Experiments with Glowing Bacteria: Ocean Glow
As a biological method for toxicity detection, luminescent bacteria are increasingly valued because of their simplicity, rapidity, sensitivity and low cost. Two methods are recommended in this experimental project. One is the luminescent bacteria method (GB/T15441-1995) issued by the State Environmental Protection Administration in 1995, which uses marine luminescent bacteriastrain。
Luminophilus lucidum T3 method (A)
This method is suitable for the acute toxicity monitoring of soluble chemicals in industrial wastewater, sewage water and laboratory conditions.
1. Principle
Due to the significant negative correlation between the relative luminosity of luminescent bacteria and the total concentration of toxic components in the water sample (P ≤ 0.05), the relative luminosity of the water sample can be determined by a bioluminescence photometer to indicate its acute level.
The acute toxicity level of water quality can be characterized by selecting the equivalent reference mercury chloride concentration (in mg/L), or by selecting the EC50 value (in half effective concentration, in percentage concentration of sample solution).
2. Sample collection and storage
① The sampling bottle is a glass bottle with a PTFE liner, which must be clean and dry. When collecting water samples, the bottle should be filled with water samples without leaving air. After sampling, seal the bottle mouth with plastic tape.
② Toxicity testing should be carried out within 6h after sampling. Otherwise, samples should be stored at 2-5 ° C, but not more than 24h. The report should state the collection time and determination time of the water sample.
③ Samples containing suspended solids must be centrifuged or filtered to avoid interfering with the determination.
3. Equipment
① Bioluminescence photometer: configure 2ml or 5ml test tubes.
When the concentration of mercury chloride standard solution is 0.10mg/L, the relative luminosity of luminescent bacteria is 50%, and the error does not exceed ± 10%.
② 2ml or 5ml test sample tubes (with standard grinding plugs, made of glass frit for the manufacture of colorimetric tubes, manufactured by professional glassware factories), are suitable for corresponding models of bioluminescence photometers.
③ Microsyringe: 10 μl.
④ Syringe: lml.
⑤ Quantitative liquid bottle: 5ml.
(6) Straw: 2ml, 10m1, 25ml.
Reagent bottle: l00ml.
Measuring cylinder: 100ml, 500ml.
Brown volumetric bottle: 50ml, 250ml, 1000ml.
Semi-micro burette (with a grinding mouth test bottle, the whole set of instruments is brown): 10ml.
4. Reagents and materials
Unless otherwise specified, the reagents used in this method shall be analytically pure reagents, distilled water or water of equivalent purity that meet national standards.
① Mercury chloride HgCl2.
② Sodium chloride NaCl, chemically pure.
③ Lyophilized powder of luminescence bacterium T3 (PhotobacteriumphosphoreumT3spp.), packed in ampoules, 0.5g per bottle, and the effective shelf life is 6 months in the refrigerator at 2-5 ° C. The density of the newly prepared luminescence bacterium dormant cells (lyophilized powder) is not less than 8 million cells per gram; when the lyophilized powder is resuscitated for 2 minutes according to step 5 (3) ④, it emits light (can be tested in the dark room, and the naked eye should see low light). After dilution into working solution, there are not less than 16,000 cells per ml of bacterial solution ((5ml test tube) or 20,000 cells (2ml test tube) (all determined by dilution plate method). The initial luminous amount measured on the bioluminescence photometer should be between 600-1900mV. Below 600mV, the magnification should be adjusted to "× 2" gear, and above 1900mV, the magnification should be adjusted to "× 0.5" gear. If it still does not meet the standard, replace the lyophilized powder.
④ Sodium chloride solution, 3g/100m1:3g of sodium chloride in a glass container, add l00ml of distilled water with a measuring cylinder.
⑤ Sodium chloride solution, 2g/l00ml sodium chloride 2g, add distilled water l00ml in the reagent bottle, store at 2-5 ° C.
(6) Reference mercury chloride standard solution: 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.18, 0.20, 0.22, 0.24mg/L.
Mercury chloride mother liquor, p = 2000mg/L: 1/10,000 analytical balance, weigh 0.1000g of well-sealed amorphous water mercury chloride in a 50ml volumetric bottle, dilute it to the scale with 3g/100m1 sodium chloride solution, and put it in a refrigerator at 2-5 ° C for backup. The shelf life is 6 months.
< unk > Mercury chloride working solution, p = 2mg/L: Pipette 2000mg/L mother liquor l0ml of mercury chloride in a l000ml volumetric bottle, with 3g/l00ml sodium chloride solution bandwidth evaluation. Then pipette 20mg/L mercury chloride solution 25ml in a 250m1 volumetric bottle, with 3g/l00m] sodium chloride solution bandwidth evaluation. Pour this solution into a test bottle equipped with a semi-micro burette, and then dilute the mercury chloride 2mg/L solution into a series of concentrations according to the table 5-3-6 with 3g/l00ml sodium chloride solution (all 50ml volumetric bottles). The shelf life of mercury chloride working solution cannot exceed 24h, and those who exceed it must be reformulated.
5. Test procedures
(1) Dilute the sample solution
① The conditions for dilution of the sample solution before determination:
Sample liquid pre-test: Take the sample mother liquor 2m1 with sodium chloride to a concentration of 3g/l00ml in advance and put it into the sample tube, and set up a CK tube (sodium chloride 3g/100m1 solution), and measure the relative luminosity according to 4 ②-③.
If the measured sample has a relative luminosity of less than 50% or even zero, it needs to be diluted to express the result at EC50.
If the measured sample has a relative luminosity of more than 1%, no dilution is required if the result is to be expressed at a concentration of mercury chloride equivalent to the relative luminosity.
② The dilution of the sample solution: The dilution of the sample solution is always distilled water, and before the bandwidth evaluation, sodium chloride or concentrated solution is added according to the concentration of 3g/100ml of sodium chloride (the mother liquor can only add solids).
③ Selection of dilution concentration of sample solution:
Pre-test: Dilute the sample solution into five concentrations in a logarithmic series: 100%, 10%, 1%, 0.1%, 0.01% (the logarithms are 0, -1, -2, -3, -4), according to 5 (2) and 5 (3). Roughly test again, depending on which concentration range the relative luminosity of 1% -100% falls.
Formal test: Add 6-9 concentrations in the concentration range where 1% -100% relative luminosity falls (for example, if it falls between 0.1% -10%, it should be diluted to 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2.5%, 5%, 7.5%, 10%; if it falls between 1% -10%, it should be diluted to 1%, 2%, 4%, 6%, 8%, 10%), and measure again according to 5 (2) - (3); these 6-9 concentrations can also be determined by looking up the logarithmic table according to the principle of equal logarithmic spacing (for example, if it falls between 1% -10%, it should be diluted to 10%, 6.31%) The logarithms of 3.98%, 2.51%, 1.58%, and 1.00% are respectively 1.00, 0.80, 0.60, 0.40, 0.20, and 0.00, and the logarithmic spacing is 0.2).
(2) Determination conditions
① Room temperature: 20-25 ° C.
During the determination of the same batch of samples, the temperature fluctuation is required to not exceed ± 1 ° C, and all test vessels, reagents, and solutions are placed in a temperature-controlled test chamber 1 hour before testing.
② pH: To determine acute toxicity, including pH effects, the pH of the sample to be tested should not be adjusted.
To determine the acute toxicity excluding the influence of pH, the pH of the sample to be tested and CK (sodium chloride 3g/100m1) should be adjusted to the following value before determination: 4.5 for water samples containing mainly Cu, 5.4 for water samples containing mainly other metals, and 7.0 for water samples containing mainly organic compounds.
Dissolved oxygen: This method can only determine acute toxicity, including the effects of dissolved oxygen.
(3) Determination steps
① Arrangement of test tubes: Arrange test tubes on plastic or iron test tube racks in the following two situations.
A. According to 5 (1) ①, the relative luminosity of the sample mother liquor is more than 1%, arranged as follows:
The reference mercury chloride series concentration solution tube is placed on the left side, and the sample tube is placed on the right side. The mercury chloride solution and sample tube are discharged in the front, the last emission control (CK) tube, and the last two discharge CK pre-test tubes. Each tube of mercury chloride or sample solution is equipped with a tube of CK (sodium chloride 3g/100m1 distilled aqueous solution). Set up three repetitions. Each batch of samples needs to be equipped with a standard solution for measuring the concentration of mercury chloride series at the same time.
B. According to 5 (1) ①, the relative luminosity of the sample mother liquor is below 50% or even zero, as follows:
Only 0.10mg/L mercury chloride solution tube is placed on the left side (as a reference for testing whether the activity of luminescent bacteria is normal, the relative luminosity after 15 minutes of reaction should be about 50%), and the diluent tube on the right side is placed (arranged in sequence from low concentration to high concentration). Others are the same a. Each batch of samples needs to be tested at the same time with 0.10mg/L mercury chloride solution.
② Add 3g/l00ml sodium chloride solution: Add 2m1 or 5ml sodium chloride 3g/l00ml to each CK tube with a 5ml quantitative liquid bottle (depending on the instrument model).
③ Add sample solution: Use 2ml or 5ml straws to add 2ml or 5ml sample solution to each sample tube (depending on 5 (3) ②). Change a straw for each sample number.
④ Luminescent bacteria lyophilized fungus recovery
Remove the ampoule and sodium chloride solution containing 0.5g luminescent bacteria lyophilized powder from the refrigerator freezer compartment at 2-5 ° C, put it in a 1-1.5 L thermos bottle with ice cubes, use an lml syringe to absorb 0.5m1 cold sodium chloride 2g/l00m1 (suitable for 5ml test tube) or lml cold 2.5% sodium chloride (suitable for 2m1 test tube) and inject it into the opened lyophilized powder ampoule, be sure to mix well. After 2min, the bacteria will recover and glow (can be inspected in a dark room, and the naked eye should see low light). Spare.
⑤ Preheating and zeroing of the instrument: turn on the power supply of the bioluminescence photometer, preheat for 15min, adjust to zero, and set aside.
< unk > Test the quality of the frozen powder of resuscitated luminescent bacteria: Take another 2m1 or 5ml test tube and add 2m1 or 5ml sodium chloride 3g/100ml (as determined by 5 (3) ②), add 10μ1 recurring photobacterial liquid, cover the bottle stopper, and turn it upside down five times by hand to achieve uniformity. Unplug the bottle stopper and put the tube into the test compartment of the respective model instrument. If the luminescence amount is displayed immediately (or rises to) 600mV or more after 5-l0min, this bottle of lyophilized powder can be used for testing. If it is lower, it will be treated as 4 ③. The luminescence amount of the bacterial liquid will rise slowly for about 5-15min, and then decrease slowly for about 4h. The luminescence amount of CK for 4h should not be less than 400mV, and if it is lower, replace the lyophil
0006 Add resuscitation bacterial solution to each test tube: After the resuscitation of the luminescent bacterial solution is stable (about 0.5h), press 5 (3), from left to right, press mercury chloride or sample tube (front) -CK tube (back) -mercury chloride or sample tube (front) -CK tube (back)... In sequence, accurately absorb 10 μl of resuscitation bacterial solution with a 10 μl micro-injector (do not use a dosing device to reduce errors), add each tube one by one, cover the cork, turn it upside down five times by hand, remove the cork, and put it back in place (the interval between adding bacterial solution to each tube should not be less than 30s. Each tube must be accurately timed at the time of adding bacterial solution, and recorded to seconds, which is the start time of the reaction between the sample and the luminescent bacteria. Immediately add 15 minutes to this time as the time for each tube reaction to terminate (i.e., the amount of light that should be read).
The reading of the termination time of the reaction between the luminescent bacteria and the sample: according to the order in which the bacterial liquid was originally added to each tube, when a tube reaches the recorded reaction termination time, without a stopper, immediately place the test tube into the instrument test compartment and read out its luminous amount (expressed in millivolts of the electrical signal converted by the optical signal).
Correction of interference in the determination of colored samples:
A. Remove the black plastic nozzle on the instrument sample compartment;
B. Take a -2m1 test tube (diameter 12mm), add sodium chloride 3g/ml solution 2m1, put the tube into a 5ml tube (diameter 20mm) containing a small amount of sodium chloride 3g/100m1 solution, and make the outer tube and the inner tube of sodium chloride 3g/l00ml level flush. This is a CK tube;
C. Take another 2ml test tube, add 2ml of sodium chloride 3g/ml solution, and put it into another 5ml tube with a small amount of colored sample solution to be tested. Make the outer tube and the inner tube of sodium chloride 3g/ml level flush. This is a CKc tube;
D. Add resuscitation luminescent bacterial solution 10 μ1 to the inner tube of CK and CKc two tubes at the same time (Note: It must be tested in this batch of samples)
Set the same bottle of resuscitation bacterial solution used), immediately time to seconds, and wait for the reaction to be completed for 15 minutes. Quickly put it into the instrument test compartment and measure the luminous amount of two 5ml test tubes with inner tubes. Note the luminous amount Ll (CK tube) and L2 (CKc tube) respectively;
E. Calculate the correction value of the amount of light emitted due to color (mV) Delta L = L1-L2;
F. Test the luminescence amount (mV) of the colored sample tube and its CK tube (sodium chloride 3g/l00ml solution) according to the conventional steps described in 5 (3) 0006 and 5 (3) 😂. The luminescence amount (mV) measured by all CK tubes must be subtracted from the correction value Delta L (mV) before it can be used as the CK luminescence amount (mV).
The correction schematic diagram of the interference in the determination of the colored sample solution is shown in Figure 5-3-1.
6. Quality assurance and quality control
① The luminous intensity and stability of each luminous bacteria are different. The same luminous bacteria should be used in the determination of the same batch of samples. If a mercury chloride standard curve is required, the same luminous bacteria should also be used with the sample. If one is not enough, two of the same batch can be mixed and used.
The general reaction time in the world is 5min or 15min. In view of the sensitivity and stability of the luminescence bacteria currently used in our country, 15min is used.
③ The relative deviation of three repeated measurements was determined to be less than or equal to 1%.
④ The measurement should be carried out immediately after the sample is collected. If it cannot be determined within 6 hours, it should be stored at low temperature, but not more than 24 hours.
⑤ Regarding the question of how to dilute the sample, it is generally determined according to the toxicity of the sample, but at least six different concentrations should be selected in the test. If the relative luminosity of the six concentrations is in addition to 1% -100%, several concentrations can be added to make it fall within the range of 1% -100% relative luminosity to find the relevant equation.
If the sample concentration is the highest limit concentration (i.e. 100%), the relative luminescence cannot reduce the luminous intensity by 50%, then the EC50 value for the sample can only be shown as > 100%.
0006 In view of the influence of the experimental factors of the luminescent bacteria method, the experimental results have certain errors. When evaluating the dose-effect relationship of poisons, a 95% confidence interval should be determined, that is, T = a + bC ± 2SE (SE is the remaining standard deviation), and the EC50 obtained from this also has a certain interval. The accuracy and authenticity of the experimental results can be determined based on these results. For the reproducibility of the experimental results, the general EC50 value has a coefficient of variation of 6% -10%.
7. Expression of test results
Calculate the relative luminosity (%) of the sample and calculate the average value.
Relative luminosity (%) = mercury chloride tube or sample tube luminescence (mV)/CK tube luminescence (mV) × 100
Relative Luminosity (%) Mean = (Repeat 1) (%) + (Repeat 2) (%) + (Repeat 3) (%)/3
2) If the relative luminosity of the sample mother liquor in 5 (1) ① is above 1%, establish and test the correlation equation between the concentration of mercury chloride (C) and its relative luminosity (T,%), and also draw the relationship curve.
① Find a (intercept), b (slope, regression coefficient) and r (correlation coefficient) of the unary linear regression equation, and list the equations:
T = a + bC mercury chloride
Check the correlation coefficient significance level (P value) table to check the significance level of the r value. If P ≤ 0.01 and EC50 mercury chloride = 0.lomg/L ± 0.02mg/L, the correlation equation is established; otherwise, it cannot be established, and the luminescence of the series of mercury chloride concentrations must be re-measured. If the mercury chloride solution is prepared overnight, it must be reformulated and then measured.
② You can also draw a relationship curve according to the above equation established. That is, specify the luminosity as 10% and 90%, substitute it into the above equation, and obtain the corresponding two mercury chloride concentrations. On the constant coordinate paper, set two points and draw a straight line, which is the relationship curve between the mercury chloride concentration and the relative luminosity that conforms to the equation.
3) If the relative luminosity of the sample mother liquor in 5 (1) ① is lower than 50% or even zero, and the result is expressed by EC50, establish and test the correlation equation between the dilution concentration (C) of the sample and its relative luminosity (T), and draw the relationship curve.
The correlation equation T = a + bC samples were established according to the method described in 7.2), and the correlation coefficient r and the significance level (P value) were tested.
If P ≤ 0.05, the correlation equation is established; otherwise, it cannot be established, and the luminous amount of the sample dilution series concentration must be re-measured.
8. Results evaluation and reporting
(1) Expression of sample toxicity with mercury chloride concentration
1) Applicable conditions: Those who meet the conditions (relative luminosity of sample mother liquor > 1%) and establish a qualified (P ≤ 0.01, EC50 mercury chloride = 0.10mg/L ± 0.02mg/L) mercury chloride concentration and its relative luminosity correlation equation.
2) Expression method:
① Substitute the measured sample relative luminosity into the correlation equation of 7 (2) to obtain the mercury chloride concentration (generally expressed in mg/L) equivalent to the acute toxicity of the sample.
② The test result report also lists the relative luminosity of the sample and its equivalent mercury chloride concentration value.
3) Applicability: It is suitable for the toxicity determination of samples with a relative luminosity above 1%, especially above 50% (i.e. EC50 values are not possible) but below 100% (i.e. medium and low levels of toxicity).
(2) Expression of sample toxicity by EC50 value
1) Applicable conditions: Those who meet the conditions (the relative luminosity of the sample mother liquor is less than 50% or even zero) and establish a qualified (P ≤ 0.01) sample dilution concentration and its relative luminosity correlation equation according to 7/3).
2) Expression method:
① Substitute T into the correlation equation established by 7 (3) to obtain the EC50 value of the sample. The EC50 value here is expressed as the dilution concentration of the sample (generally expressed as a percentage concentration).
The test result report lists the EC50 value of the sample.
3) Applicability: It is suitable for toxicity determination of samples with a relative luminosity below 50%, especially zero (i.e. high or very high toxicity level), which cannot express toxicity at a comparable concentration of mercury chloride.
(3) Measurement record format (see Table 5-3-8)
(4) Measurement result report
① Laboratory room temperature.
② Sampling location, date, and time.
③ The correlation equation between mercury chloride concentration or sample dilution percentage concentration and relative luminosity:
T=a+bC
r= P≤ EC50氯化汞= mg/L
L=a+bC a= b=
Regression equation r = P <
④ Sample EC50 value (dilution percentage concentration) or relative luminosity (%) and equivalent mercury chloride concentration (mg/L).
