274 Modern Food Microbiology
yeasts could be characterized. The latter method is one in which a microcalorimeter is filled with
a flow-through calorimetric vessel. By the use of a chemically defined medium containing seven
sugars, thermograms were produced by nine lactic acid bacteria (belonging to the genera Enterococcus,
Leuconostoc, and Lactobacillus) distinctive enough to recommend the method for their identification.
66
All cultures were run at 37
◦ C except “S. cremoris,” which was run at 30
◦ C, and results were obtained
within 24 hours.
This method has been used to study spoilage in canned foods, to differentiate between the Enterobacteriaceae, to detect the presence of S. aureus, and to estimate bacteria in ground meat. In detecting
S. aureus, results were achieved in 2 hours using an initial number of 10
7 –10
8 cells per milliliter and in
12–13 hours when only 2 cells per milliliter were used.
119 As a monitoring device, flow microcalorimetry was used to determine the viability of recovered frozen cells of S. cerevisiae within 3 hours after
thawing.
13 When applied to comminuted meat, the peak exothermic heat production rate (HPR) could
be recorded within 24 hours for meats that contained 10
5 –10
8 cfu/g, and results correlated well with
plate count results.
70 With 10
2 cfu/ml, a measurable HPR was produced after 6 hours, with a peak
HPR at 10 hours.
Flow Cytometry
Flow cytometry is the science of measuring components (cells) and the properties of individual
cells in liquid suspension. In essence, suspended cells, one by one, are brought to a detector by means
of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells
traverse the detector, and among the cell properties that can be detected are fluorescence, absorbance,
and light scatter. By use of flow sorting, individual cells can be sorted on the basis of their measured
properties, and 1–3 or more global properties of the cell can be measured.
141 Flow cytometers and cell
sorters make use of one or more excitation sources such as argon, krypton, or helium–neon ion lasers
and one or two fluorescent dyes to measure and characterize several thousand cells per second. When
a dye is used, its excitation spectrum must match the light wavelengths of the excitation source.
40
Two dyes may be used in combination to measure, for example, total protein and DNA content. In
these instances, both dyes must excite at the same wavelength and emit at different wavelengths so
that the light emitted by each dye is measured separately. The early history of flow cytometry has been
reviewed by Horan and Wheeless.
83
Although most studies have been conducted on mammalian cells, both DNA and protein have been
measured in yeast cells. Typically, yeast cells are grown, fixed, and incubated in an RNase solution for
1 hour. Cell protein may be stained with fluorescein isothiocyanate and DNA with propidium iodide.
Following necessary washing, the stained cells are suspended in a suitable buffer and are now ready
for application to a flow cytometer. The one used by Hutter et al.
84 was equipped with a 50-mW argon
laser. Yeast cells were excited at different wavelengths with the aid of special optical filters. By this
method, baker’s yeast was found to contain 4.6 × 10
−14 g of DNA per cell, and the protein content
per cell was found to be 1.1 × 10
−11 g.
Flow cytometry when combined with fluorescently labeled monoclonal antibodies detected S.
Typhimurium in eggs and milk within 40 minutes with a sensitivity of 10
3 /ml.
136 When a 6-hour
nonselective enrichment was used, the detection limit was ten cells per milliliter for milk and one cell
per milliliter for eggs.
A flow cytometric method for the detection and enumeration of bacteria in milk was developed by
enzymatically clearing the milk of lipid particles and proteins. When bacteria were added to UHTtreated milk and the numbers determined by PCA and flow cytometry, the numbers recovered by the
yeasts could be characterized. The latter method is one in which a microcalorimeter is filled with
a flow-through calorimetric vessel. By the use of a chemically defined medium containing seven
sugars, thermograms were produced by nine lactic acid bacteria (belonging to the genera Enterococcus,
Leuconostoc, and Lactobacillus) distinctive enough to recommend the method for their identification.
66
All cultures were run at 37
◦ C except “S. cremoris,” which was run at 30
◦ C, and results were obtained
within 24 hours.
This method has been used to study spoilage in canned foods, to differentiate between the Enterobacteriaceae, to detect the presence of S. aureus, and to estimate bacteria in ground meat. In detecting
S. aureus, results were achieved in 2 hours using an initial number of 10
7 –10
8 cells per milliliter and in
12–13 hours when only 2 cells per milliliter were used.
119 As a monitoring device, flow microcalorimetry was used to determine the viability of recovered frozen cells of S. cerevisiae within 3 hours after
thawing.
13 When applied to comminuted meat, the peak exothermic heat production rate (HPR) could
be recorded within 24 hours for meats that contained 10
5 –10
8 cfu/g, and results correlated well with
plate count results.
70 With 10
2 cfu/ml, a measurable HPR was produced after 6 hours, with a peak
HPR at 10 hours.
Flow Cytometry
Flow cytometry is the science of measuring components (cells) and the properties of individual
cells in liquid suspension. In essence, suspended cells, one by one, are brought to a detector by means
of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells
traverse the detector, and among the cell properties that can be detected are fluorescence, absorbance,
and light scatter. By use of flow sorting, individual cells can be sorted on the basis of their measured
properties, and 1–3 or more global properties of the cell can be measured.
141 Flow cytometers and cell
sorters make use of one or more excitation sources such as argon, krypton, or helium–neon ion lasers
and one or two fluorescent dyes to measure and characterize several thousand cells per second. When
a dye is used, its excitation spectrum must match the light wavelengths of the excitation source.
40
Two dyes may be used in combination to measure, for example, total protein and DNA content. In
these instances, both dyes must excite at the same wavelength and emit at different wavelengths so
that the light emitted by each dye is measured separately. The early history of flow cytometry has been
reviewed by Horan and Wheeless.
83
Although most studies have been conducted on mammalian cells, both DNA and protein have been
measured in yeast cells. Typically, yeast cells are grown, fixed, and incubated in an RNase solution for
1 hour. Cell protein may be stained with fluorescein isothiocyanate and DNA with propidium iodide.
Following necessary washing, the stained cells are suspended in a suitable buffer and are now ready
for application to a flow cytometer. The one used by Hutter et al.
84 was equipped with a 50-mW argon
laser. Yeast cells were excited at different wavelengths with the aid of special optical filters. By this
method, baker’s yeast was found to contain 4.6 × 10
−14 g of DNA per cell, and the protein content
per cell was found to be 1.1 × 10
−11 g.
Flow cytometry when combined with fluorescently labeled monoclonal antibodies detected S.
Typhimurium in eggs and milk within 40 minutes with a sensitivity of 10
3 /ml.
136 When a 6-hour
nonselective enrichment was used, the detection limit was ten cells per milliliter for milk and one cell
per milliliter for eggs.
A flow cytometric method for the detection and enumeration of bacteria in milk was developed by
enzymatically clearing the milk of lipid particles and proteins. When bacteria were added to UHTtreated milk and the numbers determined by PCA and flow cytometry, the numbers recovered by the