CN110441292A - A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria - Google Patents

A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria Download PDF

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CN110441292A
CN110441292A CN201910623712.1A CN201910623712A CN110441292A CN 110441292 A CN110441292 A CN 110441292A CN 201910623712 A CN201910623712 A CN 201910623712A CN 110441292 A CN110441292 A CN 110441292A
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photobacteria
value
control sample
growth
chronic
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袁楠楠
鲍安平
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Nanjing College of Information Technology
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Nanjing College of Information Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence

Abstract

The invention discloses a kind of growths of photobacteria and acute toxicity and chronic toxicity detection method, comprising steps of preparing laboratory sample, negative control sample and positive control sample；Prepare photobacteria bacterium solution；Photobacteria bacterium solution is added in 96 porocyte culture plates, microplate reader is subsequently placed in, measures Initial optical density value and initial bioluminescence value after 30min；Laboratory sample, negative control sample and positive control sample are added into photobacteria bacterium solution, the preceding every 30min of 1h surveys an OD value and bioluminescence value, and then every 1h is surveyed primary, and test is not less than for 24 hours；Obtain optical density mutation time and bioluminescence mutation time；Calculate acute luminous inhibiting rate, chronic luminous inhibiting rate and growth inhibition ratio.The present invention can assess growth, acute and chronic poisonous effect of the test substance to photobacteria simultaneously, and accuracy rate is high, realize the high pass quantization of toxotest, and testing efficiency is high.

Description

A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria
Technical field
The invention belongs to technical field of environmental detection, and in particular to a kind of growth and acute toxicity and chronic toxicity of photobacteria Detection method.
Background technique
Photobacteria be it is a kind of in normal metabolic processes can spontaneous blue green light chemosynthetic autotroph microorganism, hair Light relies primarily on the bioluminescence enzyme system being made of NAD (P) H:FMN oxidoreducing enzyme and luciferase, luminous intensity with Intracellular metabolism is closely coupled, and when the activity of photobacteria is higher, cell metabolism ATP content is higher, it is luminous then It is relatively strong.It is suppressed, whereas if photobacteria is influenced cell activity by certain factors, ATP content can then respond and under Drop, luminous so as to cause photobacteria die down, or even stop shining.Traditional photobacteria toxicity test is then according to this original Reason by detecting the luminous intensity of photobacteria, and compares with blank group, characterizes the toxicity of tested substance.
Traditional photobacteria toxicity test is often applied to environmental pollution monitoring, as application for a patent for invention one kind is based on hair (application No. is 201710407732.6, publication No. is CN 107238599 to the soil comprehensive toxicity detection method of photobacteria method A), comprising steps of being pre-processed to pedotheque；Soil sample is weighed, uses methanol Soxhlet extraction after crossing nylon mesh, decompression rotation is steamed Hair is concentrated, and continues to be concentrated by the mixture after addition ethylene glycol spare；Sample leaching is added in photobacteria culture solution after taking culture Extract, 15wt.%NaCl solution and pure water, stand after mixing, measure luminous intensity；The expression of sample toxicity: measurement is stood The luminous inhibiting rate of mixed liquor, mercury chloride equivalent concentration afterwards；As patent of invention detects copper polluted soil using fresh water photobacteria The method (application No. is 200910079402.4, Authorization Notice No. is 101487798 B of CN) of acute toxicity, specific method choosing Fresh water photobacteria is used to use bacterium as detection, comprising the following steps: the first step prepares fresh water with Soil Background solution and shines carefully The bacteria suspension of bacterium；Second step extracts soil pore water using dual chamber centrifugal process；The toxicity of third step detection pedotheque.
But current photobacteria detection method of toxicity usually only acute (5~30min) shine toxicity detection this A toxicology endpoint can only assess test substance to the acute effect of the luminous function of photobacteria, to ignore substance to hair Effect except the effect of delayed toxicity caused by photobacteria or luminous function, such as growth.In addition, existing bacterial poison Property detection method be that luminous intensity is tested using light-emitting appearance, assessment mode is single, and institute sample limited amount every time, test effect Rate is low.
Summary of the invention
It is an object of the invention to overcome deficiency in the prior art, growth and the urgency for providing a kind of photobacteria are slow Property detection method of toxicity, while growth, acute and chronic poisonous effect of the test substance to photobacteria are assessed, accuracy rate Height, realizes the high pass quantization of toxotest, and testing efficiency is high.
The present invention provides the following technical solutions:
A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria, comprising the following steps:
Prepare the laboratory sample, negative control sample and positive control sample of several various concentrations；
Aseptically prepare photobacteria bacterium solution；
The photobacteria bacterium solution is added in 96 porocyte culture plates, 96 porocyte culture plates are then placed in enzyme mark In instrument, Initial optical density value and initial bioluminescence value are measured after 30min；
Laboratory sample, negative control sample and positive control sample are added into photobacteria bacterium solution, the preceding every 30min of 1h is surveyed OD value and bioluminescence value, then every 1h is surveyed primary, and test is not less than for 24 hours, every 5~10min vibration in test process Move 96 porocyte culture plates；
The OD value and bioluminescence value for making negative control sample change over time curve, and it is prominent to obtain optical density respectively Become time and bioluminescence mutation time；
Acute luminous inhibiting rate is calculated according to the bioluminescence value of laboratory sample, according to laboratory sample in the bioluminescence The bioluminescence value of mutation time calculates the chronic luminous inhibiting rate of photobacteria, is mutated according to laboratory sample in the optical density The OD value of time calculates the growth inhibition ratio of photobacteria.
Preferably, the acute luminous inhibiting rate is calculated by formula (1):
Ra=100 (LNC-Lt)/LNC (1)
R in formula (1)aIndicate acute luminous inhibiting rate, LNCIndicate that the average organism of negative control sample when 30min shines Value, LtIndicate the average organism luminous value of synchronization laboratory sample.
Preferably, the chronic luminous inhibiting rate is calculated by formula (2):
Rc=100 (INC-It)/INC (2)
R in formula (2)cIndicate chronic luminous inhibiting rate, INCIndicate negative control sample in the flat of bioluminescence mutation time Equal bioluminescence value, ItIndicate the average organism luminous value of synchronization laboratory sample.
Preferably, the growth inhibition ratio is calculated by formula (3):
Rg=100 (ODNC-ODt)/(ODNC-OD0) (3)
R in formula (3)gIndicate growth inhibition ratio, ODNCIndicate that negative control sample is close in the average light of optical density mutation time Angle value, ODtIndicate the average optical density of synchronization laboratory sample, OD0Indicate the corresponding photobacteria bacterium solution of negative control sample Initial optical density value.
Preferably, the laboratory sample is water phase laboratory sample or organic phase laboratory sample, the corresponding negative control Sample is water phase negative control sample or organic phase negative control sample.
Preferably, the water phase laboratory sample preparation method comprising steps of
Deposit to be checked is mixed with 3%NaCl solution, 12h is vibrated under the conditions of 20 DEG C in shaking table, is obtained with membrane filtration Water phase leaching liquor mother liquor is obtained, is then diluted with 3%NaCl solution and obtains various concentration water phase laboratory sample；
Using 3%NaCl solution as water phase negative control sample.
Preferably, the organic phase laboratory sample preparation method comprising steps of
Deposit to be checked is mixed with the acetone mixed of 1:1 by volume and hexane, 115 DEG C of conditions in Hyperfrequency waves eliminating stove Lower processing 20min, with membrane filtration, filtrate is evaporated with nitrogen evaporator is done, and residue is dissolved in dimethyl sulfoxide, obtains organic phase leaching Organic phase leaching liquor is dissolved in 3%NaCl solution by extract, the organic phase leaching liquor mother liquor that concentration is 1% is obtained, with 3% NaCl solution dilution obtains various concentration organic phase laboratory sample；
Using 3%NaCl solution as solvent, the dimethyl sulphoxide solution that dose volume concentration is 1% is organic phase negative control Sample.
Preferably, the photobacteria bacterium solution preparation method the following steps are included:
Photobacteria solution is placed in agar plate culture, single bacterium colony is then chosen and is seeded in culture medium culture to turbidity and reach To 500~600FTU, residue is resuspended to culture medium, is rapidly frozen after packing with liquid nitrogen, in -80 by centrifugation removal supernatant It DEG C freezes；
The photobacteria frozen is placed in thaw at RT, in the streak inoculation of medium agar plate and overnight incubation, will be obtained Bacterial solution diluted with culture medium, obtain photobacteria bacterium solution.
Preferably, using the culture medium and the mixed liquor of the NaCl solution as blank control sample；With the 3 of 9mg/L, The chloramphenicol of 5- chlorophenesic acid and 0.1mg/L are positive control sample.
Preferably, the temperature condition that OD value and bioluminescence value are tested in microplate reader is 10~15 DEG C.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention changes over time curve by negative control sample OD value and bioluminescence value, and it is close to obtain light Mutation time and bioluminescence mutation time are spent, acute luminous inhibiting rate, chronic luminous inhibiting rate and life are further calculated Long inhibiting rate can assess growth, acute and chronic poisonous effect of the test substance to photobacteria simultaneously, avoid tradition The problem of ignoring test substance delayed toxicity in method keeps detection effect more comprehensive and accurate；
(2) present invention obtains the life that can characterize photobacteria using microplate reader and the cooperation test of 96 porocyte culture plates The data for growing effect, acute and chronic poisonous effect, can test multiple samples simultaneously, reduce the dosage of sample to be tested, realize The high pass quantization of toxotest；
(3) test method provided by the invention, process is simple, at low cost, accuracy rate is high, using self-reacting device, saves Manpower, testing efficiency are high.
Detailed description of the invention
Fig. 1 is layout of each sample in 96 porocyte culture plates in embodiment；
Fig. 2 is that the OD value of water phase negative control sample and bioluminescence value change over time curve in embodiment 1；
Fig. 3 is the growth of photobacteria and acute toxicity and chronic toxicity effect figure in positive control sample in embodiment 1；
Fig. 4 is the water phase laboratory sample of deposit 1 in embodiment 1 to the luminous inhibiting rate of the acute and chronic of photobacteria and life Long inhibiting rate tendency chart；
Fig. 5 is the water phase laboratory sample of deposit 2 in embodiment 1 to the luminous inhibiting rate of the acute and chronic of photobacteria and life Long inhibiting rate tendency chart；
Fig. 6 is the water phase laboratory sample of deposit 3 in embodiment 1 to the luminous inhibiting rate of the acute and chronic of photobacteria and life Long inhibiting rate tendency chart；
Fig. 7 is the water phase laboratory sample of deposit 4 in embodiment 1 to the luminous inhibiting rate of the acute and chronic of photobacteria and life Long inhibiting rate tendency chart；
Fig. 8 is that the OD value of organic phase negative control sample and bioluminescence value change over time curve in embodiment 2；
Fig. 9 be deposit 1 in embodiment 2 organic phase laboratory sample to the acute and chronic of photobacteria shine inhibiting rate and Growth inhibition ratio tendency chart
Figure 10 be deposit 2 in embodiment 2 organic phase laboratory sample to the acute and chronic of photobacteria shine inhibiting rate with And growth inhibition ratio tendency chart；
Figure 11 be deposit 3 in embodiment 2 organic phase laboratory sample to the acute and chronic of photobacteria shine inhibiting rate with And growth inhibition ratio tendency chart；
Figure 12 be deposit 4 in embodiment 2 organic phase laboratory sample to the acute and chronic of photobacteria shine inhibiting rate with And growth inhibition ratio tendency chart.
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention Technical solution, and not intended to limit the protection scope of the present invention.
Embodiment 1
A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria, comprising the following steps:
Step 1: acquiring soil to be measured: selection 4 different lakes, with grab sampler acquisition 0~10cm of surface layer deposition Object, cross 1.8mm mesh screen remove impurity, air-dry, be crushed to partial size less than 2mm it is spare, respectively number sediment 1, sediment 2, sink Starch 3 and sediment 4.
Step 2: preparation water phase laboratory sample: 3g deposit being mixed with 30mL 3% (m/v) NaCl solution, in shaking table In vibrate 12h under the conditions of 20 DEG C, water phase leaching liquor mother liquor is obtained with 0.45 μm of membrane filtration, using 3%NaCl solution as dilution, The water phase leaching liquor mother liquor of preparation is diluted according to 1:2 continuous gradient dilutions method, obtains 100%, 50%, 25% and 12.5% water phase laboratory sample.
Step 3: preparing control sample: using 3%NaCl solution as water phase negative control sample, with culture medium and 3%NaCl The mixed liquor of solution is as blank control sample, with the chloramphenicol of 3, the 5- chlorophenesic acid of 9mg/L and 0.1mg/L for positive control Sample, wherein positive control sample is prepared by solvent of 3%NaCl solution.
Step 4: photobacteria freezes: Fei Shi orphan's bacterium freeze-dried powder is resuspended to 0.5mL supplement seawater complete medium (SSWC) in, SSWC agar plate is transferred to by serial dilution, after cultivating 72h under the conditions of 20 DEG C, single bacterium colony is chosen and is seeded to In 50mL SSWC culture medium, overnight incubation (90rpm, 20 DEG C), can obtain the pure culture of Fei Shi orphan bacterium in constant incubator Object, Fei Shi orphan bacterium by logarithmic growth phase switchs to stationary phase after cultivating 16h, and turbidity reaches 500-600FTU, by bacterium solution in 5450g Under the conditions of be centrifuged 30min, remove supernatant, residue be resuspended to 50mL cryopreservation culture medium, it is quick with liquid nitrogen after packing Freezing, obtains Fei Shi orphan's bacterium low temperature stock solution, freezes in -80 DEG C.
Step 5: preparation photobacteria bacterium solution: the Fei Shi orphan's bacterium low temperature stock solution frozen being placed in thaw at RT, in SSWC Agar plate streak inoculation prepares pre-culture, and overnight incubation (90rpm, 20 DEG C) to turbidity reaches 500-700FTU, will To bacterial solution be diluted to 20FTU with SSWC culture medium, Fei Shi orphan's bacterium solution of this concentration is photobacteria bacterium solution.
Step 6: test: multi-function microplate reader being placed in 15 DEG C of low temperature incubators, as shown in Figure 1, first that 100 μ L are empty White control sample (" blank " in Fig. 1) and photobacteria bacterium solution are added separately in 96 porocyte culture plates, then that 96 holes are thin Born of the same parents' culture plate, which is placed in microplate reader, makes bacterium adaptive temperature, and Initial optical density value is measured after 30min (microplate reader sets λ=578nm) With initial bioluminescence value (microplate reader sets time of measuring as 500ms)；Then corresponding reality is added into photobacteria bacterium solution Testing sample, (A1-A4 is 1 laboratory sample of sediment of four kinds of concentration in Fig. 1, and B1-B4 is that the sediment 2 of four kinds of concentration tests sample Product, C1-C4 are 3 laboratory samples of sediment of four kinds of concentration, and D1-D4 is 4 laboratory sample of sediment of four kinds of concentration), it is negative right (PC a is 3, the 5- chlorophenesic acid of 9mg/L in Fig. 1, and PC b is 0.1mg/L for product (" NC " in Fig. 1) and positive control sample in the same old way Chloramphenicol), start kinetic loop test OD value and bioluminescence value, the preceding every 30min of 1h survey an OD value and Bioluminescence value, then every 1h is surveyed primary, and test is not less than for 24 hours, every 5~10min in test process, preferably 7min vibration one Secondary 96 porocyte culture plates.
Step 7: making the OD value of water phase negative control sample and bioluminescence value changes over time curve, such as Fig. 2 Shown, by OD value, it is found that the exponential phase of growth of Fei Shi orphan bacterium and the transformation for stablizing growth period occur, in 10h or so, (light is close Spend mutation time), OD value has risen to growth inhibiting analysis of enough amounts for bacterium, and bioluminescence value is most Big value then appears in 15h or so (bioluminescence mutation time), therefore 10h and 15h can be used as analysis growth inhibition and chronic hair The suitable exposure duration point of Xanthophyll cycle.
Step 8: the acute luminous inhibiting rate is calculated by formula (1):
Ra=100 (LNC-Lt)/LNC (1)
R in formula (1)aIndicate acute luminous inhibiting rate, LNCIndicate that the average organism of negative control sample when 30min shines Value, LtIndicate the average organism luminous value of synchronization laboratory sample；
The chronic luminous inhibiting rate is calculated by formula (2):
Rc=100 (INC-It)/INC (2)
R in formula (2)cIndicate chronic luminous inhibiting rate, INCIndicate negative control sample in the flat of bioluminescence mutation time Equal bioluminescence value, ItIndicate the average organism luminous value of synchronization laboratory sample；
The growth inhibition ratio is calculated by formula (3):
Rg=100 (ODNC-ODt)/(ODNC-OD0) (3)
R in formula (3)gIndicate growth inhibition ratio, ODNCIndicate that negative control sample is close in the average light of optical density mutation time Angle value, ODtIndicate the average optical density of synchronization laboratory sample, OD0Indicate the corresponding photobacteria bacterium solution of negative control sample Initial optical density value.
If Fig. 3 is the growth and acute toxicity and chronic toxicity effect figure of photobacteria in positive control sample, as seen from the figure, The chloramphenicol that 3, the 5- chlorophenesic acid (PC a) of 9mg/L is 68.1%, 0.1mg/L to the acute luminous inhibiting rate of Fei Shi orphan bacterium (PC b) is respectively 60.8% and 47.8% to the chronic luminous inhibition of Fei Shi orphan bacterium and growth inhibition inhibiting rate, illustrates this research The enough sensitivities of Fei Shi orphan's hyphopodium of middle selection, can be used for dynamics toxicity test.
As shown in Fig. 3,4,5,6, acute and chronic of the water phase laboratory sample to photobacteria of deposit 1,2,3,4 is respectively indicated Shine inhibiting rate and growth inhibition ratio trend.As seen from the figure, Fei Shi orphan bacterium is exposed to the water phase laboratory sample of deposit 1 not Have and generates any luminous inhibition and growth inhibitory effect；However when concentration is higher than 50%, the water phase of deposit 2 and deposit 4 Acute luminous generation inhibiting effect of the laboratory sample to bacterium；The water phase laboratory sample of deposit 3 under four diluent concentrations all Acute luminous inhibiting effect is produced to bacterium；Deposit 2, deposit 3 and 4 water phase laboratory sample of deposit generate acute luminous Inhibiting rate is below 10%, and does not generate chronic luminous inhibition and growth inhibition effect to Fei Shi orphan bacterium.
Embodiment 2
A kind of growth and acute toxicity and chronic toxicity detection method of photobacteria, comprising the following steps:
Step 1: preparation organic phase laboratory sample: using soil to be measured spare in 1 step 1 of embodiment, 1g being deposited Object is mixed with the 30mL acetone that 1:1 is mixed by volume and hexane, is handled 20min under the conditions of 115 DEG C in Hyperfrequency waves eliminating stove, is mentioned Liquid is taken to cross 0.45 μm of filter membrane, filtrate is evaporated with nitrogen evaporator does, and residue is dissolved in 4mL dimethyl sulfoxide (DMSO), obtains organic phase Leaching liquor；Organic phase leaching liquor is dissolved in 3%NaCl solution to final concentration of 1%, using this solution as organic phase before experiment Leaching liquor mother liquor；Using 3%NaCl solution as dilution, organic phase leaching liquor mother liquor is carried out according to 1:2 continuous gradient dilutions method Dilution, obtains 100%, 50%, 25% and 12.5% organic phase laboratory sample.
Step 2: preparing control sample: using the DMSO solution that volumetric concentration is 1% as organic phase negative control sample, with The mixed liquor of culture medium and 3%NaCl solution is as blank control sample, with 3, the 5- chlorophenesic acid of 9mg/L and 0.1mg/L Chloramphenicol is that positive control sample, wherein negative control sample and positive control sample are prepared by solvent of 3%NaCl solution.
Step 3: the photobacteria bacterium solution prepared using step 4 in embodiment 1 and step 5, using being walked in embodiment 1 Rapid six test method is to organic phase laboratory sample, organic phase negative control sample, blank control sample and positive control sample Carry out the test of kinetic loop test OD value and bioluminescence value.
Step 4: making the OD value of organic phase negative control sample and bioluminescence value changes over time curve, such as Shown in Fig. 8, by OD value it is found that the exponential phase of growth of Fei Shi orphan bacterium occurs with the transformation for stablizing growth period in 10h or so (light Density mutation time), the maximum value of bioluminescence value then appears in 14h or so (bioluminescence mutation time), therefore 10h and 14h can be used as the suitable exposure duration point of analysis growth inhibition and chronic luminous inhibition.
Step 5: using formula listed by step 8 in embodiment 1, calculates separately out acute luminous inhibiting rate, chronic shines Inhibiting rate and growth inhibition ratio.
As shown in Fig. 9,10,11,12, the organic phase laboratory sample of deposit 1,2,3,4 is respectively indicated to photobacteria The luminous inhibiting rate of acute and chronic and growth inhibition ratio trend.As seen from the figure, the organic phase laboratory sample of deposit 1 is to Fei Shi orphan bacterium Acute, chronic luminous function and growth all do not generate depression effect；Deposit 2, deposit 3 and deposit 4 have Machine phase laboratory sample all shows similar inhibiting effect, the organic phase laboratory sample of deposit 2, deposit 3 and deposit 4 The acute inhibiting rate that generates of four dilutions be no more than 10%, and without generating chronic inhibition；When dilution is higher than When 50%, the organic phase laboratory sample of deposit 2, deposit 3 and deposit 4 shows to inhibit to make to the growth of Fei Shi orphan bacterium With, but inhibiting rate is below 15%.
Therefore, detection method provided by the invention is to hydrophilic composition in substance (water phase) and hydrophobic combination (organic phase) Genotoxic potential detection be all suitable for, can synchronize, the growth and acute toxicity and chronic toxicity of high-throughput detectable substance confrontation photobacteria Effect.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations Also it should be regarded as protection scope of the present invention.

Claims (10)

1. the growth and acute toxicity and chronic toxicity detection method of a kind of photobacteria, which comprises the following steps:
Prepare the laboratory sample, negative control sample and positive control sample of several various concentrations；
Aseptically prepare photobacteria bacterium solution；
The photobacteria bacterium solution is added in 96 porocyte culture plates, 96 porocyte culture plates are then placed in microplate reader In, Initial optical density value and initial bioluminescence value are measured after 30min；
Laboratory sample, negative control sample and positive control sample are added into photobacteria bacterium solution, the preceding every 30min of 1h surveys primary OD value and bioluminescence value, then every 1h is surveyed primary, and test is not less than for 24 hours, every 5~10min vibration one in test process Secondary 96 porocyte culture plates；
The OD value and bioluminescence value for making negative control sample change over time curve, when obtaining optical density mutation respectively Between and bioluminescence mutation time；
Acute luminous inhibiting rate is calculated according to the bioluminescence value of laboratory sample, is mutated according to laboratory sample in the bioluminescence The bioluminescence value of time calculates the chronic luminous inhibiting rate of photobacteria, according to laboratory sample in the optical density mutation time OD value calculate photobacteria growth inhibition ratio.
2. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that logical It crosses formula (1) and calculates the acute luminous inhibiting rate:
Ra=100 (LNC-Lt)/LNC (1)
R in formula (1)aIndicate acute luminous inhibiting rate, LNCIndicate the average organism luminous value of negative control sample when 30min, LtTable Show the average organism luminous value of synchronization laboratory sample.
3. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that logical It crosses formula (2) and calculates the chronic luminous inhibiting rate:
Rc=100 (INC-It)/INC (2)
R in formula (2)cIndicate chronic luminous inhibiting rate, INCIndicate negative control sample in the average life of bioluminescence mutation time Object luminous value, ItIndicate the average organism luminous value of synchronization laboratory sample.
4. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that logical It crosses formula (3) and calculates the growth inhibition ratio:
Rg=100 (ODNC-ODt)/(ODNC-OD0) (3)
R in formula (3)gIndicate growth inhibition ratio, ODNCIndicate negative control sample in the average optical density of optical density mutation time Value, ODtIndicate the average optical density of synchronization laboratory sample, OD0Indicate the corresponding photobacteria bacterium solution of negative control sample Initial optical density value.
5. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that institute Stating laboratory sample is water phase laboratory sample or organic phase laboratory sample, and the corresponding negative control sample is water phase negative control Sample or organic phase negative control sample.
6. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 5, which is characterized in that institute State the preparation method of water phase laboratory sample comprising steps of
Deposit to be checked is mixed with 3%NaCl solution, vibrates 12h under the conditions of 20 DEG C in shaking table, water is obtained with membrane filtration Then phase leaching liquor mother liquor is diluted with 3%NaCl solution and obtains various concentration water phase laboratory sample；
Using 3%NaCl solution as water phase negative control sample.
7. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 5, which is characterized in that institute State the preparation method of organic phase laboratory sample comprising steps of
Deposit to be checked and the acetone mixed of 1:1 by volume and hexane are mixed, in Hyperfrequency waves eliminating stove under the conditions of 115 DEG C at 20min is managed, with membrane filtration, filtrate is evaporated with nitrogen evaporator is done, and residue is dissolved in dimethyl sulfoxide, obtains organic phase extraction Organic phase leaching liquor is dissolved in 3%NaCl solution by liquid, the organic phase leaching liquor mother liquor that concentration is 1% is obtained, with 3%NaCl Solution dilution obtains various concentration organic phase laboratory sample；
Using 3%NaCl solution as solvent, the dimethyl sulphoxide solution that dose volume concentration is 1% is organic phase negative control sample Product.
8. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that institute State the preparation method of photobacteria bacterium solution the following steps are included:
Photobacteria solution is placed in agar plate culture, single bacterium colony is then chosen and is seeded in culture medium culture to turbidity and reach 500~600FTU, centrifugation removal supernatant, is resuspended to culture medium for residue, is rapidly frozen after packing with liquid nitrogen, in -80 DEG C It freezes；
The photobacteria frozen is placed in thaw at RT, it is thin by what is obtained in the streak inoculation of medium agar plate and overnight incubation Bacterium solution is diluted with culture medium, obtains photobacteria bacterium solution.
9. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that with Culture medium and the mixed liquor of the NaCl solution are as blank control sample；With 3, the 5- chlorophenesic acid of 9mg/L and 0.1mg/L Chloramphenicol is positive control sample.
10. the growth and acute toxicity and chronic toxicity detection method of photobacteria according to claim 1, which is characterized in that The temperature condition that OD value and bioluminescence value are tested in microplate reader is 10~15 DEG C.
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CN111413329A (en) * 2020-04-30 2020-07-14 武汉科技大学 Biological acute toxicity detection method applied to detection of pollutants and actual water sample
CN111979140A (en) * 2020-07-10 2020-11-24 山东瑞泽检测评价技术服务有限公司 Bright photobacterium growth promoter for monitoring water pollutants and application thereof
CN112557353A (en) * 2020-12-17 2021-03-26 天津工业大学 Cell viability detection method and device based on delayed luminescence spectrum

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Application publication date: 20191112