294 Modern Food Microbiology
small intestine tied approximately 3 cm distal from the stomach. The intestine was injected with 1.0
ml of a suspension of 2 × 10
8 cells of adherence-positive and adherence-negative strains, followed
by closing of the abdomen. Six hours later, the animals were killed, and the small intestine was
removed and cut into four sections. Following homogenization with 3% NaCl, the number of cells in
the homogenate was determined by plating. With adherence-positive cells, larger numbers were found
in the homogenates, especially in the upper section of the intestine.
Another adhesion model consists of immobilizing soluble mucosal glycoproteins from mouse intestines on polystyrene.
28 Using this model, it was shown that two plasmid-bearing strains of E. coli
(K88 and K99) adhered readily, as do other adhering strains of this organism.
HeLa Cells
This cell line has had wide use in testing for the invasive potential of intestinal pathogens as well
as for adherence. Although HeLa cells seem to be preferred, other cell lines such as human laryngeal
carcinoma and Henle 407 human intestine may be employed. In general, monolayers of cells are
prepared by standard culture techniques on a chamber slide and inoculated with 0.2 ml of a properly
prepared test culture suspension. Following incubation for 3 hours at 35
◦ C to allow for bacterial
growth, monolayer cells are washed, fixed, and stained for viewing under the light microscope. In the
case of invasive E. coli, cells will be present in the cytoplasm of monolayer cells but not in the nucleus.
In addition, invasive strains are phagocytized to a greater extent than noninvasives and the number of
bacteria/cell is >5. According to the Bacteriological Analytical Manual (BAM),
17 at least 0.5% of
the HeLa cells should contain no less than five bacteria. Positive responses to this test are generally
confirmed by the Sereny test (see reference 17).
A modification is used for invasive Yersinia. By this method, 0.2 ml of a properly prepared bacterial
suspension is inoculated into chamber slides containing the HeLa cell monolayer. Following incubation
for 1.5 hours at 35
◦ C, the cells are washed, fixed, and stained for microscopic examination. Invasive
Y. enterocolitica are present in the cytoplasm—usually in the phagolysosome. Infectivity rates are
generally greater than 10%. Although invasive E. coli are confirmed by the Sereny test, this is not
done with Y. enterocolitica, even though invasive, because this organism may not yield a positive
Sereny test.
HeLa cells have been used to test for adherence of V. parahaemolyticus and to study the penetration
of Y. enterocolitica. Strains of the latter that gave an index of 3.7–5.0 were considered penetrating.
41
The infectivity of HeLa cells by Y. enterocolitica has been studied by use of cell monolayers in roller
tubes. The number of infecting bacterial cells is counted at random in 100 stained HeLa cells for up
to 24 hours.
14
Chinese Hamster Ovary Cells
The Chinese hamster ovary (CHO) assay was developed by Guerrant et al.
21 for E. coli enterotoxins
and employs CHO cells grown in a medium containing fetal calf serum. Upon establishment of
a culture of cells, enterotoxin is added. Microscopic examinations are made 24–30 hours later to
determine whether cells have become bipolar and elongated to at least three times their width and
whether their knoblike projections have been lost. The morphological changes in CHO cells caused
by both cholera toxin and E. coli enterotoxin have been shown to parallel the elevation of cyclic AMP.
It has been found to be 100–10,000 times more sensitive than skin permeability and ileal loop assays
small intestine tied approximately 3 cm distal from the stomach. The intestine was injected with 1.0
ml of a suspension of 2 × 10
8 cells of adherence-positive and adherence-negative strains, followed
by closing of the abdomen. Six hours later, the animals were killed, and the small intestine was
removed and cut into four sections. Following homogenization with 3% NaCl, the number of cells in
the homogenate was determined by plating. With adherence-positive cells, larger numbers were found
in the homogenates, especially in the upper section of the intestine.
Another adhesion model consists of immobilizing soluble mucosal glycoproteins from mouse intestines on polystyrene.
28 Using this model, it was shown that two plasmid-bearing strains of E. coli
(K88 and K99) adhered readily, as do other adhering strains of this organism.
HeLa Cells
This cell line has had wide use in testing for the invasive potential of intestinal pathogens as well
as for adherence. Although HeLa cells seem to be preferred, other cell lines such as human laryngeal
carcinoma and Henle 407 human intestine may be employed. In general, monolayers of cells are
prepared by standard culture techniques on a chamber slide and inoculated with 0.2 ml of a properly
prepared test culture suspension. Following incubation for 3 hours at 35
◦ C to allow for bacterial
growth, monolayer cells are washed, fixed, and stained for viewing under the light microscope. In the
case of invasive E. coli, cells will be present in the cytoplasm of monolayer cells but not in the nucleus.
In addition, invasive strains are phagocytized to a greater extent than noninvasives and the number of
bacteria/cell is >5. According to the Bacteriological Analytical Manual (BAM),
17 at least 0.5% of
the HeLa cells should contain no less than five bacteria. Positive responses to this test are generally
confirmed by the Sereny test (see reference 17).
A modification is used for invasive Yersinia. By this method, 0.2 ml of a properly prepared bacterial
suspension is inoculated into chamber slides containing the HeLa cell monolayer. Following incubation
for 1.5 hours at 35
◦ C, the cells are washed, fixed, and stained for microscopic examination. Invasive
Y. enterocolitica are present in the cytoplasm—usually in the phagolysosome. Infectivity rates are
generally greater than 10%. Although invasive E. coli are confirmed by the Sereny test, this is not
done with Y. enterocolitica, even though invasive, because this organism may not yield a positive
Sereny test.
HeLa cells have been used to test for adherence of V. parahaemolyticus and to study the penetration
of Y. enterocolitica. Strains of the latter that gave an index of 3.7–5.0 were considered penetrating.
41
The infectivity of HeLa cells by Y. enterocolitica has been studied by use of cell monolayers in roller
tubes. The number of infecting bacterial cells is counted at random in 100 stained HeLa cells for up
to 24 hours.
14
Chinese Hamster Ovary Cells
The Chinese hamster ovary (CHO) assay was developed by Guerrant et al.
21 for E. coli enterotoxins
and employs CHO cells grown in a medium containing fetal calf serum. Upon establishment of
a culture of cells, enterotoxin is added. Microscopic examinations are made 24–30 hours later to
determine whether cells have become bipolar and elongated to at least three times their width and
whether their knoblike projections have been lost. The morphological changes in CHO cells caused
by both cholera toxin and E. coli enterotoxin have been shown to parallel the elevation of cyclic AMP.
It has been found to be 100–10,000 times more sensitive than skin permeability and ileal loop assays