CN102344948B

CN102344948B - Method for determining acute toxicity of direct dyes by using saccharomyces cerevisiae

Info

Publication number: CN102344948B
Application number: CN2011102862447A
Authority: CN
Inventors: 谭之杨
Original assignee: Wuhan Textile University
Current assignee: Wuhan Textile University
Priority date: 2011-09-25
Filing date: 2011-09-25
Publication date: 2013-11-27
Anticipated expiration: 2031-09-25
Also published as: CN102344948A

Abstract

The invention relates to the field of textile safety and dye production, in particular to a novel method for rapidly detecting toxicity of direct dyes. The method is characterized in that: after a system consisting of 20 microliters of saccharomyces cerevisiae suspension with the density of 1.0 * 10<4> cells/ml, 130 microliters of a series of dye solution with concentration to be detected and 110 microliters of 2.2-time yeast peptone dextrose (YPD) liquid medium in a cover-connecting centrifugal tube is subjected to shaking culture for 6 to 7.5 hours on a constant temperature shaker at the temperature of 30 DEG at 150 r/min, the cell number is determined by using a hand-held cell counter based on a resistance method cell counting principle or a dull colony counting method, and EC50 of the dyes is calculated. A 96-well cell culture plate (or an elisa plate) replaces the cover-connecting centrifugal tube, and each 96-well plate can determine four kinds of dyes. The method can be used for detecting the comprehensive acute toxicity of various water soluble inorganic matters, water soluble organic matters or microorganisms.

Description

Method with determining acute toxicity of direct dyes by using saccharomyces cerevisiae

Technical field

The present invention relates to textiles safety and DYE PRODUCTION field, is a kind of method of substantive dyestuff to the biomass cells acute toxicity of estimating.

Background technology

Substantive dyestuff has stronger avidity to fiber, help that need not mordant just can more than dye cellulosic fibre, therefore in Dyestuffs Used for Cellulose Fibres, account for larger quantity, in this class dye molecule, all contain sulfonic group or carboxyl, commodity are made sodium salt, so water soluble.The structure of substantive dyestuff has the characteristics of line style, coplanarity, longer conjugate system, on chemical structure, is mainly that tetrazo, trisazo-etc. have sulfonic polyazo dye.Be mainly used in the dyeing of the products such as cotton yarn, knitted fabrics, viscose fiber and silk, also for the dyeing of fur and paper.

The raw material of most substantive dyestuff is aromatic amine, and aromatic amine is a class carcinogenic substance.Therefore, in Germany forbade dyestuff in the first batch, substantive dyestuff had accounted for very large proportion, and three analog derivatives such as p-diaminodiphenyl, tolidine of wherein usining account for all disabled more than 90% of substantive dyestuff as the synthetic substantive dyestuff of intermediate.

The key instrument that in current substantive dyestuff detection method, regulation is used is gas chromatograph-mass spectrometer, all rely at present import, every gas chromatograph-mass spectrometer is amounted to Renminbi 800,000 yuan of left and right, every equipment can only be checked 30 duplicate samples at most in one day, simultaneously in the Check processing process, will use the materials such as a large amount of high purity reagent and import extraction column, appropriate litigation fees is high.Numerously city-level feeler mechanism all ability do not purchase and be equipped with this test set, also just can't carry out corresponding check work, this has restricted the coverage of China's substantive dyestuff detection.

And traditional acute toxic test generally need to arrange 5-7 dosage group, stipulate that simultaneously the number of mouse, nude mice or the cavy of each dosage group is no less than 10; The number of rat, new zealand rabbit is 6-8; The number of rabbit, dog etc. is 4-6, asks its LD according to the mortality ratio of 24 h and the dead property compound of total death toll calculating speed in 14 days ₅₀(LC ₅₀), and need to observe in detail generation and the evolution of animal toxicity symptom, dead before symptom, death time, body weight change, perspiration salivation, courageous and upright secretory product, pupil change, organ has or not hyperemia, hemorrhage, oedema or other change.

The complicacy of tradition determination of acute toxicity process, the carcase that expensive, height is consuming time and residual force people to attempt developing toxicity in fish test, flea class toxotest, thermophilas toxotest, algae toxicity test, photobacterium bioassay and biosensor test method to the secondary pollution problems of environment.

The toxicity in fish testing method:24 h are adopted in the toxicity in fish experiment, 48 h, and 96 h measure the LC of toxic substance ₅₀, be applicable to the toxicity test of one matter in water, but the hydrostatic method, change water law and flowing water method measurement result is inconsistent, need simultaneously great many of experiments material and repeated experiments repeatedly.

Flea class toxotest method:The flea class is the bioorganism generally adopted in the world.Adopt mortality ratio, predation to change into index.But standard is difficult for unified.

Thermophilas toxotest method:Employing single cell gel electrophoresis technique (SCGE) and flow cytometry joint-detection thermophilas ( Tetrahymena thermophila) tail of a comet length, hangover rate, cell injury rate, DNA damage and DNA relative intensity of fluorescence, from the DNA damage angle, inquire into the toxic action of nitrogen-containing heterocycle compound common trade effluent to thermophilas.But the toxicity kind that this method is tested is limited, be difficult to dyestuff and dyeing waste water toxotest thereof.

The algae toxicity testing method:By the growth-inhibiting of scenedesmus obliquus, chlorella, salt pool spirulina, flat algae etc., as test index, measure the acute toxicity of detected material, also have lux gene is proceeded in filament Azotica-anabena, utilize it as reporter gene, by the mensuration to luminous intensity, detect the acute toxicity of analyte.But workload is large, determination period is long, and is not suitable for dyestuff.

The photobacterium bioassay method:U.S. Backman company has developed the noclilucence photometer (being the bio-toxicity determinator) of a kind of commodity Microtox by name, and wherein the sensitivity of the toxicity of the variation detection of contamination of photobacterium phosphoreum luminous intensity is suitable with fish 96 h acute toxicity tests.China classifies this method as standard method that acute toxicity of water quality detects in nineteen ninety-five.The lonely bacterium in Qinghai ( Vibrio qinghaiensisSp. nov.) also for the bio-toxicity of fresh water sample, detect.Nitrobacteria also detects for the toxicity of toxic substance.Britain PPM company has researched and developed the on-line testing instrument of a kind of AMTOX by name, and this instrument consumes the ammonia in sample based on using immobilized nitrifying bacteria, and by the wear rate of measuring ammonia nitrogen in substrate, detects the toxicity of sample.But photogenic bacterium method detection toxicity has the luminous intensity background values and differs greatly, the problem of variations in light amplitude broad between detection period, measured data are inaccurate.Simultaneously, because many dyestuffs itself are fluorescence quenchings, be unsuitable for this method.

The biosensor test method:The biosensor detected for toxicity has Enzyme sensor, microbiological sensor, DNA sensor and immunosensor etc.But the specificity of enzyme, DNA and Ag-Ab is strong, therefore, a kind of biosensor can only detect a kind of toxicity of material specifically.

Summary of the invention

Technical problem to be solved by this invention is: the method for quick of developing a kind of novel microorganism acute toxicity, and interaction that can comprehensive multiple toxic substance, judge dyestuff and the mass concentration of dyeing waste water and the relation of biological effect, it is carried out to hazard assessment, for monitoring and the comprehensive evaluation that guarantees security dye production and use, formulation treatment of dyeing wastewater qualified discharge standard, environmental pollution early warning, water quality provides scientific basis.

The present invention measures and connects that to cover in centrifuge tube be 1.0 * 10 by density by using based on the hand-held cell counter of electrical resistance method cell counting principle ⁴The yeast saccharomyces cerevisiae suspension 20 μ l of cell/ml and series concentration dye solution 130 μ l to be measured and 2.2 times of systems that YPD liquid nutrient medium 110 μ l form, the cell number after 6 h ~ 7.5 h is cultivated in concussion on 30 ℃, 150 r/min constant-temperature tables, asks the EC that calculates this dyestuff ₅₀.With 96 porocyte culture plates (or enzyme plate), replace 1.5 ml to connect and cover centrifuge tube, each 96 orifice plate can be measured 4 kinds of dyestuffs.The method also can be used for detecting the composite acuity toxicity of various water soluble inorganic substances, water soluble organic substance or microorganism.

With existing method, compare, the present invention has following major advantage.

One, trace: the dyestuff to be measured of consumption, lower than 60 mg, is specially adapted to the acute toxic test of dyestuff on textiles.

Two, quick: the roughly EC that measures a kind of material ₅₀The time of-scope is no more than 8 h, and a people can test hundreds of material in one day; Measure a kind of EC of material ₅₀-a people, can complete in one day.

Three, low consumption: measure a kind of sample consumption: 1 25 ml sterile test tube, amount to 5.28 yuan of Renminbi; 54 20-300 μ l standard suction nozzles, amount to 5.94 yuan of Renminbi; 55 1.5 ml even cover centrifuge tube, amount to 2.2 yuan of Renminbi; YEPD liquid nutrient medium 5.5 ml, amount to 0.03 yuan of Renminbi; PBS (pH 7.4) damping fluid 42.3 ml, amount to 0.04 yuan of Renminbi; 2 of Scepter sensors, amount to 44 yuan of Renminbi; 4.00 yuan of the electricity charge; Add up to 56.3 yuans.If measure simultaneously several materials, the expense of measuring is lower.

Four, convenient: only need Bechtop, high-pressure sterilizing pot, constant-temperature table, whizzer, electrical resistance method hand-held cell counter to carry out the work easily, the armamentarium total value is minimum to be reached below 40,000 yuans.

Five, general: as to be applicable to the various material of certain solubility is arranged in water acute toxic tests.

The accompanying drawing explanation

Fig. 1 is the schematic diagram by 3 multiplying factor constant gradient dilution preparation dye solution to be measured.

Fig. 2 asks to calculate dyestuff EC to be measured ₅₀The schematic diagram of-scope.

Fig. 3 asks to calculate dyestuff EC to be measured ₅₀Schematic diagram.

Fig. 4 asks to calculate dyestuff EC to be measured ₅₀The signal partial enlarged drawing.

Embodiment

Measure direct light turquoise blue GB (claim again the bright blue GL of tin profit, direct light turquoise GL, directly sun-proof is sapphire blue; Lionol Blue GB) EC ₅₀.

1) prepare the yeast saccharomyces cerevisiae bacteria suspension: in Bechtop, with aseptic toothpick, collect the dull and stereotyped yeast saccharomyces cerevisiae list bacterium colony of cultivating and proceed to label in sterilized Eppendorf pipe 1., add 1000 μ l sterile pure water suspendibles, getting this suspension 200 μ l adds in 15 ml sterile test tube, add 1.8 ml sterile purified waters, on spectrophotometer, record OD ₆₀₀=0.248, learn that the cell density of suspension in 1. number pipe is 0.248 ÷ 1.4 * 10 ⁸* 10=1.7714 * 10 ⁸Individual/ml; From 1. pipe, get 56.4 μ l and enter 2. pipe, add 944 μ l sterile purified waters 1 * 10 ⁷The bacteria suspension of individual/ml; By 1000 times of the dilutions of the bacteria suspension in 2. pipe, namely be mixed with cell density and be about 1.0 * 10 ⁴The bacteria suspension of individual/ml, put into 4 ℃ of refrigerators standby.

2) preparation direct light turquoise blue GB solution: be 20 g/L according to the solubleness of direct light turquoise blue GB in 20 ℃ of water, therefore the peak concentration of the dye solution to be measured of preparation is 20000 mg/l.With electronic balance, take 60 mg direct light turquoise blue GB and enter 5 ml and connect and to cover in centrifuge tube, add sterile pure water 3 ml and dissolve, put into 4 ℃ of refrigerators standby (dyestuff storing solution); To be numbered 0#-11# and the aseptic Eppendorf pipe of 1.5 ml that is numbered 0 ' #-11 ' #, minutes two are emitted on the multifunctional colour centrifuge tube shelf of 5 * 16=80 hole, and with the adjustable 20-300 μ of single track l range pipettor, coordinating 20-300 μ l suction nozzle to get respectively 130 μ l concentration is that the direct light turquoise blue GB solution to be measured of 20000 mg/l enters 11#, 11 ' # and 10# manages; In the 10# pipe, add sterile pure water 260 μ l, after mixing; From the 10# pipe, get respectively 130 μ l mixed solutions and enter 10 ' # and 9# pipe, and add sterile pure water 260 μ l, then mix, prepare successively 8#-1#, 9 ' #-1 ' # pipe solution, take out 130 μ l mixed solutions and discard from 1#, the effective pipettor of 1 ' #; Obtain 20000,6667,2222,741,247,82.3,27.4,9.14,3.05,1.02,0.34 mg/l totally 2 groups of the direct light turquoise blue GB solution to be measured of totally 11 kinds of concentration.

3) culturing cell: above-mentioned 24 Eppendorf pipes are placed in ice bath, and it is 3.0 * 10 that 0# and 0 ' # pipe add 130 μ l sterile purified waters, all Guan Zhongjun to add 2.2 times of YPD liquid nutrient mediums, 110 μ l and 20 μ l cell densities ⁵The bacteria suspension of individual/ml; Build pipe lid, shaking culture is 7.5 hours on 30 ℃, 150 r/min constant-temperature tables.

4) washed cell: take out each centrifuge tube and put into the small-sized high speed centrifugal machine, with centrifugal 10 min of 4000 r/min, abandon supernatant liquor; With 12 road adjustable 20-300 μ l range pipettors, add PBS (pH 7.4) damping fluid 300 μ l to wash the cell mass 2 times in each centrifuge tube.

5) counting suspension preparation: use respectively PBS (pH 7.4) damping fluid to be mixed with 240 μ l bacteria suspensions the cell mass after each washing.

6) measure cell number: with Millipore Scepter TM hand-held cell counter, measure cell number separately, the results are shown in Table 1.

The impact of table 1 direct light turquoise blue GB on brewing yeast cell propagation.

Dye strength (mg/l)	0	0.17	0.51	1.52	4.6	13.7	41	123	370	1111	3333	10000
													Average cell concentration	160000	160000	160000	160000	158000	154000	148000	140000	125000	110000	85000	670
Average cell propagation	159330	159330	159330	159330	157330	153330	147330	139330	124330	109330	84330	0
													Cell fission rate (%)	100	100	100	100	99	96	92	87	78	69	53	0

7) ask and calculate dyestuff EC ₅₀Scope: the cell concn of respectively managing bacteria suspension by measured, deduct initial cell concentration, the cell concn divided by the control tube bacteria suspension, be converted into percentage ratio, is defined as the cell fission rate; The cell fission rate of same concentrations dyestuff is average, by direct light turquoise blue GB series concentration, divided by 2 values, be that X-coordinate, average cell division rate are that ordinate zou is drawn respectively, obtain a curve, by a bit (0 on ordinate zou, 50) make the parallel lines of X-axis, hand over curve in the A point, cross the parallel lines that the A point is made Y-axis, hand over X-axis in the B point, the X-coordinate numerical value that B is ordered is the EC of required dyestuff ₅₀Scope, be designated as respectively L and H by near direct light turquoise blue GB concentration corresponding the B point, L=3333, and H=10000, be shown in accompanying drawing 2.

Accompanying drawing 2 is direct light turquoise blue GB impacts on the yeast saccharomyces cerevisiae division.

8) measure and ask and calculate dyestuff EC ₅₀: by 3333 and 10000, be expressed as respectively 3.3 * 10 respectively ³With 1 * 10 ⁴, after rounding, obtain L '=3 * 10 ³And H '=1 * 10 ⁴, therefore, the concentration range of dye solution to be tested is decided to be 3000 ~ 10000 mg/l.Press table 2 preparation steps 8) required direct light turquoise blue GB dye solution.

The preparation of table 2 step 8) required direct light turquoise blue GB dye solution.

Centrifuge tube number	0	1	2	3	4	5	6	7	8	9	10	11
													The dyestuff storing solution	0	39	48.1	57.2	66.3	75.4	84.5	93.6	102.7	111.8	120.9	130
Sterile pure water	91	81.9	72.8	63.7	54.6	45.5	36.4	27.3	18.2	9.1	0	91
													Centrifuge tube number	0’	1’	2’	3’	4’	5’	6’	7’	8’	9’	10’	11’
The dyestuff storing solution	0	39	48.1	57.2	66.3	75.4	84.5	93.6	102.7	111.8	120.9	130
													Sterile pure water	91	81.9	72.8	63.7	54.6	45.5	36.4	27.3	18.2	9.1	0	91

At above-mentioned each Guan Zhongjun, add 2.2 times of YPD (YEPD) liquid nutrient medium, 110 μ l respectively, all add yeast saccharomyces cerevisiae suspension 20 μ l again, shake up, build the centrifuge tube lid, concussion was cultivated 6 hours on 30 ℃ of temperature, rotating speed 150 r/min constant-temperature tables.

Culture tube is put into to the small-sized high speed centrifugal machine, with centrifugal 10 min of 4000 r/min, abandon supernatant liquor, (each washing is all by cell suspension with PBS (pH 7.4) damping fluid 300 μ l, to wash each cell mass 2 times, then centrifugal and abandon supernatant liquor immediately), use respectively PBS (pH 7.4) damping fluid to be mixed with 260 μ l bacteria suspensions the cell mass after each washing.

The cell concn of respectively managing bacteria suspension by measured, deduct initial cell concentration, and the cell concn divided by the control tube bacteria suspension, be converted into percentage ratio, is defined as the cell fission rate; The cell fission rate of same concentrations dyestuff is average, by dye strength, be that X-coordinate, average cell division rate are that ordinate zou is drawn divided by 2 values, obtain a curve, by a bit (0 on ordinate zou, 50) make the parallel lines of X-axis, hand over curve in the A1 point, cross the parallel lines that the A1 point is made Y-axis, hand over X-axis in the B1 point.

Upper and lower two concentration values of B1 are distributed and are designated as L ₁And H ₁, that is: L ₁=4400, H ₁₌5100, by line segment L ₁H ₁Be divided into 10 equal portions, visible down scale numerical value corresponding to B1 is about b=8.5, according to formula (H ₁-L ₁) ÷ 10 * b=(5100-4400) ÷ 10 * 8.5, see accompanying drawing 3 and accompanying drawing 4, calculate EC ₅₀=4995 mg/l.With the mouse mouth of bibliographical information through LC ₅₀=5 g/kg are consistent.

Accompanying drawing 3 EC ₅₀The impact of scope direct light turquoise blue GB on the yeast saccharomyces cerevisiae division.

The EC of accompanying drawing 4 direct light turquoise blue GB ₅₀Ask and calculate schematic diagram (amplify the part of accompanying drawing 3).

Claims

1. by the method for determining acute toxicity of direct dyes by using saccharomyces cerevisiae, it is characterized in that: by following steps, realized under aseptic condition:

1) prepare sterilised yeast suspension: by the yeast saccharomyces cerevisiae lyophilized powder or the dull and stereotyped yeast saccharomyces cerevisiae bacterium colony of cultivating prepares with aseptic pure water or by the cell density adjustment of the yeast saccharomyces cerevisiae of liquid culture, making cell density is 1.0 * 10 ⁴The yeast saccharomyces cerevisiae suspension of individual/ml, a kind of dyestuff need 1000 μ l bacteria suspensions at least, put into 4 ℃ of refrigerators standby;

2) preparation dye solution: with 1.5 ml of numbering, connect cover centrifuge tube, single track is adjustable, and 20-300 μ l range pipettor coordinates 20-300 μ l suction nozzle to prepare; The peak concentration of dye solution is equal to or slightly less than the solubleness of this substantive dyestuff, but is not more than 20000 mg/l; Consumption is every pipe 130 μ l; And carry out the constant gradient dilution by 3 multiplying factors, and dilute altogether 10 times, obtain totally 2 groups of the substantive dyestuff solution to be measured of 11 kinds of concentration, see Fig. 1;

3) set up culture system: by 2 groups every group 12 1.5 ml, even cover centrifuge tube and form, every group of front 11 centrifuge tubes are respectively by dye solution to be measured: 2.2 times of YPD liquid nutrient mediums: yeast saccharomyces cerevisiae suspension=130 μ l: 110 μ l: 20 μ l form; Every group of last 1 effective sterile purified water replaces dye solution to be measured to manage in contrast; All adopt the adjustable 20-300 μ of single track l range pipettor to add;

4) carry out cell cultures: build the centrifuge tube lid, 6-7.5 hour is cultivated in concussion under 30 ℃ of temperature, rotating speed 150 r/min conditions;

5) counting suspension preparation: culture tube is put into to compact centrifuge, with centrifugal 10 min of 4000 r/min, abandon supernatant liquor, with the PBS damping fluid 300 μ l of pH 7.4, wash each cell mass 2 times, each washing is all by cell suspension, then centrifugal and abandon supernatant liquor immediately, the cell mass after each washing is mixed with to 300 μ l bacteria suspensions with the PBS damping fluid respectively;

6) measure cell number; Use based on the hand-held cell counter of electrical resistance method cell counting principle and measure the cell concn of respectively managing bacteria suspension;

7) ask and calculate dyestuff EC ₅₀: the cell concn of respectively managing bacteria suspension by measured, deduct initial cell concentration, the cell concn divided by the control tube bacteria suspension, be converted into percentage ratio, is defined as the cell fission rate; The cell fission rate of same concentrations dyestuff is average, take dye strength as X-coordinate, average cell division rate be that ordinate zou is drawn, obtain a curve, by a bit (0 on ordinate zou, 50) make the parallel lines of X-axis, hand over curve in the A point, cross the parallel lines that the A point is made Y-axis, hand over X-axis in the B point, the X-coordinate numerical value that B is ordered is the EC of required dyestuff ₅₀, see Fig. 2;

8) repeating step 2) to step 7), but step 2) in the gradient dilution coefficient need be according to dyestuff EC ₅₀Scope redefines, and respectively a is rounded, and obtains L ' and H ', take the concentration range of L ' and the H ' dye solution in mensuration process again; The X-coordinate numerical value that Bl is ordered is the EC of required dyestuff ₅₀Coarse value, distribute upper and lower two concentration values of Bl to be designated as L ₁And H ₁, by line segment L ₁H ₁Be divided into 10 equal portions, observe down scale numerical value corresponding to B1, and be designated as b; Then according to formula (H ₁-L ₁) ÷ 10 * b calculates EC ₅₀Value.