Anal. Chem. 1998, 70, 1755-1760
Bacterial Endospore Detection Using Terbium Dipicolinate Photoluminescence in the Presence of Chemical and Biological Materials Paul M. Pellegrino,*,† Nicholas F. Fell, Jr.,‡ David L. Rosen,† and James B. Gillespie†
U.S. Army Research Laboratory, AMSRL-IS-EE, 2800 Powder Mill Road, Adelphi, Maryland 20783-1197, and Physical Sciences Laboratory, New Mexico State University, P.O. Box 30002, Las Cruces, New Mexico 88003-8002
A determination of the viability of an endospore detection technique using terbium dipicolinate photoluminescence in the presence of other chemical and biological materials was performed. The compounds and organisms examined, possible environmental constituents, covered three broad categories: organic compounds, inorganic compounds, and biological materials. Each substance was tested for a false positive, which occurs if the intrinsic terbium photoluminescence is enhanced in the absence of a bacterial endospore. The detection technique was also investigated for false negatives, which occur if a known positive endospore signal is inhibited significantly. Although several materials may give rise to false negative signals, none caused a false positive signal to be observed. Bacterial endospores can be formed by bacteria of the genera Bacillus and Clostridium during times of stress or lack of food.1 This dormant bacterial form can endure unusually harsh conditions that would kill normal active bacteria. Due to their virtually indestructible coating and low water content, endospores can survive extended periods of boiling, heating, freezing, and desiccation.2 In fact, B. stearothermophilus and B. subtilis endospores are used to check the performance of autoclaves.1 Even powerful disinfectant solutions have difficulty with this particular bacterial growth stage. The EPA, FDA, and USDA are interested in determining the amount of harmful bacteria in the air, as a means of monitoring indoor environments, water quality, or food quality.1,3,4 Many of the bacteria in the atmosphere are in endospore form, including those that cause food spoilage and poisoning. Measurements of this type require aerosol sampling, and these samples would contain many materials other than the bacteria of interest. An efficient and specific test for endospore detection would provide †
U.S. Army Research Laboratory. New Mexico State University. (1) Dart, R. K. Microbiology for the Analytical Chemist; The Royal Society of Chemistry: Cambridge, UK, 1996; pp 84-88. (2) Keeton, W. T. Biological Science, 3rd ed.; W. W. Norton & Co.: New York, 1980; p 925. (3) Alcamo, I. E. Fundamentals of Microbiology; Addison-Wesley: Reading, MA, 1984. (4) Salle, A. J. Fundamentals Principles of Bacteriology, 7th ed.; McGraw-Hill: New York, 1973. ‡
S0003-2700(97)01232-8 CCC: $15.00 Published on Web 03/24/1998
© 1998 American Chemical Society
health and safety workers with an invaluable tool for ensuring the safety of the food supply and environment. Recently, a new method for detecting bacterial endospores has been demonstrated.5 It relies on the presence of calcium dipicolinate (Ca(dpa)) in the endospore casing and its release into solution. Endospores contain 2-15 wt % of dpa.6 If Tb3+ is in the solution, it will complex with the dpa released from the spore casing. The complex [Tb(dpa)]+ exhibits a greatly enhanced luminescence compared to Tb3+ alone when irradiated with UV light at the dpa absorption maximum. Previous research has determined that energy transfer from the ligand (dpa) to the terbium luminescence excited states leads to this enhancement.7,8 In this new method, a small amount of an aqueous suspension of the analyte is added to a TbCl3 solution. Any samples containing particulates are filtered to isolate the water-soluble compounds. The sample is then irradiated with a wavelength corresponding to the dpa absorption maximum, and the luminescence emission spectrum is collected. Any sample that exhibits a stronger emission intensity than Tb3+ alone contains bacterial endospores. The focus of this study was to determine the viability of this test in the presence of potential environmental constituents. Since the atmosphere contains appreciable quantities of inorganic, organic, and biological materials, samples from all three of these categories were examined. This study centered around investigating the effect of these materials on the photoluminescence of terbium and terbium dipicolinate. A positive response was defined as a luminescent signal level that exceeds that of terbium alone by 3 times the standard deviation of the system’s inherent noise. All materials were tested for a positive response by adding them to an aqueous solution of TbCl3. Their effect on a known positive response was tested by adding them to a suspension of Tb3+ and powdered B. globigii endospores (BG). A false positive was defined as a positive response for a sample that does not contain bacterial endospores, and a false negative was defined as a sample known to contain bacterial endospores that did not yield a positive response. (5) Rosen, D. L.; Sharpless, C.; McGown, L. B. Anal. Chem. 1997, 69, 10821085. (6) Gould, G. W.; Hurst, A. The Bacterial Spore; Academic Press: New York, 1969. (7) Richardson, F. S. Chem. Rev. 1982, 82, 541-552. (8) Metcalf, D. H.; Bolender, J. P.; Driver, M. S.; Richardson, F. S. J. Phys. Chem. 1993, 97, 553-564.
Analytical Chemistry, Vol. 70, No. 9, May 1, 1998 1755
Table 1. Chemicals Utilized in Experiments substance name DL-tryptophan D-phenylalanine L-glucose riboflavin tryptone NaCl KCl NH4NO3 K2HPO4, 1 M solution
sourcea
symbol trp phe glu rib nbt nac kcl amn pho
Fluka Sigma Sigma Fisher Sigma Fisher Mallinckrodt Sigma
substance name L-tyrosine D(+)-malic
acid sodium benzoate nutrient broth β-NAD CaCO3 NaNO3 (NH4)2SO4
symbol
sourcea
tyr mal nab nub nad cac nan ams
Fluka Sigma Fisher Sigma Sigma Fisher Matheson Alfa Products
a Detailed source information: Sigma, Sigma Chemical Co., St. Louis, MO; Fluka, Fluka Chemika, Ronkonoma, NY; Fisher, Fisher Scientific Co., Fair Lawn, NJ; Matheson, Matheson, Coleman, & Bell, Norwood, OH; Mallinckrodt, Mallinckrodt, St. Louis, MO; Alfa Products, Alfa Products, Danvers, MA; Aldrich, Aldrich Chemical Co., Milwaukee, WI.
Table 2. Biological Materials Used in Experiments material
symbol
sourcea
Bacillus globigii powder Bacillus globigii in nutrient broth Bacillus subtilis in nutrient broth Erwinia herbicola in nutrient broth Acinetobacter haemolyticus in nutrient broth Micrococcus luteus in enriched nutrient broth Bacillus globigii suspension yeast Candida utiliz lyophilized Bacillus subtilis lyophilized Aerobacter aerogenes Type I lyophilized Azotobacter Vinelandii lyophilized Pseudomonas fluorescens Type IV Thuricide insecticide containing 0.8% Bacillus thuringensis var. Berliner defatted German cockroach nondefatted Eastern cottonwood pollen nondefatted cultivated wheat pollen nondefatted desert ragweed pollen defatted Aspergillus flavus mold defatted Cladosporium herbarum mold
BG ebg ebs eeh eah eml sbg cuy lbs laa lav lpf btb gci ecp cwp drp afm chm
ERDEC Dugway lot 10-125 ERDEC grown from lot 10-125 ERDEC grown from ATCC no. 9372 ERDEC grown from ATCC no. 33243 ERDEC grown from ATCC no. 17906 ERDEC grown from ATCC no. 4698 Steris Corp., Mentor, OH Sigma Chemical Co., St. Louis, MO Sigma, ATCC no. 6633 Sigma Sigma, ATCC no. 12518 Sigma, ATCC no. 12633 Rigo Co., Buckner, NY Greer Laboratories, Lenier, NC Greer Laboratories, Lenier, NC Greer Laboratories, Lenier, NC Greer Laboratories, Lenier, NC Greer Laboratories, Lenier, NC Greer Laboratories, Lenier, NC
a ERDEC, U. S. Army Edgewood Research, Development, Engineering Center, Aberdeen Proving Ground, MD; ATCC, American Type Culture Collection.
EXPERIMENTAL SECTION Sample Preparation. Tables 1 and 2 list all the substances used in these experiments and their symbols. These materials were chosen as substances that might be present in a sample collected from the atmosphere. Unless otherwise specified, all solutions and suspensions were prepared in Trizma buffer (6.36 g/L Trizma-HCl (Sigma Chemical Co.), 1.18 g/L Trizma base (Sigma Chemical Co.), and 42 mL/L ethanol (Sigma Chemical Co.) in distilled water) at a pH of 7.6. The concentrations of the analyte solutions were chosen to match the dipicolinic acid (dpa, Aldrich) solutions used in the previous study.5 Solutions of the organic compounds were prepared at a concentration of 650 µM. A sample of nutrient broth with tryptone, a standard microbiological growth medium, was made with 8 g/L nutrient broth and 10 g/L tryptone in distilled water. The inorganic components were made at a concentration of 1 mM, except for the K2HPO4. The K2HPO4 was examined at both 1 mM and 1 M, the standard phosphate buffer concentration. Powdered bacteria suspensions were prepared with 0.5 g/L; the yeast suspension was made with 0.75 g/L; and the pollens, molds, and insect samples were prepared with 1.25 g/L. A commercial insecticide, Thuricide, which contains 0.8% B. thuringensis endospores, was diluted by a 1756 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
factor of 40 with buffer. A commercially prepared 40% ethanol suspension of BG endospores was obtained with a concentration of 1.3 × 109 CFU/mL (Steris Corp., Mentor, OH). Bacteria in growth medium were obtained to test the responses of several different genera of bacteria. When it was found that the growth medium interfered with the observation of terbium luminescence, the samples were centrifuged at 4000g for 10 min to remove the bacteria. These samples were then resuspended in buffer. This process was repeated twice to ensure that the samples were free from growth medium. This treatment was also applied to an aliquot of the 6 × 108 CFU/mL BG as a control. Several stock solutions were prepared in Trizma buffer. Terbium chloride (TbCl3‚6H2O, Aldrich) stock solutions were prepared at a concentration of 30 µM. An initial standard solution of BG powder was prepared at a concentration of 6 × 109 CFU/ mL in 40% ethanol to match the composition of the BG stock solution in the previous study.5 However, this was found to give an intensity much higher than that of an equivalent amount of dpa, so a second stock solution at a concentration of 6 × 108 CFU/ mL in 40% ethanol was made and used as a standard. Solutions of terbium with BG (Tb/BG stock) were prepared by taking 200 mL of the 30 µM TbCl3 and adding 400 or 800 µL of the 6 × 109
CFU/mL BG in order to match the final concentration of the BG standard when added to the TbCl3 solution. All measurements were performed on a final solution prepared from the stock and the analyte solutions listed above. These solutions were made by adding 400 µL of the analyte solution to 10 mL of the 30 µM TbCl3 or Tb/BG stock solution, except where otherwise noted. The TbCl3 stock solution was used to test for positive responses, and the Tb/BG stock was used to test for signal reductions due to chemical effects (test material complexation with Tb3+ or dpa2-) or luminescence quenching. The measurement was repeated, and smaller amounts of the analyte solution were added if the absorption was greater than 0.15, significant spectral interference occurred, or a false negative was obtained. If a biological organism or other insoluble component was present in the final solution, it was filtered through a sterile 0.22-µm PTFE (poly(tetrafluoroethylene)) or PVDF (poly(vinylidene difluoride)) syringe filter into a 1-cm2 quartz cuvette. The filters were wetted with ethanol in the case of the PTFE filters or distilled water in the case of the PVDF filters. Tests showed that the luminescence intensity obtained was unaffected by which type of filter was used. It was also determined that wetting of the filters is necessary to minimize retention of material in the filter. Data Acquisition. Absorption and luminescence emission spectra were obtained for each sample. Absorption measurements were performed with a Varian Cary 5E UV-visible spectrophotometer, using buffer as the reference. The absorption spectrum was collected from 240 to 300 nm, with a spectral bandwidth of 0.25 nm. The photoluminescence emission spectra measurements were performed on the system outlined below. The output of a UVenhanced Xe arc lamp (Oriel, model 6254) was focused with quartz optics on the entrance slit of a monochromator (Acton Research Corp., SpectraPro 275) set at 280 nm with a holographic 1800 grooves/mm grating and 2.475-mm slit width, resulting in a bandwidth of 5 nm. The output beam was focused onto the sample cuvette using a quartz lens. The luminescence emission at 90° was collected and focused onto the entrance slit of a second identical monochromator with glass lenses. This monochromator was set at 530 nm with a 600 grooves/mm grating and 100-µm entrance slit width, resulting in a resolution of four pixels, or 0.6 nm. A 1024-element intensified linear diode array detector (Princeton Instruments, IRY-1024G/RB) was placed at the exit image plane of the second monochromator. With the use of these settings, a section of the luminescence emission spectrum with a width of 146.7 nm was viewed on the array. The signal was integrated for 1 s for each spectrum, and 120 individual spectra were accumulated to improve the signal-to-noise ratio (S/N). Since three replicate measurements of the luminescence emission were collected, all reported intensity values are the average of the replicates. An emission spectrum of the buffer was used to correct each sample spectrum for dark count and array background effects. Unless noted, all spectra were analyzed without further correction. RESULTS AND DISCUSSION Equilibrium Calculations. Equilibrium calculations were performed to determine which terbium dipicolinate complexes
were present at the concentrations examined in these experiments. Based on the acid dissociation constants for dipicolinic acid9 and the measured pH, 99.8% of the dipicolinic acid was in the completely deprotonated form. Since the vast majority of the available dpa was in the dpa2- form, the total concentration of dpa was used in further calculations for the total amount of dpa2-. Depending on the relative concentrations of the terbium and dipicolinic acid, one, two, or three dipicolinate anions would complex with each terbium cation.7,8,10 With the use of the terbium dipicolinate formation constants of Grenthe10 and the concentrations of Tb and dpa used in these experiments, a series of equilibrium calculations were performed. These calculations show that the predominant form of the terbium dipicolinate complex was [Tb(dpa)]+ up to a concentration of 3500 µL of 65 µM dpa in 10 mL of 30 µM TbCl3. A series of measurements was performed by adding progressively more 65 µM dipicolinic acid to 10 mL of 30 µM TbCl3 in order to establish the approximate amount of dpa released by the bacterial endospores and the primary form(s) of the complex present in solution. When 400 µL of 6 × 108 CFU/mL BG endospores was added to 10 mL of 30 µM TbCl3, the intensity was equal to that obtained from the sample where 300-400 µL of 65 µM dpa was added to 10 mL of 30 µM TbCl3. Since virtually all of the dpa in solution is completely dissociated and the equilibrium calculations show that the dpa in solution is completely complexed, it can be assumed that the concentration where the luminescence emission intensities for dpa and BG solutions were equal had equal concentrations of [Tb(dpa)]+. Calculations indicate that dpa/BG dry weight percentage of the BG at this point is 0.61%. The typical dry weight percentage for B. globigii ranges from 7 to 9%, implying that only a fraction of the dpa was released into the solution. This conclusion is not unexpected, considering no extra steps were taken to extract the dpa. The accuracy of this determination is limited by the reproducibility of the intensity of the [Tb(dpa)]+ from the solutions of Tb and BG. Quenching in the Tb with BG solutions is considered unimportant since the spores are not dissolved and their interior contents would remain intact. These measurements and supporting calculations indicate that the predominant form the terbium dipicolinate complex formed upon addition of the standard amount of BG to TbCl3 was [Tb(dpa)]+. Limit of Detection. The limit of detection (LOD) is defined as the concentration for which a signal is obtained equal to the TbCl3 luminescence plus 3 times the standard deviation of the noise in the signal. For our system, the LOD is 1.21 × 105 CFU/ mL BG in a final test solution that contained only TbCl3 and the BG. Our LOD represents a 3.6-fold improvement over the value previously reported.5 Under the conditions of this experiment, contributions from the blank signal and the noise level on that signal were of comparable magnitudes, suggesting further improvement in the limit of detection can be obtained by improving the S/N of the TbCl3 solution luminescence. There are several ways this can be accomplished. To decrease the noise in the luminescence, a more stable source could be used, the detector could be cooled to a lower temperature, or an inherently quieter detector, such as a CCD, could be used. Alternatively, more (9) Tichane, R. M.; Bennett, W. E. J. Am. Chem. Soc. 1957, 79, 1293-1296. (10) Grenthe, I. J. Am. Chem. Soc. 1961, 83, 360-364.
Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
1757
Figure 1. Suppression of luminescence by organic compounds. The squares mark the absorbance of the final solution, and the circles mark the intensity of the Tb peak at 545 nm after addition of the compound normalized to the Tb/BG stock plus 400 µL of buffer. All solutions were made with 10 mL of Tb/BG stock solution and 400 µL of the test compound, except for (1) addition of 10 µL of tryptophan and 300 µL of buffer, (2) 15 µL of riboflavin, and (3) 200 µL of nutrient broth with tryptone. Compound abbreviations are defined in Table 1. Points are connected only for clarity. No functional relationship is implied.
accumulations could be collected, effectively averaging the random noise out of the signal. Chemical Materials. A wide variety of organic and inorganic materials of biological and environmental interest were tested. To determine if a false positive signal would be observed, 400 µL of each compound was mixed with 30 µM TbCl3. Except for dpa itself, none of the substances listed in Table 1 yielded a positive response. This was not surprising, since not only was it necessary for the molecule to complex with Tb, but also a very specific energy level distribution was required for successful energy transfer from the ligand to Tb.7,8 Several of the organic materials did, however, exhibit spectral overlap with the Tb luminescence bands, most notably riboflavin and tryptophan. Each of the compounds was also mixed with the Tb/BG stock solution to examine its effect on a known positive response. The observed signal intensity of the Tb/BG stock, plus 400 µL of the analyte solutions normalized to that of the Tb/BG stock, plus 400 µL of buffer for the 545-nm emission peak, is shown for the organic compounds in Figure 1 and the inorganic materials in Figure 2. From these results, it was clear that only three materials had an appreciable effect on the [Tb(dpa)]+ luminescence: β-NAD (nicotinamide adenine dinucleotide), nutrient broth with tryptone, and dibasic potassium phosphate. Two effects were primarily responsible for the observed signal decreases. In the cases of the nutrient broth with tryptone and β-NAD, some of the observed signal decrease was caused by solution absorbance greater than 0.1. In addition, these materials or their components might have formed stronger complexes with Tb than dpa, reducing the amount of terbium dipicolinate complexes formed. Barela and Sherry11 have studied the effect of various buffer salts on the luminescence of the [Tb(dpa)3]3- complex and found that phosphate buffer significantly reduces the intensity of the luminescence. Measurements on the intrinsic luminescence of Tb in solution with phosphate showed suppression of the luminescence (11) Barela, T. D.; Sherry, A. D. Anal. Biochem. 1976, 71, 351-357.
1758 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
Figure 2. Suppression of luminescence by inorganic compounds. The squares mark the absorbance of the final solution, and the circles mark the intensity of the Tb peak at 545 nm after addition of the compound normalized to the Tb/BG stock plus 400 µL of buffer. All solutions were made with 10 mL of Tb/BG stock solution and 400 µL of the test compound with the phosphate testing measured at 1 M and (*) 1 mM concentrations. Compound abbreviations are defined in Table 1. Points are connected only for clarity. No functional relationship is implied.
but was difficult to quantitate due to the low signal levels. Complexation with phosphate or the presence of phosphate in solution would have led to luminescence quenching due to an unknown mechanism, in addition to simply displacing the dpa from Tb. When the standard 400 µL of the 1 M potassium phosphate was added, no luminescence signal was observed. Even with the addition of only 10 µL of 1 M K2HPO4, the intensity was severely attenuated to approximately 0.5% of the Tb/BG stock signal intensity. When 400 µL of 1 mM K2HPO4 was added, the signal was still reduced to 2.25% of the Tb/BG stock signal intensity. Since β-NAD is an organophosphate, it was quite plausible that it would complex with Tb more strongly than dpa did and, thus, reduce the observed luminescence. Due to spectral interference, only 200 µL of nutrient broth with tryptone, 15 µL of riboflavin, and 100 µL of tryptophan were used. Even at this lower level, the nutrient broth with tryptone still severely attenuated the signal. Biological Samples. A variety of biological samples were examined, including bacteria, pollens, molds, and powdered insects. Again, the samples were first tested for a positive response with 30 µM TbCl3. Only three of the samples yielded a positive response, as shown in Figure 3: the Thuricide, which contains B. thuringensis; the powdered BG; and the Steris Corp. B. globigii. When 400 µL of the Thuricide solution was added to 10 mL of the TbCl3, no signal was observed, so the experiment was repeated with 10 µL. Three of the other samples, B. subtilis and B. globigii in growth medium and the lyophilized B. subtilis, were expected to give positive responses but did not. There are several explanations for their failure to exhibit enhanced Tb luminescence. Since two of the samples were in nutrient broth, which significantly reduces the luminescence of the Tb complex, the signal from the endospores might have been suppressed. However, on washing by centrifugation and resuspension in buffer, the samples still did not show a positive response. A BG powder sample in 40% ethanol was used as a control for the washing procedure. When it was examined, the washed and resuspended bacteria did not give an enhanced Tb luminescence. The
Figure 3. Emission spectra from the bacteria giving positive test results. (Top) Powdered B. globigii 6.165 × 108 CFU/mL, 20 µL, intensity offset by +4000. (Middle) Thuricide, containing 0.8% B. thuringensis, diluted by a factor of 40, 10 µL, intensity offset by +2000. (Bottom) Steris B. globigii 1.3 × 109 CFU/mL, 400 µL.
supernatant was then checked, and it did yield a positive result, indicating that the available dpa from the endospore casing was in solution and did not precipitate with the bacteria on centrifugation. This suggests that the dpa from any endospores in the samples in growth medium was also in the supernatant: however, this cannot be confirmed due to the presence of the nutrient broth. Since they were washed to remove them from their growth medium, a similar line of reasoning would explain why the signal obtained from the Steris Corp. B. globigii was so small compared to that obtained for a similar concentration of powdered BG. In the case of the lyophilized sample, it is possible that the bacteria were freeze-dried before most of them reached the endospore stage, and thus the number of endospores in the sample was below the detection limit. The biological samples were also checked for their effect on the luminescence of the Tb/BG stock solution. Figure 4 shows the signal intensity of the 545-nm luminescence peak normalized to that of the Tb/BG stock plus 400 µL of buffer and the absorbance of each sample. The wide range of suppression of the luminescence by the biological substances, in contrast to the chemical species, indicates some of the difficulty in quantitative use of this test. As was observed with the chemical materials, the worst suppression occurred when the absorbance was high. Due to the complexity of biological systems, it was difficult to determine which other causes contributed to the reduction in luminescence observed. There are numerous organophosphates in biological organisms that could have leached into the solutions and interfered with Tb complexation with dpa or quenched the luminescence. Other components of the organisms being tested could also have complexed with Tb, reducing the amount of [Tb(dpa)]+. Prefiltering of the pollens, molds, and crushed insect did not improve the situation, indicating that the interfering material was in solution. Since many of these materials could have been expected in an atmospheric sample, their suppression of Tb luminescence was an important concern. Given that most bacteria are significantly smaller than pollens, size selection during atmospheric sampling might have alleviated some of these problems.
Figure 4. Suppression of luminescence by biological materials. The squares mark the absorbance of the final solution, and the circles mark the intensity of the Tb peak at 545 nm after addition of the compound normalized to the Tb/BG stock plus 400 µL of buffer. All solutions were made with 10 mL of Tb/BG stock solution and 400 µL of the analyte suspension or solution. The superscripts note exceptions: (1) 100 µL added, (2) samples washed by centrifugation and resuspension in buffer, (3) sample solutions filtered before addition of 400 µL to TbCl3, and (4) sample solutions prefiltered as described in (3) and only 100 µL added to TbCl3. Material abbreviations are given in Table 2. Points are connected only for clarity. No functional relationship is implied.
CONCLUSIONS We have examined a number of chemical and biological materials for their effect on a terbium test for the presence of bacterial endospores. We have found that only bacterial endospores yield a positive response to this test; in other words, no false positive responses were observed. Unfortunately, there were cases where false negative results were obtained. Several factors gave rise to the false negatives. The presence of significant amounts of phosphate-containing compounds resulted in suppression of the Tb and [Tb(dpa)]+ luminescence. One reason may be that Tb has higher affinity for phosphates than dpa and results in a reduction in the amount of terbium dipicolinate complexes formed. To determine the viability of the technique for newly grown samples, several bacteria were tested in growth medium. The presence of nutrient broth was found to be particularly harmful to the successful application of this test. Washing of the samples or separation by centrifugation proved unsuccessful in defeating this problem due to the removal of the dpa in the supernatant. Although certain compounds effect the sensitivity, the detection of endospores using Tb can accomplished with the proper precautions. Because biological organisms (e.g., pollens, molds, vegetative bacteria) other than bacterial endospores do not contain dpa, this method was relatively immune to their presence. This test would, therefore, be extremely useful for rapid detection of bacterial endospore contamination. Current research efforts to improve the Tb test are being pursued, such as minimizing the effect of phosphates, improvements in dpa extraction techniques, and chemical and instrumental improvements to increase the photoluminescence signal-to-noise ratio. Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
1759
ACKNOWLEDGMENT The authors acknowledge several sources of samples and support for this work. This work was performed while P.M.P. held a National Research Council-U.S. Army Research Laboratory Research Associateship. N.F.F.’s participation was supported by U.S. Army Research Laboratory Contract DAAD07-91-C-0139. We also thank the U.S. Army Edgewood Research, Development, and
1760 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
Engineering Center, especially Ms. Dottie Paterno, for providing the bacteria samples used in this study.
Received for review November 10, 1997. February 13, 1998. AC971232S
Accepted
