Membrane Filter-Fluorescent-Antibody Method for Detection and Enumeration of Bacteria in Water Page: 399
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APPLIED MICROBIOLOGY, Mar. 1969, p. 399-401
Copyright @ 1969 American Society for Microbiology
Vol. 17, No. 3
Printed in U S.A.
Membrane Filter-Fluorescent-Antibody Method for
Detection and Enumeration of Bacteria in Water
RUFUS K. GUTHRIE AND DENNIS J. REEDER
Department of Biology, North Texas State University, Denton, Texas 78213
Received for publication 13 January 1969
A technique which employs nonfluorescing membrane filters and specific
fluoresceinisothiocynate-labeled antiserum has been successfully used in the identi-
fication and enumeration of known species of Escherichia coli which have been
added to natural populations of bacteria found in water. The quantitative results
compared favorably with those of standard tests. The use of a dissecting micro-
scope with an external lighting arrangement provided a simple requirement for
equipment. This method may be useful in monitoring specific bacterial types from
waters which were being monitored for specific pollution.
A method is described for the combined use of
membrane filter and fluorescent-antibody tech-
niques to detect and quantitate bacteria in water.
The method has been tested as a means of detect-
ing the presence of Escherichia coli in water
supplies and has been compared with standard
methods with respect to sensitivity and time
required. Limitations and the potential of the
method to rapidly detect pollution of water sup-
plies have been discussed.
The use of membrane filters with differential
media preparations which allow direct specific
determination of coliforms has been well estab-
lished as a means to determine sanitary quality of
water (1). In general, these methods require 18 to
24 hr for quantitative counts.
Fluorescent-antibody techniques have been
used for the identification of microorganisms from
various sources. These techniques, however, re-
quire precise microscopic methods, particularly
when dark-field microscopy is used. Danielsson
(4) and Danielsson and Laurell (4a) reported a
membrane-filter method for the demonstration
of bacteria by fluorescent-antibody techniques.
That method employed filters mounted under the
high-power objectives of a microscope, a method
requiring manipulation of the membranes and,
for quantitation, mathematical calculation. Carter
and Leise (3) reported a similar method for
enumeration of anthrax colonies.
In water pollution, more rapid detection and
enumeration of specific contaminating organisms
may be of prime importance. For this reason, our
study was initiated to make use of the advantages
of the membrane-filter methods for concentration
and growth of bacteria and fluorescent-antibody
techniques for rapid identification and enumera-
tion of bacteria with the use of low-power,
MATERIALS AND METHODS
Production of antiserum. Antiserum to 11 strains of
E. coli was produced in rabbits essentially by the
methods described by Edwards and Ewing (5).
Serum was used to identify specific E. coli strains or
selectively pooled to give a polyvalent serum.
Conjugation procedure. The sera were conjugated
with fluorescein isothiocyanate by the methods de-
scribed by Spendlove (8), and further purified after
conjugation by means of a diethylaminoethyl cellu-
lose column. The final conjugate was checked for
purity and absence of albumin by cellulose acetate
strip electrophoresis. Serum was stored at -15 C in
stocks of 10 mg of protein per ml. Protein determina-
tions were made by the method of Lowry et al. (6).
The stocks were thawed and diluted to a protein con-
centration of 1 mg/ml prior to use.
Filter equipment. Black, gridded filters (HABG
047, Millipore Corp., Bedford, Mass.) in glass filter
bases and funnels were used for this study. White
filters and filters from other sources autofluoresce
under the conditions of illumination and mask the
Fluorescence microscopy. Dark-field preparations
were observed with a Nikon SKE microscope (Nikon
Inc., Garden City, N.J.) fitted with an ultradark-field
condenser (NA 1.50). Illumination was provided by
a Bausch and Lomb Illuminator (Bausch & Lomb,
Inc., Rochester, N.Y.) with a 200-w mercury arc
lamp. Green, neutral-density filters were used for
illumination with visible light, and Comrning 5-58 or
7-60 filters were used for ultraviolet illumination.
Barrier filters were either the appropriate Y-8 or T-2,
or the yellow filter supplied with the Nikon equip-
ment. These filter combinations are equivalent to the
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Guthrie, R. K. & Reeder, Dennis J. Membrane Filter-Fluorescent-Antibody Method for Detection and Enumeration of Bacteria in Water, article, March 1969; [Washington, D.C.]. (https://digital.library.unt.edu/ark:/67531/metadc284541/m1/1/: accessed June 19, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.