246 Modern Food Microbiology products,88,227 microbial quality of raw fish,199 and cooked turkey rolls In the last case, LAL titers and numbers of Enterobacteriaceae in vacuum-packaged rolls were found to have a statistically significant linear relationship.45 LAL titers for foods can be determined either by direct serial dilutions or by MPN, with results by the two methods being essentially similar.185 To extract endotoxins from foods, the Stomacher has been found to be generally better than the Waring blenders or the shaking of dilution bottles.93 In this test, the proclotting enzyme of the Limulus reagent has been purified It is a serine protease with a molecular weight of about 150,000 daltons When activated with Ca2+ and endotoxin, gelation of the natural clottable protein occurs The Limulus coagulogen has a molecular weight of 24,500 When it is acted upon by the Limulus clotting enzyme, the coagulogen releases a soluble peptide of about 45 amino acid residues and an insoluble coagulin of about 170 amino acids The latter interacts with itself to form the clot, which involves the cleavage of –arg–lys– or –arg–gly– linkages.204 The process, as summarized from reference 144, may be viewed as noted below Commercial substrates are available that contain amino acid sequences similar to coagulogen The chromogenic substrates used for endotoxin consist of these linked to p-nitroaniline When the endotoxin-activated enzyme attacks the chromogenic substrate, free p-nitroaniline results and can be read at 405 nm The amount of the chromogenic compound liberated is proportional to the quantity of endotoxin in the sample Employing a chromogenic substrate, Tsuji et al.210 devised an automated method for endotoxin assay, and the method was shown to be sensitive to as little as 30 pg of endotoxin per milliliter Assuming that the quantity of endotoxin per Gram-negative bacterial cell is fairly constant, and assuming further that cells of all genera contain the same given quantity, it is possible to calculate the number of cells (viable and nonviable) from which the experimentally determined endotoxin was derived With a further assumption that the ratio of Gram-negative to Gram-positive bacteria is more or less constant for given products, one can make a 1-hour estimate of the total numbers of bacteria in food products such as fresh ground beef.92 Low values by this procedure are more meaningful than high values, and the latter need to be confirmed by other methods Overall, the value of the LAL test lies in the speed at which results can be obtained Foods that have high LAL titers can be candidates for further testing by other methods; those that have low titers may be placed immediately into categories of lower risk relative to numbers of Gram-negative bacteria Chemical, Biological, and Physical Methods 247 Adenosine Triphosphate Measurement Adenosine triphosphate (ATP) is the primary source of energy in all living cells It disappears within hours after cell death, and the amount per cell is generally constant,208 with values of 10−18 to 10−17 mole per bacterial cell, which corresponds to around × 104 M ATP/105 cfu of bacteria.208 Among procaryotes, ATP in exponentially growing cells is regularly around 2–6 nmole ATP/mg dry weight regardless of mode of nutrition.105 In the case of rumen bacteria, the average cellular content was found to be 0.3 fg per cell, with higher levels found in rumen protozoal cells.151 The complete extraction and accurate measurement of cellular ATP can be equated to individual groups of microorganisms in the same general way as endotoxins for Gram-negative bacteria One of the simplest ways to measure ATP is by use of the firefly luciferin–luciferase system In the presence of ATP, luciferase emits light, which is measured with a luminometer The amount of light produced by firefly luciferase is directly proportional to the amount of ATP added.157 The application of ATP measurement as a rapid method for estimating microbial numbers has been used in clinical microbiology In the clinical laboratory, it has been employed to screen urine specimens The successful use of the method for bacteriuria and for assessing biomass in activated sludge.157 suggested that it should be of value for foods It lends itself to automation and represents an excellent potential method for the rapid estimation of microorganisms in foods The major problem that has to be overcome for food use is the removal of nonmicrobial ATP The method was suggested for food use by Sharpe et al.187 Thore et al.208 used Triton X-100 and apyrase selectively to destroy nonbacterial ATP in urine specimens and found that the resultant ATP levels were close to values observed in laboratory cultures with detection at 105 bacteria per milliliter In meats, the problem of nonmicrobal ATP was addressed by Stannard and Wood194 by use of a three-stage process consisting of centrifugation, use of cation exchange resin, and filtration to get rid of food particles and collect bacteria on 0.22-µm filters ATP analyses were carried out on bacteria eluted from the filter membranes, and 70–80% of most microorganisms were recovered on the filters A linear relationship was shown between microbial ATP and bacterial numbers over the range 106 –109 cfu/g By the methods employed, results on ground beef were obtained in 20–25 minutes In another study, 75 samples of ground beef were evaluated, and a high correlation was found between log10 APC and log10 ATP when samples were incubated at 20◦ C.108 In this study, the amount of ATP/cfu ranged from 0.6 to 17.1 fg, with 51 of the 75 samples containing ≤5.0 fg of ATP The ATP assay has been employed successfully for seafoods and for the determination of yeasts in beverages The ATP assay has been adapted for the determination of microbial load on chicken carcasses12 as well as pork and beef.190 Chicken carcasses were rinsed and results were obtained within 10 minutes, but the method could not reliably detect