292 Modern Food Microbiology
of toxins and enterotoxins. Some examples of these models are summarized in Table 12–2, and brief
descriptions are presented below.
Human Mucosal Cells
As employed by Ofek and Beachey,
35 human buccal mucosa cells (about 2 × 10
5 in phosphatebuffered saline) are mixed with 0.5 ml of washed E. coli cells—2 × 10
8 /ml. The mixture is rotated
for 30 minutes at room temperature. Epithelial cells are separated from the bacteria by differential
centrifugation, followed by drying and staining with gentian violet. Adherence is determined by the
microscopic counting of bacteria per epithelial cell. As employed by Thorne et al.,
47 E. coli cells are
labeled with
3 H-amino acids (alanine and leucine) or fluorescein isothiocyanate. In another use of this
method, V. parahaemolyticus cells were mixed with mucosal epithelial cells and incubated at 37
◦ C for
5 minutes followed by filtering. The unbound cells were washed off, and the culture dried, fixed, and
stained with Giemsa. Adherence was quantitated by counting the total number of V. parahaemolyticus
adhering to 50 buccal cells as compared to controls. Best results were obtained when approximately 10
9
bacterial cells and 10
5 buccal cells were suspended together in phosphate-buffered saline at pH 7.2 for
5 minutes. All 12 strains tested adhered. Adherence apparently bears no relationship to pathogenicity
for V. parahaemolyticus.
Human Fetal Intestine
By this adherence model, human fetal intestine (HFI) cells are employed in monolayers. The monolayers are thoroughly washed, inoculated with a suspension of V. parahaemolyticus, and incubated
at 37
◦ C for up to 30 minutes. Adherence is determined by the microscopic examination of stained
cells after washing away unattached bacteria. All strains of V. parahaemolyticus tested adhered, but
those from food-poisoning cases had a higher adherence ability than those from foods.
22 By use of
this method, the adherence of an enteropathogenic strain of E. coli of human origin was found to be
plasmid mediated.
50
Human Ileal and Intestinal Cells
To study adherence of enterotoxigenic E. coli (ETEC), Deneke et al.
13 used ileal cells from adult
humans in a filtration–binding assay. The cells were mixed with bacteria grown in
3 H-alanine and
leucine. The amount of binding was determined with a scintillation counter. ETEC strains of human
origin bound to a greater extent than controls. Binding to human ileal cells was 10- to 100-fold greater
than to human buccal cells.
Monolayers of human intestine cells were used by Gingras and Howard
20 to study adherence of
V. parahaemolyticus. The bacterium was grown in the presence of
14 C-labeled valine, and the labeled
cells were added to monolayers and incubated for up to 60 minutes. Following incubation, unattached
cells were removed, and those adhering were counted by radioactive counts of monolayers. The
adhered cells were also enumerated microscopically. The Kanagawa-positive and -negative organisms
adhered similarly. No correlation was found between hemolysis production and adherence.
Guinea Pig Intestinal Cells
To study adherence of V. parahaemolyticus, Iijima et al.
25 employed adult guinea pigs weighing
about 300 g and fasted them for 2 days before use. Under anesthesia, the abdomen was opened and the
of toxins and enterotoxins. Some examples of these models are summarized in Table 12–2, and brief
descriptions are presented below.
Human Mucosal Cells
As employed by Ofek and Beachey,
35 human buccal mucosa cells (about 2 × 10
5 in phosphatebuffered saline) are mixed with 0.5 ml of washed E. coli cells—2 × 10
8 /ml. The mixture is rotated
for 30 minutes at room temperature. Epithelial cells are separated from the bacteria by differential
centrifugation, followed by drying and staining with gentian violet. Adherence is determined by the
microscopic counting of bacteria per epithelial cell. As employed by Thorne et al.,
47 E. coli cells are
labeled with
3 H-amino acids (alanine and leucine) or fluorescein isothiocyanate. In another use of this
method, V. parahaemolyticus cells were mixed with mucosal epithelial cells and incubated at 37
◦ C for
5 minutes followed by filtering. The unbound cells were washed off, and the culture dried, fixed, and
stained with Giemsa. Adherence was quantitated by counting the total number of V. parahaemolyticus
adhering to 50 buccal cells as compared to controls. Best results were obtained when approximately 10
9
bacterial cells and 10
5 buccal cells were suspended together in phosphate-buffered saline at pH 7.2 for
5 minutes. All 12 strains tested adhered. Adherence apparently bears no relationship to pathogenicity
for V. parahaemolyticus.
Human Fetal Intestine
By this adherence model, human fetal intestine (HFI) cells are employed in monolayers. The monolayers are thoroughly washed, inoculated with a suspension of V. parahaemolyticus, and incubated
at 37
◦ C for up to 30 minutes. Adherence is determined by the microscopic examination of stained
cells after washing away unattached bacteria. All strains of V. parahaemolyticus tested adhered, but
those from food-poisoning cases had a higher adherence ability than those from foods.
22 By use of
this method, the adherence of an enteropathogenic strain of E. coli of human origin was found to be
plasmid mediated.
50
Human Ileal and Intestinal Cells
To study adherence of enterotoxigenic E. coli (ETEC), Deneke et al.
13 used ileal cells from adult
humans in a filtration–binding assay. The cells were mixed with bacteria grown in
3 H-alanine and
leucine. The amount of binding was determined with a scintillation counter. ETEC strains of human
origin bound to a greater extent than controls. Binding to human ileal cells was 10- to 100-fold greater
than to human buccal cells.
Monolayers of human intestine cells were used by Gingras and Howard
20 to study adherence of
V. parahaemolyticus. The bacterium was grown in the presence of
14 C-labeled valine, and the labeled
cells were added to monolayers and incubated for up to 60 minutes. Following incubation, unattached
cells were removed, and those adhering were counted by radioactive counts of monolayers. The
adhered cells were also enumerated microscopically. The Kanagawa-positive and -negative organisms
adhered similarly. No correlation was found between hemolysis production and adherence.
Guinea Pig Intestinal Cells
To study adherence of V. parahaemolyticus, Iijima et al.
25 employed adult guinea pigs weighing
about 300 g and fasted them for 2 days before use. Under anesthesia, the abdomen was opened and the