244 Modern Food Microbiology
increased in ham sandwiches, there was an increase in the amount of extractable thermostable nuclease
of staphylococcal origin. They suggested that the presence of 0.34 unit of nuclease indicated certain
staphylococcal growth and that at this level, it was unlikely that enough enterotoxin was present to
cause food poisoning. The 0.34 unit was shown to correspond to 9.5 × 10
−3
µg of enterotoxin by
S. aureus strain 234. The reliability of the thermostable nuclease assay as an indicator of S. aureus
growth has been shown by others.
50 It has been found to be as good as coagulase in testing for enterotoxigenic strains,
146 and in another study, all foods that contained enterotoxin contained thermostable
nuclease, which was present in most foods with 1 × 10
6 S. aureus cells per gram.
156 On the other
hand, thermostable nuclease is produced by some enterococci. Of 728 enterococci from milk and milk
products, about 30% produced nuclease, with 4.3% of the latter (31 of the 728) being positive for
thermostable nuclease.
11
The mean quantity of thermostable nuclease produced by enterotoxigenic strains is less than that for
nonenterotoxigenic strains, with 19.4 and 25.5 µg/ml, respectively, as determined in one study.
146 For
detectable levels of nuclease, 10
5 –10
6 cells are needed, whereas for detectable enterotoxin, >10
6 cells
per milliliter are needed.
147 During the recovery of heat-injured cells in trypticase soy broth (TSB),
nuclease was found to increase during recovery but later decreased.
228 The reason for the decrease was
found to be proteolytic enzymes, and the decrease was reversed by the addition of protease inhibitors.
Among the advantages of testing for heat-stable nuclease as an indicator of S. aureus growth and
activity are the following:
1. Because of its heat-stable nature, the enzyme will persist even if the bacterial cells are destroyed
by heat, chemicals, or bacteriophage or if they are induced to l-forms.
2. The heat-stable nuclease can be detected faster than enterotoxin (about 3 hours versus several
days).
115
3. The nuclease appears to be produced by enterotoxigenic cells before enterotoxins appear (Figure
11–1).
4. The nuclease is detectable in unconcentrated cultures of food specimen, whereas enterotoxin
detection requires concentrated samples.
5. The nuclease of concern is stable to heat, as are the enterotoxins.
Although S. epidermidis and some micrococci produce nuclease, it is not as stable to heating as that
produced by S. aureus.
118 Thermostable nuclease will withstand boiling for 15 minutes. It has been
found to have a D value (D 130 ) of 16.6 minutes in brain–heart infusion (BHI) broth at pH 8.2, and a
z value of 51.
50
Limulus Lysate for Endotoxins
Gram-negative bacteria are characterized by their production of endotoxins, which consist of a
lipopolysaccharide (LPS) layer (outer membrane) of the cell envelope and lipid A, which is buried in
the outer membrane. The LPS is pyrogenic and responsible for some of the symptoms that accompany
infections caused by Gram-negative bacteria.
The Limulus amoebocyte lysate (LAL) test employs a lysate protein obtained from the blood
(actually hemolymph) cells (amoebocytes) of the horseshoe crab (Limulus polyphemous). The lysate
protein is the most sensitive substance known for endotoxins. Of six different LAL preparations tested
from five commercial companies, they were found to be 3–300 times more sensitive to endotoxins
than the U.S. Pharmacopeia rabbit pyrogen test.
214 The LAL test is performed by adding aliquots
increased in ham sandwiches, there was an increase in the amount of extractable thermostable nuclease
of staphylococcal origin. They suggested that the presence of 0.34 unit of nuclease indicated certain
staphylococcal growth and that at this level, it was unlikely that enough enterotoxin was present to
cause food poisoning. The 0.34 unit was shown to correspond to 9.5 × 10
−3
µg of enterotoxin by
S. aureus strain 234. The reliability of the thermostable nuclease assay as an indicator of S. aureus
growth has been shown by others.
50 It has been found to be as good as coagulase in testing for enterotoxigenic strains,
146 and in another study, all foods that contained enterotoxin contained thermostable
nuclease, which was present in most foods with 1 × 10
6 S. aureus cells per gram.
156 On the other
hand, thermostable nuclease is produced by some enterococci. Of 728 enterococci from milk and milk
products, about 30% produced nuclease, with 4.3% of the latter (31 of the 728) being positive for
thermostable nuclease.
11
The mean quantity of thermostable nuclease produced by enterotoxigenic strains is less than that for
nonenterotoxigenic strains, with 19.4 and 25.5 µg/ml, respectively, as determined in one study.
146 For
detectable levels of nuclease, 10
5 –10
6 cells are needed, whereas for detectable enterotoxin, >10
6 cells
per milliliter are needed.
147 During the recovery of heat-injured cells in trypticase soy broth (TSB),
nuclease was found to increase during recovery but later decreased.
228 The reason for the decrease was
found to be proteolytic enzymes, and the decrease was reversed by the addition of protease inhibitors.
Among the advantages of testing for heat-stable nuclease as an indicator of S. aureus growth and
activity are the following:
1. Because of its heat-stable nature, the enzyme will persist even if the bacterial cells are destroyed
by heat, chemicals, or bacteriophage or if they are induced to l-forms.
2. The heat-stable nuclease can be detected faster than enterotoxin (about 3 hours versus several
days).
115
3. The nuclease appears to be produced by enterotoxigenic cells before enterotoxins appear (Figure
11–1).
4. The nuclease is detectable in unconcentrated cultures of food specimen, whereas enterotoxin
detection requires concentrated samples.
5. The nuclease of concern is stable to heat, as are the enterotoxins.
Although S. epidermidis and some micrococci produce nuclease, it is not as stable to heating as that
produced by S. aureus.
118 Thermostable nuclease will withstand boiling for 15 minutes. It has been
found to have a D value (D 130 ) of 16.6 minutes in brain–heart infusion (BHI) broth at pH 8.2, and a
z value of 51.
50
Limulus Lysate for Endotoxins
Gram-negative bacteria are characterized by their production of endotoxins, which consist of a
lipopolysaccharide (LPS) layer (outer membrane) of the cell envelope and lipid A, which is buried in
the outer membrane. The LPS is pyrogenic and responsible for some of the symptoms that accompany
infections caused by Gram-negative bacteria.
The Limulus amoebocyte lysate (LAL) test employs a lysate protein obtained from the blood
(actually hemolymph) cells (amoebocytes) of the horseshoe crab (Limulus polyphemous). The lysate
protein is the most sensitive substance known for endotoxins. Of six different LAL preparations tested
from five commercial companies, they were found to be 3–300 times more sensitive to endotoxins
than the U.S. Pharmacopeia rabbit pyrogen test.
214 The LAL test is performed by adding aliquots