548 ◾ Fundamental Food Microbiology
is added to the sample and allowed to bind to the colloidal gold or latex particle–conjugated
antibody. This complex flows to the other end of the strip through a porous membrane by capillary action that contains two captured zones, one specific for the bacterial pathogen and another
specific for unbound antibodies coupled to the color reagent (control line). The presence of only
one (control) line on the membrane indicates a negative sample, and the presence of two lines
indicates a positive result. The lateral flow assay gives results in 5–10 minutes, is easy to perform,
is economical, and is suitable as an initial screening test. 12
Flow Cytometry
Flow cytometry permits rapid characterization of a cell population based on a number of parameters such as morphology, nucleic acid content, and surface antigenicity. Cells are labeled with
appropriate fluorophores and passed rapidly in suspension on a cell-by-cell basis through a tunnel
and detected by a laser beam. By analysis of interactions of each cell (light scatter or fluorescence)
with the beam, a representation of the distribution of desired parameter within the population
is acquired. A fluorescent-labeled antibody can be used to detect bacterial cells directly by flow
cytometry. One of its disadvantages for use in food microbiology is the lack of discrimination
between live and dead cells and interference with the food matrix. Application of reporter bacteriophage can overcome this problem by detecting only live pathogens (see below).
Bacteriophage for Detection of Pathogens 15–17
As discussed in Chapter 38, bacteriophages are viruses that infect and kill target bacteria. Thus
it was used as a biocontrol to kill pathogens and spoilage bacteria. In recent years, however, there
is an increasing emphasis on the use of bacteriophages as detection tools for foodborne bacterial
pathogens because of their natural affinity toward its host. Moreover, bacteriophages are becoming
attractive tools because of their ability to detect only the live pathogens, and only viable pathogens
are of major concern in processed foods. Another advantage is that assays consisting of bacteriophages have a built-in amplification step because bacteriophages, after infection, can produce a large
number of progeny phages in one to two hours (Figure 14.2). Bacteriophages have been applied for
pathogen detection in various forms: Binding of phages with the target pathogen can be detected
visually, spectrophotometrically, or by employing biosensor tools, some of which are described here:
Culture or Staining Methods
Bacteriophages are currently being used for phage typing of bacteria. This is determined by measuring the turbidity of liquid culture spectrophotometrically or by monitoring the lysis of bacterial
cells on lawn cultures on agar plates visually. Lysis of bacterial cells will result in less turbid bacterial suspension or a clearing zone (plaque formation) on a bacterial lawn (Figure 38.2).
Bacteriophages prelabeled with fluorescence dye or quantum dots are added to test samples, and
binding of labeled phages to bacteria is monitored by flow cytometry or epifluorescence microscopy.
Reporter Phages
In this approach, phages are genetically engineered to carry a “reporter gene,” such as genes for
bioluminescence (lux), green fluorescence protein (gfp), or β-galactosidase (lacZ). Once a bacteriophage infects a target pathogen host, the phage gene is integrated into the host genome and
is added to the sample and allowed to bind to the colloidal gold or latex particle–conjugated
antibody. This complex flows to the other end of the strip through a porous membrane by capillary action that contains two captured zones, one specific for the bacterial pathogen and another
specific for unbound antibodies coupled to the color reagent (control line). The presence of only
one (control) line on the membrane indicates a negative sample, and the presence of two lines
indicates a positive result. The lateral flow assay gives results in 5–10 minutes, is easy to perform,
is economical, and is suitable as an initial screening test. 12
Flow Cytometry
Flow cytometry permits rapid characterization of a cell population based on a number of parameters such as morphology, nucleic acid content, and surface antigenicity. Cells are labeled with
appropriate fluorophores and passed rapidly in suspension on a cell-by-cell basis through a tunnel
and detected by a laser beam. By analysis of interactions of each cell (light scatter or fluorescence)
with the beam, a representation of the distribution of desired parameter within the population
is acquired. A fluorescent-labeled antibody can be used to detect bacterial cells directly by flow
cytometry. One of its disadvantages for use in food microbiology is the lack of discrimination
between live and dead cells and interference with the food matrix. Application of reporter bacteriophage can overcome this problem by detecting only live pathogens (see below).
Bacteriophage for Detection of Pathogens 15–17
As discussed in Chapter 38, bacteriophages are viruses that infect and kill target bacteria. Thus
it was used as a biocontrol to kill pathogens and spoilage bacteria. In recent years, however, there
is an increasing emphasis on the use of bacteriophages as detection tools for foodborne bacterial
pathogens because of their natural affinity toward its host. Moreover, bacteriophages are becoming
attractive tools because of their ability to detect only the live pathogens, and only viable pathogens
are of major concern in processed foods. Another advantage is that assays consisting of bacteriophages have a built-in amplification step because bacteriophages, after infection, can produce a large
number of progeny phages in one to two hours (Figure 14.2). Bacteriophages have been applied for
pathogen detection in various forms: Binding of phages with the target pathogen can be detected
visually, spectrophotometrically, or by employing biosensor tools, some of which are described here:
Culture or Staining Methods
Bacteriophages are currently being used for phage typing of bacteria. This is determined by measuring the turbidity of liquid culture spectrophotometrically or by monitoring the lysis of bacterial
cells on lawn cultures on agar plates visually. Lysis of bacterial cells will result in less turbid bacterial suspension or a clearing zone (plaque formation) on a bacterial lawn (Figure 38.2).
Bacteriophages prelabeled with fluorescence dye or quantum dots are added to test samples, and
binding of labeled phages to bacteria is monitored by flow cytometry or epifluorescence microscopy.
Reporter Phages
In this approach, phages are genetically engineered to carry a “reporter gene,” such as genes for
bioluminescence (lux), green fluorescence protein (gfp), or β-galactosidase (lacZ). Once a bacteriophage infects a target pathogen host, the phage gene is integrated into the host genome and