Conventional, Molecular, and Biosensor-Based Detection Methods ◾ 543
by the microslide precipitation method. This method is sensitive at the level of 0.05 μg enterotoxin
per milliliter of the extract (food poisoning probably can occur from the consumption of less than
1.0 μg of Sta. aureus enterotoxin A). Methods, such as radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA), and reverse passive latex agglutination assay (RPLA), can also be
used in place of the microslide precipitation method. Biosensor-based surface plasmon resonance
(SPR) sensors and fiber optic array sensors can detect this toxin in 1–20 ng quantities.
To detect botulinum neurotoxin produced by Clo. botulinum strains, the recommended procedure is to first extract toxin from a food. To activate toxin of nonproteolytic types (B and E), a
portion of the extract is then treated with trypsin. Both trypsinized and nontrypsinized (for proteolytic types A and B) extracts in 0.5-mL portions are then injected into mice intraperitoneally
(two mice for each sample). Heated (100°C for 10 minutes) extracts are injected similarly (control).
The mice are then observed up to 48 hours for botulism symptoms and death. Typical botulism
symptoms in mice, in sequence, are ruffling of fur, rapid and gasping breathing, weakness of
limbs, and death from respiratory failure.
Specific methods for testing toxins of some other pathogenic bacteria and molds have also been
developed and listed in the recommended books as well as available from commercial vendors.
Rapid Methods and Automation
Conventional culture-based methods are considered a gold standard for pathogen detection. These
methods rely on specific microbiological media to isolate and enumerate viable bacterial cells in
foods as discussed above. It consists of five steps involving preenrichment, selective enrichment,
selective plating for colony identification, a biochemical presumptive test, and a serological confirmation test. The preenrichment step is beneficial because this step not only increases the populations of the target organism but also allows recovery of sublethally injured or stressed bacteria
resulting from exposure to processing and storage conditions, namely heating, drying, freezing,
cooling, preservatives, and so forth. Injured microbes, under favorable environments, are capable
of recovering in food and causing disease in humans.
The culture method is highly sensitive, relatively inexpensive, and can give both qualitative and
quantitative information on the number and the nature of the microorganisms. 8–13 However, it
is labor intensive and lengthy, requiring 7–10 days. To overcome these difficulties, different rapid
methods, many of which are automated, have been developed to detect microbial loads, foodborne
pathogens, and their toxins. 3,8,11 Some of these methods have been approved by the regulatory agencies. Furthermore, rapid methods are highly desirable for use in the food processing plant for monitoring pathogens in raw materials, ready-to-eat food products, and to verify manufacturing process
control. Bacteria-specific generic rapid methods are also needed for monitoring cleaning and hygiene
practices employed in a processing plant. In addition to being rapid, they are quite specific, sensitive,
relatively accurate, and less labor intensive. However, their effectiveness in food systems with a low
level of contamination needs further evaluation. Many new methods are constantly being developed,
which differ mainly in technique. The principles, procedures, and applications in food of some of
these methods are briefly presented. Exact methods and steps are provided by the commercial farms.
Metabolic Fingerprinting 8,10
Each species of bacterium, mold, or yeast has unique carbon or nitrogen requirements for growth
and survival. When a panel of appropriate sugars (carbon source) or amino acids (nitrogen source)
by the microslide precipitation method. This method is sensitive at the level of 0.05 μg enterotoxin
per milliliter of the extract (food poisoning probably can occur from the consumption of less than
1.0 μg of Sta. aureus enterotoxin A). Methods, such as radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA), and reverse passive latex agglutination assay (RPLA), can also be
used in place of the microslide precipitation method. Biosensor-based surface plasmon resonance
(SPR) sensors and fiber optic array sensors can detect this toxin in 1–20 ng quantities.
To detect botulinum neurotoxin produced by Clo. botulinum strains, the recommended procedure is to first extract toxin from a food. To activate toxin of nonproteolytic types (B and E), a
portion of the extract is then treated with trypsin. Both trypsinized and nontrypsinized (for proteolytic types A and B) extracts in 0.5-mL portions are then injected into mice intraperitoneally
(two mice for each sample). Heated (100°C for 10 minutes) extracts are injected similarly (control).
The mice are then observed up to 48 hours for botulism symptoms and death. Typical botulism
symptoms in mice, in sequence, are ruffling of fur, rapid and gasping breathing, weakness of
limbs, and death from respiratory failure.
Specific methods for testing toxins of some other pathogenic bacteria and molds have also been
developed and listed in the recommended books as well as available from commercial vendors.
Rapid Methods and Automation
Conventional culture-based methods are considered a gold standard for pathogen detection. These
methods rely on specific microbiological media to isolate and enumerate viable bacterial cells in
foods as discussed above. It consists of five steps involving preenrichment, selective enrichment,
selective plating for colony identification, a biochemical presumptive test, and a serological confirmation test. The preenrichment step is beneficial because this step not only increases the populations of the target organism but also allows recovery of sublethally injured or stressed bacteria
resulting from exposure to processing and storage conditions, namely heating, drying, freezing,
cooling, preservatives, and so forth. Injured microbes, under favorable environments, are capable
of recovering in food and causing disease in humans.
The culture method is highly sensitive, relatively inexpensive, and can give both qualitative and
quantitative information on the number and the nature of the microorganisms. 8–13 However, it
is labor intensive and lengthy, requiring 7–10 days. To overcome these difficulties, different rapid
methods, many of which are automated, have been developed to detect microbial loads, foodborne
pathogens, and their toxins. 3,8,11 Some of these methods have been approved by the regulatory agencies. Furthermore, rapid methods are highly desirable for use in the food processing plant for monitoring pathogens in raw materials, ready-to-eat food products, and to verify manufacturing process
control. Bacteria-specific generic rapid methods are also needed for monitoring cleaning and hygiene
practices employed in a processing plant. In addition to being rapid, they are quite specific, sensitive,
relatively accurate, and less labor intensive. However, their effectiveness in food systems with a low
level of contamination needs further evaluation. Many new methods are constantly being developed,
which differ mainly in technique. The principles, procedures, and applications in food of some of
these methods are briefly presented. Exact methods and steps are provided by the commercial farms.
Metabolic Fingerprinting 8,10
Each species of bacterium, mold, or yeast has unique carbon or nitrogen requirements for growth
and survival. When a panel of appropriate sugars (carbon source) or amino acids (nitrogen source)