256 Modern Food Microbiology
by other methods. In one study, immunomagnetic separation was combined with flow cytometry for
the detection of E. coli 0157:H7. The antigens were labeled with fluorescent antibody, which was
measured by flow cytometry, and the combined method could detect <10
3 cfu/g of pure culture or
10
3 –10
4 cfu/g in ground beef.
186 This method may be used for a number of other organisms including
viruses and protozoa.
Hemagglutination
Whereas gel diffusion methods generally require at least 24 hours for results, two comparable
serologic methods yield results in 2–4 hours: hemagglutination–inhibition (HI) and reverse passive
hemagglutination (RPH). Unlike the gel diffusion methods, antigens are not required to be in precipitable form for these two tests.
In the HI test, specific antibody is kept constant and enterotoxin (antigen) is diluted out. Following
incubation for about 20 minutes, treated sheep red blood cells (SRBCs) are added. Hemagglutination
(HA) occurs only when antibody is not bound by antigen. HA is prevented (inhibited) where toxin is
present in optimal proportions with antibody. The sensitivity of HI in detecting enterotoxins is noted
in Table 11–1.
In contrast to HI, antitoxin globulin in RPH is attached directly to SRBCs and used to detect toxin.
When diluted toxin preparations are added, the test is read for HA after incubation for 2 hours. HA
occurs only where optimal antigen antibody levels occur. No HA occurs if no toxin or enterotoxin is
present. The levels of two enterotoxins detected by RPH are indicated in Table 11–1.
MOLECULAR GENETIC METHODS
Although phenotypic methods for the identification and characterization of foodborne bacteria continue to be widely used, the trend is clearly in the direction of making more use of genotypic methods,
especially for foodborne pathogens. This trend is aided by the availability of test kits produced by a
number of companies, and some are listed below under Polymerase Chain Reaction. The importance
of 16S rRNA in classifying bacteria along genotypic lines is noted in Chapter 2, and a glimpse of how
this is achieved is presented below.
As noted in Chapter 2, the 70S bacterial ribosome contains three definable rRNA fractions: 5S,
16S, and 23S. The 5S contains ca. 120 nucleotides while 16S and 23S contain ca. 1,500 and ca. 3,000,
respectively. The 16S rRNA has been shown to be an excellent chronometer of life forms, especially
bacteria. It was by the use of 16S rRNA sequence and hybridization data that the class Proteobacteria
was established, as noted in Chapter 2. By use of these sequences, new bacterial genera can be
defined on a genotypic rather than a phenotypic basis. As a result of this and other molecular genetic
information, the genus Pseudomonas has been reduced by the transfer of over 50 species to 10 new
genera (see Chapter 2). The existence of a number of gene banks for many other bacterial taxa of
importance in foods suggests that generic and species realignments will continue.
The extraction and amplification of 16S rRNA from bacterial cells is relatively simple. With ca.
1 g of wet cells, they are broken open in the presence of DNase (to destroy all DNA), and RNA may
be extracted by use of commercial kits. With the addition of nucleotide primers, reverse transcriptase
(RT), and deoxyribonucleotides, RT reads the rRNA template and makes DNA (cDNA) copies. The
original 16S rRNA sequence can be deduced after a series of manipulations.
by other methods. In one study, immunomagnetic separation was combined with flow cytometry for
the detection of E. coli 0157:H7. The antigens were labeled with fluorescent antibody, which was
measured by flow cytometry, and the combined method could detect <10
3 cfu/g of pure culture or
10
3 –10
4 cfu/g in ground beef.
186 This method may be used for a number of other organisms including
viruses and protozoa.
Hemagglutination
Whereas gel diffusion methods generally require at least 24 hours for results, two comparable
serologic methods yield results in 2–4 hours: hemagglutination–inhibition (HI) and reverse passive
hemagglutination (RPH). Unlike the gel diffusion methods, antigens are not required to be in precipitable form for these two tests.
In the HI test, specific antibody is kept constant and enterotoxin (antigen) is diluted out. Following
incubation for about 20 minutes, treated sheep red blood cells (SRBCs) are added. Hemagglutination
(HA) occurs only when antibody is not bound by antigen. HA is prevented (inhibited) where toxin is
present in optimal proportions with antibody. The sensitivity of HI in detecting enterotoxins is noted
in Table 11–1.
In contrast to HI, antitoxin globulin in RPH is attached directly to SRBCs and used to detect toxin.
When diluted toxin preparations are added, the test is read for HA after incubation for 2 hours. HA
occurs only where optimal antigen antibody levels occur. No HA occurs if no toxin or enterotoxin is
present. The levels of two enterotoxins detected by RPH are indicated in Table 11–1.
MOLECULAR GENETIC METHODS
Although phenotypic methods for the identification and characterization of foodborne bacteria continue to be widely used, the trend is clearly in the direction of making more use of genotypic methods,
especially for foodborne pathogens. This trend is aided by the availability of test kits produced by a
number of companies, and some are listed below under Polymerase Chain Reaction. The importance
of 16S rRNA in classifying bacteria along genotypic lines is noted in Chapter 2, and a glimpse of how
this is achieved is presented below.
As noted in Chapter 2, the 70S bacterial ribosome contains three definable rRNA fractions: 5S,
16S, and 23S. The 5S contains ca. 120 nucleotides while 16S and 23S contain ca. 1,500 and ca. 3,000,
respectively. The 16S rRNA has been shown to be an excellent chronometer of life forms, especially
bacteria. It was by the use of 16S rRNA sequence and hybridization data that the class Proteobacteria
was established, as noted in Chapter 2. By use of these sequences, new bacterial genera can be
defined on a genotypic rather than a phenotypic basis. As a result of this and other molecular genetic
information, the genus Pseudomonas has been reduced by the transfer of over 50 species to 10 new
genera (see Chapter 2). The existence of a number of gene banks for many other bacterial taxa of
importance in foods suggests that generic and species realignments will continue.
The extraction and amplification of 16S rRNA from bacterial cells is relatively simple. With ca.
1 g of wet cells, they are broken open in the presence of DNase (to destroy all DNA), and RNA may
be extracted by use of commercial kits. With the addition of nucleotide primers, reverse transcriptase
(RT), and deoxyribonucleotides, RT reads the rRNA template and makes DNA (cDNA) copies. The
original 16S rRNA sequence can be deduced after a series of manipulations.