Journal of Applied Bacteriology 1991 71, 277-284 ADONIS 0021884791001237

The adsorption of bacteria to immobilized lectins

R.A. Patchett, Alison F. Kelly & R.G. Kroll
AFRC lnsfitute of Food Research, Reading Laboratory, Shinfield, Reading, UK
3580/01/91:accepted 2 March 1991

R . A . PATCHETT, A.F. K E LLY AND R.G. K R O L L . 1991. T h e agglutination of a selection of bacteria

by some lectins was examined. T h e lectin from Codium fragile agglutinated seven strains of
Salmonella typhimurium. T h e lectin from Helix pomatia agglutinated eight of 12 strains o f
Listeria monocytogenes and a further two strains gave a weak agglutination reaction. Helix
pomatia lectin conjugated to magnetic microspheres enabled t h e adsorption of L.
monocytogenes from suspension with subsequent elution by the competing ligand N-acetyl
galactosamine. Affinity chromatography of a suspension of L. monocytogenes through a
column of H . pomatia lectin immobilized on agarose, also adsorbed cells a n d enabled
subsequent elution with N-acetyl galactosamine. T h e column technique enabled the m o r e
rapid adsorption of bacteria perhaps because of improved interactions between bacteria a n d
immobilized lectin.

INTRODUCTION for the concentration of salmonellas prior to detection by

impedance techniques (Bird et al. 1989).
Methods for the rapid detection and/or counting of bacteria
Lectins are proteins with specific carbohydrate binding
can be highly sensitive when applied to aqueous suspen-
activities. They may be isolated from a wide variety of bio-
sions. They can be of restricted value, however, when
logical sources and can be used for labelling cell surface
applied directly to samples such as foods. For example,
components for tissue typing (Lis & Sharon 1986). Fur-
assay of microbial metabolites such as ATP is subject to
thermore, the selective agglutination of immature thy-
high background levels of non-microbial origin (Webster et
mocytes by peanut lectin allows their separation from
al. 1988). Particle-counting methods based on microscopy
mature thymocytes (Reisner 8i Sharon 1984) and affinity
(Pettipher 1983) or flow cytometry can similarly suffer from
chromatographic techniques have been used to separate dif-
interference by particulate matter (Patchett et al. 1991).
ferent human lymphocytes (Hellstrom et al. 1976). Some
Many of these problems might be reduced if the bacteria
lectins have been shown to bind to a number of bacteria
were separated and concentrated from the sample before
and a range of bacterial components such as teichoic acids,
analysis (Wood & Gibbs 1982). Food samples can be
lipopolysaccharides and other bacterial polysaccharides
complex and varied matrices and simple centrifugation and
(Pistole 198 1). Lectins can also sometimes distinguish
filtration methods cannot generally be successfully applied.
between bacterial species, e.g. Neisseria gonorrhoeae may be
As alternative approaches, the adsorption of bacteria to ion
differentiated from other Neisseria spp. and related bacteria
exchange resins (Daniels 1972; Wood & Gibbs 1982) or
by its agglutination with wheat germ lectin (Doyle et al.
electropositive filters (Hirsh & Martin 1984; Kroll 1985;
1984). This work examines the agglutination of some food-
Thomas 1988) have been examined. However, another
borne bacteria by a selection of lectins and the application
possibility is to exploit the specific binding activities of bio-
of lectin affinity methods to adsorb some of these bacteria
logical molecules. Enzyme-linked immunsorbent assays
from aqueous suspensions.
exploit the ability of immobilized antibodies to bind and
retain a specific antigenic molecule or cell during sub-
sequent washing, prior to detection by a secondary
antibody-enzyme complex (Morris 1985). Antibodies MATERIALS AND METHODS
immobilized on magnetic microspheres have been used for
the separation of encapsulated strains of Staphylococcus Growth of bacterial cultures
aureus from mastitic milk samples Uohne et al. 1989) and
The Listeria spp. were grown on coryneform broth which
Correspondence r o : Dr R . C . Kroll, AFRC Insrrrute of’Food Research, consisted of (g/l): tryptone (Difco), 10; yeast extract
Reading 1.uboratory. Shinjeld, Readrng, Berks R C 2 9AT, U K . (Difco), 5; sodium chloride, 5 ; glucose, 5; p H 7.2 at 37°C.
278 R . A . PATCHETT E r A L
The Salmonella spp. were grown on yeast dextrose broth
which consisted of (g/l): Nutrient Broth No. 2 (Oxoid), 25;
yeast extract (Oxoid), 3; glucose, 5; pH 6.8 at 37°C. The
other cultures were grown on Nutrient Broth No. 2 (Oxoid)
a t 30°C. All the cultures were grown overnight in shake
flasks.
Screening of lectln blndlng
Cultures of listerias, salmonellas, Bacillus spp., Escherichia
coli K 12 and Staph. aureus were harvested by centrifugation
(5000 g, IOmin), washed and resuspended in phosphate-
buffered saline (PBS; 5 mmol/l potassium phosphate, 150
mmol/l NaCl, pH 7.0) containing 0.1 mmol/l CaCl, and 0.1
mmol/l MnCI, to give a final optical density (600 nm) of
approx. 10. They were then stored on ice. The ability of
the lectins to bind to the bacteria was determined by an
agglutination assay. Bacterial suspensions (25 pi) were
mixed with lectin solutions (25pl of 200 pg/ml in PBS con-
taining 0.1 mmol/l CaCI, and 0.1 mmol/l MgCI,) on
microscope cavity slides. In control experiments lectins
were replaced with PBS. The slides were incubated in a
humid box at room temperature for 30 min. Agglutination
of bacteria was assessed by the formation of clumps visible
to the naked eye or under a low power stereo-microscope.
Adsorption of Listerla monocytogenes to lmmoblllzed
lectln
Preparation of Helix pornatia lectin-magnetic
microspheres
Tosyl-activated magnetic microspheres (DynabeadsR;
Dynal (UK) Ltd, Wirral, Merseyside, UK), 0.5 ml, were
placed into a microfuge tube, precipitated with a magnet,
and the supernatant fluid removed. The DynabeadsR were
resuspended in 0.5 ml of distilled water. Helix pomatia
lectin (0.5 ml of 200 ,ug/ml in 0.1 mol/l sodium tetraborate
buffer (pH 9.5) containing N-acetyl galactosamine at 20
mg/ml) was added and incubated at 25°C for 24 h with
end-over-end mixing. The H . pomatia lectin-DynabeadR
conjugate was washed twice with 1 ml of bovine serum
albumin (BSA; 0.1% in PBS), resuspended in BSA (0.1%
in PBS) and incubated for 18 h at 4°C with end-over-end
mixing. The lectin-DynabeadR conjugate was washed twice
with 1 ml PBS, resuspended in 0.5 ml PBS to give approx-
imately 4 x lo8 beads/ml and stored at 4°C.
Adsorption to Helix pornatia lectin-magnetic
microspheres
Suspensions of washed cells of L. monocytogenes C200 at
various cell densities in PBS were incubated with H .
pomatia lectin-DynabeadR conjugate at 25°C for 1 h with
mixing. The beads were precipitated with a magnet, the
supernatant fluid was removed and the beads washed with
1 ml of PBS. The washed beads were incubated with 0.2 ml
of N-acetyl galactosamine (10 mg/ml in PBS) at 25°C for
30 min, precipitated with a magnet and the supernatant
fluid removed. The bacteria were counted by plating of
samples on coryneform agar (coryneform broth solidified
with 2% agar, Oxoid No. 2) after serial dilution in PBS.
Colonies were counted after 24 h incubation at 37°C.
Binding to Helix pomatia lectin-agarose conjugate
Batch method. Suspensions (2 ml) of washed cells of of L.
monocytogenes C200 in PBS at approximately 1 x lo4
cfu/ml were incubated with H . pomatia lectin-agarose con-
jugate (Sigma; 0.25 ml packed volume) with mixing at
25°C for 1 h. The agarose was allowed to settle for 10 min
and the supernatant fluid removed. The agarose was
washed with PBS (1 mi) and then incubated with N-acetyl
galactosamine (0.2 ml of 50 mg/ml solution in PBS) at 25°C
for 30 min. The agarose was allowed to settle for 10 min
and the supernatant fluid removed. The bacteria were
counted by plating samples of fractions on coryneform agar.
Column method. Affinity columns were prepared by placing
H . pomatia lectin-agarose conjugate into 1 ml disposable
syringes (Sabre, Reading, UK). Columns were packed to
the 0.5 ml mark and elution of the agarose prevented by a
small disc of 25 pm pore size nylon mesh at the base of the
syringe. The agarose was washed with PBS before use.
Suspensions of washed cells (2 ml) of L. monocytogenes
C200 (1 x lo4 cfu/ml) were applied to the column and
eluted by gravity. T h e eluent was collected and the column
washed with successive applications of PBS (2 x 1 ml) and
N-acetyl galactosamine (1 ml of 10 mg/ml and then 1 ml of
20 mg/ml solution). T h e number of bacteria in the fractions
was determined by plating on coryneform agar.
RESULTS
Agglutlnatlon of bacteria by lectins
T h e ability of various lectins to bind to the bacteria was
examined by their ability to cause agglutination of bacterial
suspensions. In initial experiments, a number of bacterial
suspensions were screened against a range of commercially
available lectins. These results identified some lectins that
could bind with a degree of specificity (results not shown).
Selected lectins were then examined in more detail with a
number of different bacterial strains. Helix pomatia lectin
was able to agglutinate L. monoc-ytogenes; therefore a
 IMMOBILIZED LECTINS AND BACTERIA 279

number of L. monocytogenes strains and Listeria spp. were
examined for binding to this lectin (Table 1). Strong agglu-
tination was observed with eight of 12 strains of L. mono-
cytogenes and a further two strains gave weak and variable
reactions. Two of the 12 strains failed to agglutinate with
this lectin under the conditions of this assay. Agglutination
was also observed with five of the six strains of L . innocua
tested and both the strains of L. murrayi. This lectin also
caused agglutination of one of the three strains of L. welsh-
imeri but failed to agglutinate any of the L. ivanovi or L .
seeligeri strains. T h e H . pomatia lectin failed to agglutinate
many of the other bacteria tested, although positive agglu-
tinations were observed with E. coli NCDO 2070 and a
weak reaction was observed with Bacillus licheniformis.
Seven strains of Salmonella typhimurium were agglutinat-
ed by the lectin from the marine alga Codium fragile (Table
2). The agglutination of other Salmonella spp. was less reli-
able with only one of three Salm. enteritidis strains being
agglutinated. The reaction of C. fragile lectin with other
bacteria was also variable, with five of the 1 1 strains of
Bacillus spp. tested giving positive agglutinations. T h e
three E. coli strains gave negative results.
A more thorough examination of the agglutination of L.
monocytogenes by a group of lectins was undertaken. T h e
lectin from H. pomatia binds to N-acetyl galactosamine and
oligosaccharides containing N-acetyl galactosamine (Wu et
al. 1987). T o determine whether other, possibly more effi-
cient, lectins could be found that bound to L. mono-
cytogenes, the agglutination of a number of Listeria spp.
with some lectins reported to bind to N-acetyl galactos-
amine was examined (Table 3). T h e lectin from H. pomatia
proved the most successful for agglutinating Listeria spp.
but lectins from Dolichos bijorus, Maclura pornifera, Bau-
hinia purpurea and Arachis hypogea failed to cause agglutin-
ation despite a reported binding activity for
oligosaccharides containing N-acetyl galactosamine. T h e
lectin from the soya bean (Glycine max) caused agglutina-
tion of one of the L. monocytogenes strains tested, whilst the
lectin of Bandeiraea simpli$cola (BS 11) caused agglutina-
tion of L. innocua C614 and two strains of L. monocytogenes

Table 1 The agglutination of some bacteria by lectin from Helix pomatia

Listeria monocytogenes c200* + L. welshimeri CIP 88 -
c 2 0 1* + CIP 89 -
c202* + CIP 8149 +
C203* +
C231* + L. seeligeri SLCC 3990 -
C213* + SLCC 3794 -
C226* +
C227* - L. murrayi NCTC 10814 +
C228* f NCTC 10815 +
C52* +
NCTC 10888 - Salmonella typhimurium 010 34014t -
NCTC 10890 +
-
Salm. enteritidis 010 37782t -
L. innocua c645 -
c535 + Bacillus cereus 818: -
C532 +
C644 + B. licheniformis NCTC 1772 k
c530 +
C614 + Escherichia coli K 12 AB 61218 -
E. coli B NCDO 2328 -
L. ivanovi C1173 - E . coli NCDO 2070 +
C659 -
C614 -

* From Dr D. Jones, University of Leicester, UK.

t From Dr D. Gibson, Torry Research Station, Aberdeen, UK.
$ From Mr H. Underwood, AFRC Institute of Food Research, Norwich, UK.
5 From Dr S. Hill, AFRC Institute of Food Research, Norwich, UK.
+, Strong agglutination; -, no visible agglutination; +, variable agglutination; CIP, Collection Institut Pasteur; SLCC, Seeliger
Listeria Culture Collection.
280 R . A . PATCHETT ET A L

Table 2 The agglutination of some bacteria by the lectin from Codium fragile

Salmonella typhimurium 010 34014t B . pumilis NCFB 1788

010 379387 B . sphaericus NCFB 1767
010 34590t B . megaterium DSM 32
010 378741. B . lichenijormis KRM 007#
010 37340t
NCTC 0074 Escherichia coli K12 AB 61214
NCTC 8391 E . coli B NCDO 2328
E. coli NCDO 2070
Salm. enteritidis NCTC 4444
NCTC 5188 Staphylococcus aureus NCDO 949
010 137782t
Listeria monocytogenes C203*
Salm. gallinarum NCTC 9240 C213*
Salmonella sp. ex. pepperamit +
Bacillus cereus 818$ +
DSM 487 0
DSM 336 0
DSM 351 +
NCDO 626 +
NCIB 9373 0
DSM 360 +
* From Dr D. Jones, University of Leicester, UK.
t From Dr D. Gibson, Torry Research Station, Aberdeen, UK.
1From Mr H. Underwood, AFRC Institute of Food Research, Norwich, UK.
4 From Dr S. Hill, AFRC Institute of Food Research, Norwich, UK.
# From M.R. Griffiths, Hannah Research Institute, Ayr, UK.
+, Strong agglutination; -, no visible agglutination; 0, agglutination difticult to assess due to natural agglutination.

including L. monocytogenes NCTC 10888 which failed to
agglutinate with H. pomatiu lectin.
Adsorption of L. monocytogenes to lmmoblllzed lectin
Adsorption to H. pomatia lectin-magnetic
microsphere conjugate
When H . pomatiu lectin conjugated to magnetic micro-
spheres was added to a suspension of L. monocytogenes
C200, the cell density in the suspension decreased with
incubation over a period of 2 h (Fig. 1). After precipitation
of the DynabeadsR and washing in buffer, subsequent
washing with N-acetyl galactosamine caused release of L.
monocytogenes from the immobilized lectin. T h e number of
L. monocytogenes cells released increased with longer incu-
bation time with the lectin-DynabeadR conjugate. T h e
release of adsorbed cells by N-acetyl galactosamine
appeared to be incomplete; after initial incubation for 60
min the cell density in suspension decreased by 75% but
only 29% of the apparently adsorbed cells were released on

Table 3 Agglutination of Listeria spp. with N-acetyl galactosamine-binding lectins

L. monocytogenes L . innocua

Lectin source C200 C52 C228 C201 C226 C231 NCTC 10888 NCTC 10890 C614

7000 c low affinity binding site and this was susceptible to release
2000 1 2.76 x lo7 beads were required. At higher initial concen-
0 30 60 90 I20
Time ( m i d
Fig. 1 Effect of incubation time on adsorption of Listeria
monocytogenes to Helix pomatia lectin-DynabeadsR. Suspensions of
washed bacteria in phosphate-buffered saline (PBS) (1 ml) at
6.4 x lo3 cfu/ml (A, A)or 5 x 10' cfu/ml (0, 0 )were
incubated with H . pornaria lectin-DynabeadsR (4 x 10' beads)
with mixing at 25°C. At intervals beads were precipitated with a
magnet, the supernatant fluid removed for counting bacteria (open
symbols) and the beads washed in PBS ( 1 ml). The washed beads
were incubated with N-acetyl galactosamine (0.2 ml of 10 mg/ml
solution) for 30 min at 25"C, precipitated and the supernatant
fluid removed for counting bacteria (closed symbols). The bacteria
were counted by plating of fractions on coryneform agar
washing with N-acetyl galactosamine. Control experiments
indicated a small loss in viability (5%) over a 60 min incu-
bation period in buffer (results not shown), but this was
insufficient to account for the discrepancy. The failure to
recover all of the bacteria may be due to incomplete release
of all of the cells from the beads or loss of bacteria in the
intermediate washing stage. Experiments which started
with lo4 cfu/ml showed very low levels of bacteria in the
wash ( < 5 % total). At high cell densities (lo5 cfu/ml),
higher concentrations of bacteria were found (e.g. at an
initial cell density of 1.5 x 10' cfu/ml, 2 x 10' cfu/ml were
present in the wash; results not shown). These levels were
higher than could be accounted for by the dilution of a
residual amount of liquid and suggested that some of the
adsorbed bacteria were released from the DynabeadR.
These results suggested that at high concentrations of bac-
teria, a portion of the bound population was attached to a
IMMOBILIZED LECTINS AND BACTERIA 281
by washing. Alternatively, it may indicate the breaking up
of clumps of bacteria at high cell densities. Binding of bac-
teria to the beads was observed with both high and low
initial concentrations of bacteria (Fig. l), and higher con-
centrations of N-acetyl galactosamine or longer incubation
times did not result in a greater release of adsorbed cells
(not shown).
Adsorption of L. monocytogenes to immobilized lectin was
dependent on the amount of lectin-DynabeadR conjugate
present (Fig. 2). At 1 x lo2 cfu/ml, approximately 8 x lo6
beads were required to adsorb 75% of the bacteria, whilst
at an initial density of 4.8 x lo2 cfu/ml approximately
trations of bacteria it was not possible to follow the absorp-
tion of bacteria by the decline in cell density as the
proportion of cells adsorbed became too small to give sig-
nificant decreases in cell density. Under these conditions
the release of bacteria from the lectin-DynabeadR conjugate
l 800
oo0l
h
3
.t
1
u
I
b
n
'c
0
Number of lectin-dynobeads ( x lo-')
Fig. 2 Effect of the Helix pomatia lectin-DynabeadR
concentration on binding of Listeria monocytogenes. Suspensions of
washed bacteria in phosphate-buffered saline (PBS) (1 ml) at
4.8 x 10' cfu/ml (0) or 1 x 10' cfu/ml (A) were incubated at
25°C for 1 h with various concentrations of H . pomatiu
lectin-DynabeadsR. The beads were precipitated, the supernatant
fluid removed and the beads washed in PBS (1 ml). The beads
were incubated with N-acetyl galactosamine (0.2 ml of 10 mg/ml
solution) for 30 min at 25"C, precipitated and the supernatant
fluid removed. The bacteria were counted by plating on
coryneform agar
282 R . A . P A T C H E T T E r A L .

only on the concentration of the conjugate, but also on the

initial density of the bacteria suspension (Fig. 3). This sug-
gests that adsorption may be limited by the effective colli-
sion frequency between the bacteria and the conjugate.

Adsorption to lectin immobilized on agarose

Batch adsorption. Incubation of a suspension of L. mono-

cytogenes C200 with H . pomatia lectin-agarose conjugate in
batch fashion and end-over-end mixing failed to achieve
significant binding of the bacteria. The majority of the bac-
teria were recovered from the supernatant fluid of the
beads, with decreasing numbers recovered in subsequent
washes with buffer and with N-acetyl galactosamine. There
was no significant difference in recovery pattern when the
bacteria were applied in a solution of the competing ligand
N-acetyl galactosamine. (Results not shown.)

Column chromatography. Passage of suspensions of L. mono-

0 2
Number of lectin-dynabeods ( x lo-')
cytogenes C200 through columns of H. pomatia lectin
immobilized on agarose resulted in partial adsorption of the
bacterial population to the column (Fig. 4a). After washing
the columns with buffer, the adsorbed bacteria could be
eluted with N-acetyl galactosamine. However, the overall
recovery of bacteria from the columns was highly variable
(between 33 and 215%; results not shown). The low
recovery values may indicate irreversible binding of bac-
teria to the column. The high recovery values probably
indicated disruption of clumped bacteria. In control experi-
ments, L. monocytogenes C200 was suspended in PBS buffer
containing N-acetyl galactosamine (10 mg/ml) (Fig. 4b). All
the bacteria were recovered in the initial eluent, indicating
that the retention of L. monocytogenes C200 by H . pomatia
lectin-agarose was a specific binding activity.
4 6 8

Fig. 3 Desorption of Listeria monocytogenes from Helix pomatia

lectin-Dynabead?. Suspensions of washed bacteria in
phosphate-buffered saline (PBS) (1 ml) at 3 x lo3 cfu/ml (A),
1 x lo4 cfu/ml (0) or 4.9 x lo4 cfu/ml(O) were incubated with
various concentrations of H. pomatia lectin-Dynabead? for 1 h at
25°C. The beads were precipitated, the supernatant fluid removed
and the beads washed in PBS (1 ml). The beads were incubated
with N-acetyl galactosamine (0.2 ml of 10 mg/ml solution) for 30
min at 25"C, precipitated and the supernatant fluid removed. The
bacteria were counted by plating on coryneform agar
upon washing with N-acetyl galactosamine was monitored.
However, it must be noted that release was probably
incomplete (cf. Fig. 1) and thus gave an underestimate of
the total numbers adsorbed. The number of bacteria rel-
eased from the lectin-DynabeadR conjugate depended not
DISCUSSION
T h e lectin from H. pomatia agglutinated the majority of
strains of L. monocytogenes tested, suggesting the wide-
spread occurrence of N-acetyl galactosamine-containing oli-
gosaccharides on the cell surface. However, a detailed
examination of the agglutination of L. monocytogenes by a
range of lectins did not show a direct relationship between
the reported binding activity of the lectins for N-acetyl
galactosamine and the ability to agglutinate these bacteria,
T h e reason for this is unclear although it may be due i o
higher levels of specificities of the lectins for oligosac-
charides with different sequences of carbohydrate residues
(Wu et al. 1987). Alternatively, different surface character-
istics of the bacteria may impede the lectins' access tq
binding sites.

Fig. 4 Adsorption of Listeria - 6000
monocytogenes to a Helix pomatia
lectin-agarose column. (a) A suspension of
washed cells of L. monocytogenes (2 ml) in
h
4000
phosphate buffered saline (PBS) at
5 x lo3 cfu/ml was passed through a
column of H. pomatia lectin-agarose
(packed bead volume 0.5 ml) under 2000
gravity. The column was washed with
2 x 1 ml PBS and subsequently with 1 ml
of N-acetyl galactosamine (GaL/NAC) at
20 mg/ml and 1 ml of N-acetyl 0 0
galactosamine at 20 mg/ml. Bacteria were
counted by plating of fractions on
coryneform broth. In a control experiment
(b) the initial suspension of bacteria
contained N-acetyl galactosamine (10
mg/ml)
T h e immobilized H. pomatia lectin was able to adsorb L.
monocytogenes from aqueous suspension. This lectin immo-
bilized on magnetic microspheres enabled the adsorption of
bacteria in a batch system. However, the adsorption was
highly dependent on the concentration of beads used. Sig-
nificant adsorption of L. monocytogenes was not achieved
with H. pomatia lectin-agarose in batch systems. T h e H.
pomatia lectin-agarose was more difficult to maintain in
suspension compared with the magnetic microspheres, thus
failure to achieve significant binding may have been due to
poor mixing. Helix pomatia lectin-agarose proved more
successful in a column system, probably providing better
contact between lectin and bacteria and enabling better
adsorption.
For application of these methods to the adsorption of
bacteria from suspension, the column technique has the
advantage of enabling all bacteria to come into contact with
the solid matrix. I n batch systems the amount of solid
matrix required to ensure sufficient distribution through
the liquid phase may be prohibitive. T h e alternative
approach of increased incubation times to enable mixing
and binding to occur may lead to cell damage or death with
prolonged incubation periods.
Techniques for the adsorption of bacteria could prove
useful for increasing the sensitivity of methods for detecting
bacteria by removing background interference and by
potentially concentrating the organisms. I n our experiments
the efficiency of adsorption and recovery was low.
However, the present work shows that lectins will adsorb
bacteria and they may be used for the extraction of bacteria
from aqueous environments.
I M M O B I L I Z E D LECTINS AND BACTERIA 283
00 00
6000
4000
2000
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