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
2 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 4 × 104 M ATP/10
5 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 10
5 bacteria per milliliter. In meats, the problem of
nonmicrobal ATP was addressed by Stannard and Wood
194 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 10
6 –10
9 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 log 10 APC and log 10 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 carcasses
12 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 <1 ×10
4 /ml due to carcass ATP.
12 A 500-cm
2 area for beef
carcasses, and a 50-cm
2 area for pork were surface-wiped with an ATP-free sponge. The entire test
could be completed in about 5 minutes with the minimum detectable number being log 2.0 cfu/cm
2
for beef and log 3.2/cm
2 for pork.
190
The ATP assay is widely used as a rapid and on-the-spot method for monitoring food handling
surfaces by swabbing designated areas and reading the relative light units (RLU) from a luminometer.
Since nonmicrobial ATP can contribute to RLU readings, these methods, while valuable for monitoring
purposes, should not be used to indicate numbers of microorganisms.
Radiometry
The radiometric detection of microorganisms is based on the incorporation of a
14 C-labeled metabolite in a growth medium so that when the organisms utilize this metabolite,
14 CO 2 is released and
247
Adenosine Triphosphate Measurement
Adenosine triphosphate (ATP) is the primary source of energy in all living cells. It disappears within
2 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 4 × 104 M ATP/10
5 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 10
5 bacteria per milliliter. In meats, the problem of
nonmicrobal ATP was addressed by Stannard and Wood
194 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 10
6 –10
9 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 log 10 APC and log 10 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 carcasses
12 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 <1 ×10
4 /ml due to carcass ATP.
12 A 500-cm
2 area for beef
carcasses, and a 50-cm
2 area for pork were surface-wiped with an ATP-free sponge. The entire test
could be completed in about 5 minutes with the minimum detectable number being log 2.0 cfu/cm
2
for beef and log 3.2/cm
2 for pork.
190
The ATP assay is widely used as a rapid and on-the-spot method for monitoring food handling
surfaces by swabbing designated areas and reading the relative light units (RLU) from a luminometer.
Since nonmicrobial ATP can contribute to RLU readings, these methods, while valuable for monitoring
purposes, should not be used to indicate numbers of microorganisms.
Radiometry
The radiometric detection of microorganisms is based on the incorporation of a
14 C-labeled metabolite in a growth medium so that when the organisms utilize this metabolite,
14 CO 2 is released and