4. P SEUDOMONAS P UTIDA R ESPIRATION I NHIBITION T EST P ERFORMED IN
4.5. D OSE -R ESPONSE C URVES
4.5.2. pH Measurement
4.5.2.3. Influence of the Sample Composition on Dose-Response Curves
A disadvantage of the addition of TRIS buffer is that, even while the pH value at the start of the measurement is not exactly within its dynamic range, the pH decrease due to respiration is more buffered by this system than without the TRIS buffer.
Fig. 4.26 shows the respiration of 6.7108 cfu/mL of P. putida detected with the pH-sensitive MTP using test solution with and without 5 mM of TRIS buffer.
Without TRIS, the pH decreases
more rapidly (-0.0104 / min) than with TRIS buffer (-0.0067 / min). This may lead to difficulties using lower bacteria concentrations due to bad reproducibility of the signals.
Fig. 4.26. Respiration of 6.7108 cfu/mL of P.
putida using dilution water with 5 mM TRIS and without TRIS. The change in pH per minute is -0.0067 (with TRIS) and -0.0104 (without TRIS).
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
Another aspect to be considered is that adding extraordinary components to the test solution may influence the sensitivity towards inhibitors. Dose-response curves with pure dilution water, dilution water with 100 mM NaCl and dilution water containing 100 mM NaCl and 5 mM TRIS buffer were recorded with the oxygen-sensitive MTP at a bacteria concentration of 2.0108 cfu/mL, using Cu2+ as inhibitor. This test was not performed with the pH-sensitive MTP because the effect on the biological system should be tested, not the sensory one.
The resulting dose-response curves differ considerably. Whereas the EC50
value of the test using the original sample composition (6.3·10-7 M) agrees the most with the value given in the DIN test (9.6·10-7 M), addition of 100 mM NaCl (EC50 = 1.3·10-6 M) and 5 mM TRIS (EC50 = 2.2·10-6 M) leads to lower sensitivity of the bacteria towards the inhibitor. One reason for this tendency may be that the results of a toxicity test depends on factors like temperature, pH as well as the ionic strength [11]. Furthermore, with 100 mM of NaCl, which is the concentration used for cultivation, the bacteria are more robust and not so easily stressed by the inhibitor, leading to a lower sensitivity. Regarding TRIS buffer, which tends to complex heavy metals, lowers the actual concentration of inhibitor in solution. Therefore lower sensitivity is simulated because the concentrations given on the x-axis are the employed ones. However, the best correctness of the result is not associated with the best reproducibility: Repeating the experiment gave the largest deviations using the pure dilution water, whereas the other two compositions where highly reproducible.
0 20 40 60 80 100
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 log(cCu2+/M)
Inhibition [%]
A B
C
Fig. 4.27. Dose-response curves using (A) pure dilution water, (B) dilution water with 100 mM NaCl and (C) dilution water with NaCl mM and 5 mM TRIS (detected with the oxygen-sensitive MTP).
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
Table 4.13. EC50 values and ranges of the dose-response curves obtained with the oxygen-sensitive MTP using Cu2+ as inhibitor and a bacteria concentration of 2.0108 cfu/mL. (A) Pure dilution water, (B) dilution water with 100 mM NaCl and (C) dilution water with NaCl mM and 5 mM TRIS.
EC50 [M] 'EC50 (min) [M] 'EC50 (max) [M] range [M]
A 6.3 · 10-7 -1.4 · 10-7 0.6 · 10-7 2.5 · 10-7 – 1.3 · 10-6 B 1.4 · 10-6 -0.1 · 10-6 0.09 · 10-6 3.8 · 10-7 – 3.6 · 10-6 C 2.2 · 10-6 -0.6 · 10-6 0.06 · 10-6 7.4 · 10-7 – 6.4 · 10-6
The experiment was also performed with different bacteria concentrations to investigate if the influence of sample composition on the sensitivity is proportional to the total amount of bacteria. Concentrations of 4.2107, 2.0108, 3.3108 cfu/mL and 6.7108 cfu/mL were used. Only the two extremal solutions, dilution water with and without addition of 5 mM TRIS and 100 mM NaCl were investigated to save time and labour. The dose-response curves of the measurements with pure dilution water were shifted towards lower sensitivity with growing bacteria concentration, as expected.
The EC50 of the bacteria concentration of 2.0108 cfu/mL (10-6 M) was in best accordance to the literature value (9.6·10-7M). Surprisingly enough, the dose-response curves of the experiment with addition of TRIS and NaCl seemed not to be influenced much by the bacteria concentration. The curves with the lowest and the highest concentration (A and D) even match rather well. Whereas the EC50 values with pure dilution water span a decade, the range of the EC50 values with TRIS/NaCl are distributed rather homogeneously, lacking any tendencies. An explanation for this behaviour may be that the negatively charged bacteria attract the positively charged Cu2+, thereby detoxicating the sample. This means that with higher bacteria concentration, there are less assessable dissolved Cu2+ ions than with a lower bacteria concentration, which is not accounted for on the x-axis (left). In contrast, TRIS complexes Cu2+ independent of the bacteria concentration. Therefore, the Cu2+
concentration given on the x-axis is too low for all bacteria concentrations.
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
Fig. 4.28. Dose-response with different bacteria concentrations: (A) 4.2107, (B) 2.0108, (C) 3.4108, and (D) 6.7108 cfu/mL. Left: Pure dilution water; Right: Dilution water containing 5 mM TRIS and 100 mM NaCl.
Table 4.14. EC50 values and ranges of Cu2+ using 4 different bacteria activities and two different compositions of test solutions.
cfu/mL EC50 [M] 'EC50 (min) [M] 'EC50 (max) [M] range [M] Dilution water containing 5 mM TRIS and 100 mM NaCl
4.2107 5.2 · 10-6 -1.6 · 10-6 0.7 · 10-6 9.1 · 10-7 – 1.4 · 10-5 2.0108 2.3 · 10-6 -0.4 · 10-6 0.7 · 10-6 5.6 · 10-7 – 6.9 · 10-6 3.4108 2.9 · 10-6 -0.6 · 10-6 0.2 · 10-6 2.9 · 10-7 – 1.2 · 10-5 6.7108 4.7 · 10-6 -0.6 · 10-6 0.7 · 10-6 2.9 · 10-6 – 8.6 · 10-6
Better reproducibility and the fact that a certain ionic strength is inevitable for pH measurements with respect to the influence of the IS of the toxin, militates in favour for adding 100 mM NaCl to the dilution water. The resulting EC50 is still in good accordance to the literature value. TRIS buffer, which shifts the dose-response curve
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
4.5.2.4. Bacteria Concentrations
Due to the dependency of the bacteria concentration on the sensitivity of the test, different concentrations were investigated with respect to the reproducibility and accordance of the results to those obtained with the oxygen measurements. Bacteria concentrations of 2.0108 cfu/mL, 3.4108 cfu/mL, 5.0108 cfu/mL and 6.7108 cfu/mL were used. The results are depicted in Fig. 4.29. The dose-response curves of all 4 concentrations agree satisfactorily with those obtained with the oxygen-sensitive MTP. The EC50 values obtained with the pH-sensitive MTP tend to be somewhat higher than the ones obtained with the oxygen sensor, especially at high bacteria concentrations, but provide sufficient accuracy. The EC50 for the lowest bacteria concentration is in best accordance with the value given in literature. Unfortunately, too small changes in pH lead to enormous standard deviations. Repeatability proved to be rather poor using this low concentration. Therefore the highest bacteria concentration of 6.7108 cfu/mL, which featured the best reproducibility, was chosen for all following experiments using the pH sensor.
-20
Fig. 4.29. Dose-response curves with different bacteria concentrations, obtained with the pH- and the oxygen-sensitive MTP. A: 2.0108, B: 3.4108, C: 5.0108 cfu/mL, D:
6.7108 cfu/mL.
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
Table 4.15. EC50 values and range obtained with the pH- and the oxygen-sensitive MTP using different bacteria concentrations.
cfu/mL EC50 [M] 'EC50 (min) [M] 'EC50 (max) [M] range [M]
2.0108
O2 1.5 · 10-6 -0.1 · 10-6 0.08 · 10-6 5.6 · 10-7 – 5.5 · 10-6 pH 1.7 · 10-6 -0.7 · 10-6 1.3 · 10-6 5.7 · 10-7 – 6.0 · 10-6 3.4108
O2 2.1 · 10-6 -0.2 · 10-6 0.2 · 10-6 6.9 · 10-7 – 7.8 · 10-6 pH 2.4 · 10-6 3.5 · 10-6 6.6 · 10-6 9.1 · 10-7 – 5.9 · 10-6 5.0108
O2 3.6 · 10-6 0.7 · 10-6 1.5 · 10-6 9.9 · 10-7 – 1.3 · 10-5 pH 4.7 · 10-6 -0.8 · 10-6 0.8 · 10-6 1.8 · 10-6 – 1.1 · 10-5 6.7108
O2 4.6 · 10-6 -0.3 · 10-6 0.6 · 10-6 1.5 · 10-6 – 1.6 · 10-5 pH 6.5 · 10-6 -0.6 · 10-6 2.5 · 10-6 1.6 · 10-6 – 2.4 · 10-5
4.5.2.5. Summary
The application of the P. putida respiration inhibition test to the pH-sensitive MTP stipulates certain modifications of the standard procedure. Due to the dependence of optical pH measurement on the ionic strength of the sample, 100 mM NaCl were added to the dilution water to minimise the influence of the IS of the test substance towards the overall IS.
Furthermore, the rather long response time of 30 min of the dry sensor towards samples of small buffer concentrations, as used for this test, has to be considered. Adding 5 mM TRIS to the dilution water to increase the buffer concentration shortens the response time to 8.5 min. However, this procedure was abandoned because it decreased the sensitivity of the test by shifting the dose-response curves towards higher concentrations. Moreover, although the buffer
Chapter 4: Pseudomonas Putida Respiration Inhibition Test
The long response time of the test without TRIS buffer was accounted for by incubating the samples with the toxins in the MTP instead of glass vials on a shaker.
The MTP was shaken during the incubation time of 30 min in the reader with a shaking mode of 600 rpm / 1 mm. During this time, the sensor has nearly equilibrated.
The proton increase per time was calculated with a simulation program Berkeley-Madonna using the Henderson-Hasselbalch equation instead of the pH decrease per time. This provided correct results and improved the reproducibility of the test (see chapter 4.3.5.2, page 123, and Appendix B).
A bacteria concentration of 6.7108 cfu/mL was chosen for the screening tests regarding the pH measurements, although the concentration used in the DIN test was lower (2.0108 cfu/mL). This was necessary because the pH decrease was too small to give reproducible results. Moreover, varying response times of the sensor of sometimes more than 30 min at low buffer concentrations adulterate the kinetic and can even lead to a signal increase instead of a decrease at slow kinetics (near 100 % inhibition). This effect is less pronounced at faster kinetics, i.e. with higher bacteria activities, because the pH decrease is less compensated by the signal increase due to the response time. The oxygen screening tests are performed with both 6.7108 cfu/mL for to ensure the correctness of the results obtained with the pH sensor, and with 2.0108 cfu/mL for comparison with literature values. Direct comparison of the results of the pH measurements with literature was not possible due to the higher bacteria concentration and the addition of NaCl, which are both indispensable for the pH tests.