CN102634564A - Method for detecting toxicity of antibiotic-type substances by utilizing luminous bacteria - Google Patents
Method for detecting toxicity of antibiotic-type substances by utilizing luminous bacteria Download PDFInfo
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- CN102634564A CN102634564A CN2012101137017A CN201210113701A CN102634564A CN 102634564 A CN102634564 A CN 102634564A CN 2012101137017 A CN2012101137017 A CN 2012101137017A CN 201210113701 A CN201210113701 A CN 201210113701A CN 102634564 A CN102634564 A CN 102634564A
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Abstract
本发明提供一种用发光细菌对抗生素类物质毒性进行检测的方法：抗生素类物质与发光细菌作用时间延长至12-36个小时，优选延长至24个小时。本发明技术方案中增加抗生素类物质与发光细菌的作用时间，保证发光细菌在此期间发生增殖，该方法可以实现对抗生素生物毒性的准确表征具有较好的时效性，在保证时效性的基础上使检测结果更加准确，实现了抗生素类污染物延迟毒性的检测。
The invention provides a method for detecting the toxicity of antibiotic substances by using luminescent bacteria: the interaction time between antibiotic substances and luminescent bacteria is extended to 12-36 hours, preferably extended to 24 hours. In the technical solution of the present invention, the action time between antibiotic substances and luminescent bacteria is increased to ensure that the luminescent bacteria proliferate during this period. This method can realize accurate characterization of antibiotic biological toxicity and has better timeliness. On the basis of ensuring timeliness The detection result is more accurate, and the detection of the delayed toxicity of antibiotic pollutants is realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to photogenic bacterium detection technique field, particularly a kind of method that antibiotics material toxicity is detected with photogenic bacterium.
Background technology
Photogenic bacterium is a kind of bacterium that can send the blue-greenish colour visible light of extensive existence, and it mainly is distributed in the ocean, and perhaps free living perhaps parasitizes ocean or terrestrial animal body.
The luminescence process of photogenic bacterium is a kind of photorespiration process, is that a side on the electron transport chain is propped up, the vitamin B2 phosphate (FMNH of reduced form
2) serving as the substrate of reaction, luciferase plays katalysis, and long-chain fat aldehyde works " modification " effect of making to change the luciferase structure.Luciferase is through molecular oxygen catalysis (FMNH
2) and the oxidation of alkanoic (RCHO), this redox reaction result just produces phosphorescence, and is more between wavelength 465～490nm.
The variation of external environmental condition such as pH, temperature, oxygen concn all can influence the luminous intensity of photogenic bacterium; And deleterious chemical substance; Can influence the luminous situation of photogenic bacterium equally like heavy metal ion, microbiotic, chemotherapeutic, agricultural chemicals etc.; Therefore, the bio-toxicity that adopts photogenic bacterium to measure the antibiotics pollutent as the sensitive indicator thing is possible in research practice.
Through several generations scientist's effort, successfully extract and turn out the photogenic bacterium preparation and develop supporting with it modern optical electro-detection equipment, thereby formed sophisticated photogenic bacterium toxicity detection architecture and method.Compare with traditional bio-toxicity experiment; The photogenic bacterium toxicity test reaction times is short, highly sensitive; And owing to be the biological monitoring method with luminous quantity quantitatively characterizing contaminate environment, thereby can make rapid reaction to the variation of environment, help environmental quality is carried out comprehensive evaluation.
The photogenic bacterium acute toxicity test of standard is short because of its action time; Only be applicable to that those quick performance go out the mensuration of toxic compound; Influence the toxicity of the compound of biological growth reproductive process for measuring those, the experiment time of going through must surpass the generation time of bacterium.The antibiotics material is because of its special mechanism of action (mainly be disturb to transcribe, proteinic synthetic and reproductive process), the bio-toxicity that the possibility of result that simple establishing criteria photogenic bacterium acute toxicity test method obtains can substantially understate antibiotics pollutent.Standard toxicity test result mainly reflects the variation of cell interior energy state, and the toxicity variation in the so short time that influences cellular material synthetic compound is very little, so detected result and the real situation of unreacted.
In addition, the photogenic bacterium acute toxicity test of standard is only applicable to the detection of the hazardous and noxious substances of solubility, detailed standard is not made in detection insoluble or the slightly soluble chemical substance instructed, and makes that its range of application is limited.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of application more extensively to improve one's methods with photogenic bacterium detect antibiotics class is toxic accurately, solved in the prior art can't the detect antibiotics pollutant delayed toxicity, insoluble or slightly soluble chemical substance detected problems such as out of true.
In order to solve these problems of the prior art, technical scheme provided by the invention is: a kind of with photogenic bacterium to the method that antibiotics material toxicity detects, comprise the steps;
1) preparation microbiotic mother liquor and mother liquor is diluted to the experiment desired concn;
2) preparation photogenic bacterium lyophilized powder resuscitation fluid;
3) blank preparation: for deliquescent microbiotic, with the photogenic bacterium resuscitation fluid as blank; For the microbiotic of insoluble or microsolubility, will contain have the same amount DMSO 99.8MIN. with corresponding testing sample resuscitation fluid as blank;
4) testing sample solution preparation: the testing sample of getting different concns joins in the clean testing tube, shakes up;
5) be positioned on the testing jig blank is alternate with testing sample;
6) in each testing tube, add the photogenic bacterium lyophilized powder (regulating add-on) of recovery in order according to luminous quantity and instrument requirement, the two pipe joining days 15-30s of being separated by, vibration gently makes it even,
7) calculating of EC50 value: the logarithm with testing sample concentration is an X-coordinate, and corresponding relative luminosity factor is the ordinate zou mapping, tries to achieve its EC50 value, and promptly relative luminosity factor is in the concentration of 50% sample;
It is characterized in that, after the placement of said step 6), in each pipe, add the perfect medium of photogenic bacterium in proportion successively, antibiotics material and photogenic bacterium are extended to behind the 12-36h at water quality toxicity detector mensuration luminous quantity action time.
Preferably, said antibiotics material and photogenic bacterium extend to 24 hours action time.
Preferably, antibiotics material and photogenic bacterium are cultivated under 20-25 ℃ of condition.
Preferably, said antibiotics material comprises PCs, cephalosporins, aminoglycoside, Macrolide, tetracyclines, chloromycetin, lincosamide class.
Preferably, said antibiotics material comprises tetracyclines and sulfamido microbiotic.
Preferably, said TCs specifically comprises tsiklomitsin (CTC), terramycin (OTC) or duomycin (TC), vibra-.
Preferably, described antibiotics material is insoluble or during the slightly soluble material, in the antibiotics mother liquor of configuration, adds DMSO 99.8MIN., and add-on is no more than the per mille of TV.
The a spot of DMSO 99.8MIN. of experiment proof does not exert an influence to the luminous quantity of photogenic bacterium; And after adopting the insoluble or slightly soluble antibiotics material of dmso solution; Experimentize after more insoluble or slightly soluble antibiotics toxic material being processed solution, enlarged the range of application of photogenic bacterium.
Preferably, described insoluble or slightly soluble antibiotics material is the sulfamido microbiotic.
Preferably, described sulfamido microbiotic comprises Sulphadiazine Sodium (SD), Sulphamerazine (SM), sulfamethoxazole (SMZ).
In this article; Term " photogenic bacterium " has implication well-known to those skilled in the art; Photogenic bacterium is the one type of bacterium that under normal physiological condition, can launch visible fluorescence, and this visible fluorescence wavelength is between 450-490nm, and naked eyes are visible at the dark place.At present, the named photogenic bacterium in the whole world has following several kinds: 1. belong to the luminous different tyrothricin of having of different brevibacterium sp (Xenorhabdus) (Xenorhabdus luminescens); What 2. belong to Photobacterium (Photobacterium) has photobacterium phosphoreum (Photosbacterium phosphoreum) and an abalone luminous bacillus (P.1eiognathi); What 3. belong to Shiva Bordetella (Shewanella) has a plumage field Shiva Salmonella (Shezoanella hanedai), and also once classifying as it alternately in the past, the Hai Shi of zygosaccharomyces (Alteromonas) replaces Zymomonas mobilis (Alteromonas hanedia); What 4. belong to Vibrio (Vibrio) has Vibrio harveyi (Vibrio harveyi), beautiful vibrios biotype I (V.splendidus biotype I), Fei Shi vibrios (V.fischeri), god of fire vibrios (V.1ogei) and a vibrio orientalis (V.orientalis).Some bacterial strain in vibrio cholerae (V.cholerae) and the Mediterranean Sea vibrios (V.mediterranei) has luminescence phenomenon.
In this article; Term " microbiotic " refers to some other pathogenic micro-organism is had suppresses or one type of chemical substance of killing action is called microbiotic; Concrete kind comprises the PCs in the common beta-lactam, the class microbiotic such as Oxacyclotetradecane,erythromycin deriv of macrolide, the tetracyclines Broad spectrum antibiotics that actinomycetes produce, the Streptomycin sulphate microbiotic that streptomycete extracts, the Broad spectrum antibiotics of paraxin; Certainly the for example sulfamido, quinolones, furans, nitre imidazoles etc. that also comprise other kinds well known to a person skilled in the art and technology just repeat no more at this.
Than solution of the prior art, advantage of the present invention is:
1. increase the action time of antibiotics material and photogenic bacterium in the technical scheme of the present invention; Guarantee that photogenic bacterium breeds during this period; This method can realize having ageing preferably to the accurate sign of microbiotic bio-toxicity; Guaranteeing to make detected result more accurate on the ageing basis, realized the detection of antibiotics pollutent delayed toxicity.
2. first insoluble or slightly soluble antibiotics material in the technical scheme of the present invention with a small amount of dmso solution; Improved measuring method insoluble or slightly soluble antibiotics material, thus provide a kind of application more extensively with photogenic bacterium bio-toxicity detection method accurately.
Description of drawings
Fig. 1 is the influences of different antibiotics pollutent effect different times to the photogenic bacterium luminous quantity.
Embodiment
Below in conjunction with specific embodiment such scheme is further specified.Should be understood that these embodiment are used to the present invention is described and are not limited to limit scope of the present invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in the normal experiment.
Below will describe with form for example, if any not detailed part, can be referring to laboratory manual commonly used, and the manufacturers instruction of agents useful for same and instrument.Wherein, all chemical reagent all adopt AG, and experimental water filters through Milli-XQ; Each reagent all can obtain by commercial channel with material, and particularly: used microbiotic comprises Sulphamerazine (SM) in the experiment, SULPHAMETHOXAZOLE USP (SMZ); Sulphadiazine Sodium (SD), tetramycin hydrochloride (OTC), TETRACYCLINE HYDROCHLORIDE (TC); Isphamycin (CTC) is chromatographically pure, available from sigma company.3, and the 5-NSC 2879 (3,5-DCP), HgCl2, analytical pure is available from Aladdin reagent company.Photobacterium phosphoreum lyophilized powder (being called for short 502 lyophilized powders), the recovery diluent, the osmotic pressure regulator solution, water quality toxicity rapid detection appearance (BHP9511) is all available from Beijing Hamamatsu Technology Co., Ltd..
Embodiment
1.1 the preparation of relevant medicine
1.1.1 the configuration of detection material antibiotics mother liquor: tetracyclines dissolves with dissolved in distilled water surely, adds water after sulfamido adding DMSO 99.8MIN. (being no more than the per mille of the TV) dissolving and dissolves surely.
1.1.2 the preparation of toxicity test related concentrations: mother liquor is diluted to desired concn with zero(ppm) water.
1.1.3 photogenic bacterium perfect medium: yeast extract paste 5g/L, Tryptones 5g/L, glycerine 3ml/L, sodium-chlor 30g/L, Sodium phosphate, dibasic 5g/L, potassium hydrogenphosphate 1g/L, pH are 6.5.
1.2 toxicity test experimental procedure
1) start preheating toxicity detector 30min is subsequent use;
2) preparation microbiotic mother liquor and mother liquor is diluted to the experiment desired concn;
3) preparation of the recovery of photogenic bacterium lyophilized powder and resuscitation fluid is with reference to corresponding product regulation;
4) operating process of toxicity detector is carried out according to the operational manual of respective model instrument;
5) blank preparation: for deliquescent microbiotic, with the photogenic bacterium resuscitation fluid as blank; For the microbiotic of insoluble or microsolubility, will contain have the same amount DMSO 99.8MIN. with corresponding testing sample resuscitation fluid as blank.
6) testing sample solution preparation: the testing sample 2ml (comprising 0.3ml osmotic pressure regulator solution) that gets different concns joins in the clean testing tube, and it is subsequent use to shake up the back.
7) be positioned on the testing jig blank is alternate with testing sample.
8) in each testing tube, add the photogenic bacterium lyophilized powder (regulating add-on) that 10 μ L recover in order according to luminous quantity and instrument requirement; The two pipes 15-30s of the joining days being separated by; Vibration gently; Make it evenly, go up the mensuration luminous quantity at water quality toxicity detector (BHP9511, Beijing Hamamatsu Technology Co., Ltd.) behind the placement 30min.The 30min here is exactly the action time that is action time of standard toxicity test before improving.
9) then in each pipe in proportion (substratum/test fluid=1: 4) add perfect medium successively, measure luminous quantity at water quality toxicity detector (BHP9511, Beijing Hamamatsu Technology Co., Ltd.) after placing 20 ℃ constant incubator to cultivate 24h.
1.3 result's processing
1.3.1 the calculating of relative luminosity factor
Formula: relative luminosity factor=testing sample luminous quantity/blank luminous quantity * 100%
1.3.2EC50 the calculating of value
Logarithm with testing sample concentration is an X-coordinate, and corresponding relative luminosity factor is the ordinate zou mapping, tries to achieve its EC50 value (relative luminosity factor is in the concentration of 50% sample).
The bio-toxicity that the photogenic bacterium acute toxicity test is used to monitor waste water and compound has obtained using very widely.With photogenic bacterium as the mensuration result of the acute toxicity of study subject and delayed toxicity shown in Fig. 1 and table 1.Fig. 1 (a) and (b) reflected that respectively sulfamido and TCs are to the inhibition of photogenic bacterium influence.No matter can be known by figure, be sulfamido or tetracyclines, and the delayed toxicity result of 24h compares with the standard acute toxicity result of 0.5h, and simulation curve all is moved to the left, and promptly the EC50 value reduces; And every kind of degree difference that microbiotic is moved to the left, i.e. the degree of toxicity increase is different.Can know by Fig. 1 (a) and table 1, among the standard photogenic bacterium toxicity test result (0.5h), three kinds of antibiotic EC50 of sulfamido, the ascending order of 0.5h value is SD＜SMZ＜SM, relevant numerical is respectively 62,73 and 104mg/L; But EC50 among the delayed toxicity result, the ascending order of 24h value is SMZ＜SD＜SM, and relevant numerical is respectively 0.0023,1.57 and 5.69mg/L, and the three kinds of antibiotic acute and chronic EC50 of sulfamido ratios are respectively 32119,40 and 19.Hence one can see that; The prolongation bio-toxicity test duration not only causes the EC50 value of test-compound that great change has taken place; Simultaneously antibiotic bio-toxicity order has also been produced obvious influence; Wherein the maximum of the toxicity of SMZ increase explains that SMZ is the most obvious to the delay phenomenon of the bio-toxicity of photogenic bacterium.
Equally, can know by Fig. 1 (b) and table 1, among the standard photogenic bacterium toxicity test result (0.5h), the EC50 of three kinds of TCses, the ascending order of 0.5h value is CTC＜OTC＜TC, relevant numerical is respectively 0.034,0.068 and 37mg/L; But EC50 among the delayed toxicity result, the ascending order of 24h value is CTC＜TC＜OTC, the acute and chronic EC50 ratio of three kinds of TCses is respectively 31,18586 and 18.Wherein the degree of TC toxicity increase is maximum, and the toxicity delayed effect is the most obvious.
The EC that table 1 antibiotics pollutent suppresses photogenic bacterium
50Value
Tab.1?EC
50?value?of?the?bioluminescence?inhibition?assay
In addition, can be known by table 1 result that photogenic bacterium is to 3 of the toxicity reference substance, the EC50 value of 5-DCP is not obvious over time, and EC50,0.5h and EC50,24h are respectively 2.66 and 2.20mg/L.The reason that this species diversity occurs maybe be with to be tried toxic effect of chemical mechanism and TET relevant.The acute photogenic bacterium experiment of standard only is applicable to that those show the mensuration of toxic compound very soon, influences the toxicity of the compound of biological growth reproductive process for measuring those, and the prerequisite that must increase is the generation time that experiment lasts needs to surpass bacterium.Therefore according to acute toxicity data extrapolation chronic toxicity may substantially understate antibiotics pollutent bio-toxicity; The unusual big reason of acute and chronic EC50 ratio occurring maybe be relevant with the special mechanism of action of antibiotics material, they mainly be disturb transcribe, proteinic synthetic and reproductive process.0.5h the toxicity test result mainly reflects the variation of cell interior energy state; The toxicity variation in the so short time that influences cellular material synthetic compound is very little, therefore prolongs experimental period and guarantees that propagation takes place bacterium is that this method need improved important content.This shows that the toxicity that photogenic bacterium standard acute toxicity method possibly cause the antibiotics pollutent is by substantially understate.
Above-mentioned instance only is explanation technical conceive of the present invention and characteristics, and its purpose is to let the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (9)
- One kind with photogenic bacterium to the method that antibiotics material toxicity detects, comprise the steps;1) preparation microbiotic mother liquor and mother liquor is diluted to the experiment desired concn;2) preparation photogenic bacterium lyophilized powder resuscitation fluid;3) blank preparation: for deliquescent microbiotic, with the photogenic bacterium resuscitation fluid as blank; For the microbiotic of insoluble or microsolubility, will contain have the same amount DMSO 99.8MIN. with corresponding testing sample resuscitation fluid as blank;4) testing sample solution preparation: the testing sample of getting different concns joins in the clean testing tube, shakes up;5) be positioned on the testing jig blank is alternate with testing sample;6) in each testing tube, add the photogenic bacterium lyophilized powder (regulating add-on) of recovery in order according to luminous quantity and instrument requirement, the two pipe joining days 15-30s of being separated by, vibration gently makes it even,7) calculating of EC50 value: the logarithm with testing sample concentration is an X-coordinate, and corresponding relative luminosity factor is the ordinate zou mapping, tries to achieve its EC50 value, and promptly relative luminosity factor is in the concentration of 50% sample;It is characterized in that, after the placement of said step 6), in each pipe, add the perfect medium of photogenic bacterium in proportion successively, antibiotics material and photogenic bacterium are extended to behind the 12-36h at water quality toxicity detector mensuration luminous quantity action time.
- 2. according to claim 1 a kind of with photogenic bacterium to the method that antibiotics material toxicity detects, it is characterized in that said antibiotics material and photogenic bacterium extend to 24 hours action time.
- 3. toxic the improving one's methods of photogenic bacterium detect antibiotics class material according to claim 1 is characterized in that antibiotics material and photogenic bacterium are cultivated under 20-25 ℃ of condition.
- 4. the method that antibiotics material toxicity is detected with photogenic bacterium according to claim 1; It is characterized in that said antibiotics material comprises PCs, cephalosporins, aminoglycoside, Macrolide, tetracyclines, chloromycetin, lincosamide class.
- 5. according to claim 1 with photogenic bacterium to the method that antibiotics material toxicity detects, it is characterized in that said antibiotics material is tetracyclines and sulfamido microbiotic.
- 6. according to claim 5 with photogenic bacterium to the method that antibiotics material toxicity detects, it is characterized in that said TCs specifically comprises tsiklomitsin CTC, terramycin OTC, duomycin TC, vibra-.
- 7. the method that antibiotics material toxicity is detected with photogenic bacterium according to claim 1; It is characterized in that; Described antibiotics material is insoluble or during the slightly soluble material, in the antibiotics mother liquor of configuration, adds DMSO 99.8MIN., and add-on is no more than the per mille of TV.
- 8. according to claim 7 with photogenic bacterium to the method that antibiotics material toxicity detects, it is characterized in that described insoluble or slightly soluble antibiotics material is the sulfamido microbiotic.
- According to claim 5 or 8 described with photogenic bacterium to the method that antibiotics material toxicity detects, it is characterized in that described sulfamido microbiotic is Sulphadiazine Sodium SD, Sulphamerazine SM, sulfamethoxazole SMZ.
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