator that is documented
to hold the temperature at 44.5oC "1- 0.2C thrcughout the
chamber over a 24-h pericd. The best type of incubator is a
gable-covered circulating watel bath. In general, this methoC is
applicable under the same circumstances as the multiple-tube
tirermotolerant coliform procedures (see Section 922 lE).
There are limitations to the interprctation of a thermctolerant
coliform result from thermal waters (e.g., the tropics) and pulp
anci pa,per mril effluent samples where thermotolerant Klebsiella
ha-,,e predominated and not been indicaiive of a sewerage source.
As with all coliform results, a sanitary survey should be con-
ducted to identify the most plausible source and public health
risk interpretation.
1. Laboratory Apparatus
o. Samitle bottles: See Section 90308.19.
b. Dilution boules: See Section 90308.13.
c. Pipets and graduated cltlln4nrt, See Section 90308.9.
d. Cotteiners.for culture nrcdium: See 92228.1d.
9-89
tered during transit.
b. Transfer und incubation.' At the laboratory, transfer filter
from holding medium on which it was shipped to a second sterile
Petri dish containing m-Endo or Endo LES medium and incubate
at 35 4- 0.5'C fbr 20 to 22 h.
4. Estimation of Coliform Density
Proceed as described in 92228.5. Record times of collection,
filtration, and laboratory examination, and calculate the elapsed
time. Report elapsed time with coliform results.
5. Bibliography
Grr-onErcn, E.8., P.W. Keslrn, H.L. Jsrrn & H.F. Cunr. 1955. A
delayed incubation nrembrane filter test for coliform bacteria in
water. Anrer. J. Pub. Health 45:1462.
PeNszet, A.K., T.J. MecrlrN & H.G. Cous. 1965. Coli-aerogenes and
Escherichia coli counts on water samples by means of transported
membranes. Proc. Soc. Water Treat. Exam. 14:179.
BRFTFNsKT, F.T. & J.A. Wrxren. 1969. Use of the delayed incubation
membrane filter test for determining coliform bacteria in sea water.
Water Res.3:583.
CsrN, IVi. & P.J. Hlcrcv. 1986. Elimination of overgrowth in delayed-
incubation mernbrane filter test for total coliforms by M-ST holding
medium. Apol. Envirorl Microbiol. 52:-778.
e. Culture dishes: Tight-fitting plastic dishes (see 92228.1e)
are preferred when MF culture plates will be submerged in a
water bath during incubation. Place thermoiolerant coliform
cukure plmes in plastic bags {remrve as much air as possib}e} or
seal individual dishes with waterproof (freezer) tape to prevent
leakage during submersion.
f. Filtation rorlts.' See 92228.1f.
g. lulernbrane filters: See 92228.19.
h. Absorbenr pnd.s: See 9222R.lh.
i. force1ts: See 92228.1i.
j. Water bath or incubator: The specificity of the therrnotol-
erant coliform rest is related directly to incubation temperarure.
To meet the need for greater temperature control, use a gable-
covered water bath, a heat-sink incubator, or any properly de-
signed and constructed incubator that can maintain a temperature
tolerance of -0.2"C. Most circulating water baths equippeC with
a gable top to reduce water and heat loss can maintain a tem-
Derature of M5 -f 0.2oC. However, static air incubation may be
a problem in some types of incubators because <lf potential heat
layering in the chamber, slower heat transfer from air to the
medium, and slow temperature recovery each time the incubator
is opened during daily operations.
2. Materials and Culture Medium
mFC mediunt' The need for uniformity dictates the use of
dehydrated media. Never prepare metiia from basic ingredients
when suitable dehydrated media are available. Follow the man-