CN105132515A - Experiment method for determining toxicity of Bacillus subtilis - Google Patents

Abstract

The invention belongs to the technical field of environmental pollution detection, and relates to an experiment method for determining the toxicity of Bacillus subtilis. The method comprises the following steps: 1, carrying out bacterial strain recovery and passage; 2, carrying out working bacterium liquid culture and balancing; 3, testing the toxicity of the balanced bacterium liquid obtained in step 2 by using a compound; and 4, detecting the OD620 of a sample by using ELIASA 12h after a toxicity reaction, and characterizing the toxicity by adopting the inhibition rate as an index. The experiment method has the advantages of fast sample detection, simple operation, few environment interferences, difficult pollution by competitors, tiny amount of the sample to be detected, saving of experiment consumables, and reduction of result errors brought by manual operation.

Description

A kind of experimental technique measuring bacillus subtilis mushroom toxin

Technical field

The invention belongs to environment pollution detection technical field, relate to a kind of test method measuring bacillus subtilis mushroom toxin.

Background technology

The biological effect of carrying out compound under laboratory condition detects, and the environmental emission standard that can be compound provides scientific basis.Because the microorganism growth cycle is short, testing method is simple, and testing cost is low, has again the physicochemical property similar with higher organism and enzyme mechanism, and the toxotest method therefore using microorganism as biological subject has a wide range of applications at environmental area.

Subtilis (Bacillussubtilis) is extensively present in the body surface of soil, lake, ocean and animal and plant body, self is not pathogenic, only there is monolayer cell adventitia, free from environmental pollution to person poultry harmless, there is high temperature resistant, the characteristic such as acid and alkali-resistance and anti-extrusion, at present by the type strain as gram-positive microorganism toxotest, be widely used in the toxicity detection [1-3] of classes of compounds.

Subtilis toxotest not yet has unified standard method, current common method [4-7] mainly contains: (1) colony counting method, namely the principle of a bacterium colony can be grown according to each viable bacteria, different bacteria suspension is carried out slat chain conveyor, calculates the viable count in culture according to the colony number grown; (2) inhibition zone assay method (agar diffusion paper disk method), coats bacteria suspension on agar plate according to mensuration and cultivates rear colony diameter size deterministic compound to the suppression of microorganism growth; (3) turbidimetry, measuring principle be in bacteria suspension bacterial number and bacterium liquid turbidity linear; (4) spectrophotometry, its principle and turbidimetry similar, according to bacterial number in bacteria suspension and bacterium liquid light absorption value linear, by measuring bacterium liquid light absorption value and then drawing the quantity of viable bacteria in bacterium liquid.

Above subtilis toxicity test method comparatively generally uses at present, but all there is certain defect.As colony counting method, choose the flat board of colony number between 30 ~ 300 count, too much or very few all inaccurate, and this method is only limitted to measure and forms the microorganism of bacterium colony, during operational cost and resultant error is larger; Inhibition zone assay method requires higher to experiment aseptic technique, measures colony diameter error larger; Turbidimetry and spectrophotometry instantaneity poor, complex operation, required sample size is large, and experiment reproducibility is comparatively difficult.

At environmental area, the toxic effect research that compound on organism is carried out in laboratory has become compound ecological risk assessment and has formulated the important scientific basis of environmental standard.Subtilis is as the model animals of gram-positive microorganism, and Chang Zuowei model animals detects for the toxic effect of classes of compounds, but not yet forms standard method for the toxotest of subtilis at present.Therefore, a kind of subtilis toxotest method need be set up to realize: (1) is few by environmental factors interference, not easily bacteria infection; (2) detect fast, simple to operate, save working hour and space; (3) sample size is reduced to save experiment material; (4) resultant error etc. because manual operation brings is reduced.

Summary of the invention

The object of the invention is to the defect for overcoming above-mentioned prior art and a kind of experimental technique measuring bacillus subtilis mushroom toxin is provided.

For achieving the above object, the present invention adopts following technical scheme:

In the present invention, adopt microplate reader to detect sample, its measuring principle is based upon on material absorbing spectrum and visible ray Colorimetric techniques basis.

Measure an experimental technique for bacillus subtilis mushroom toxin, it comprises the following steps:

(1) bacterial classification is recovered and is gone down to posterity;

(2) work the cultivation of bacterium liquid and balance

(3) the balance bacterium liquid of compound to step (2) carries out toxotest;

(4) after toxic reaction, test samples OD620 carries out Toxicological Characterization.

Bacterial classification in described step (1) is recovered and is gone down to posterity

Solid slant culture base carries out, is dissolved by subtilis lyophilized powder (purchased from culture presevation management committee of Microbe Inst., Chinese Academy of Sciences), being inoculated into, inclined-plane goes down to posterity obtains the bacterial strain F of stable growth for 3 times afterwards ₃, be placed in 4 DEG C of Refrigerator stores for subsequent use;

Wherein, solid slant culture based formulas is: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g, NaCl5.0g, distilled water 1.0L, agar 15.0g; Described solid medium 1mol/LNaOH adjusts pH to 7.0.

Work bacterium liquid in described step (2) is cultivated and balance, specifically comprises the following steps:

(2a) training bacterium liquid

F will be passaged to ₃bacterial strain transfering loop be inoculated in 5mL liquid nutrient medium, be 37 DEG C in temperature, under incubator rotating speed 180r/min condition cultivate 6 ~ 8h, using the work bacterium liquid as toxotest;

Liquid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g, NaCl5.0g, distilled water 1.0L; Described liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0;

(2b) balancing work bacterium liquid

According to 100,000/ the work bacterium liquid of amount of dilution removing step (2a) in sterilized water, controlling work bacterium liquid bacterial count after dilution is 10 ³individual/mL, magnetic stirrer 40min, stablize to make bacterium liquid.

Toxicity of compound reaction in described step (3) is carried out in 96 hole microwell plates, specifically comprises the following steps:

96 orifice plate Loading sequence are set, controlling every hole overall accumulated amount of application of sample is 200 μ L, namely 80 μ L test compounds are (according to the compound different concns gradient of preparation compound mother liquid concentration and setting, the compound solution of different volumes is added in orifice plate, 80 μ L are supplemented to sterilized water less than 80 μ L), the balance bacterium liquid of 80 μ L high power liquid nutrient mediums and 40 μ L steps (2b).

Described high power substratum (2.5 times) is for ensureing normal nutrition amount needed for thalli growth in chronic toxicity (12h) process.

High power liquid culture based formulas: extractum carnis 12.5g, peptone 25.0g, yeast extract 12.5g, glucose 12.5g, NaCl12.5g, distilled water 1.0L; High power liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0.

96 described orifice plate Loading sequence are:

Make a circle outward and add 200 μ L sterilized waters to prevent orifice plate rims effect;

2nd is classified as blank group (120 μ L sterilized waters, 80 μ L high power liquid nutrient mediums), to record substratum substrate OD under same culture conditions ₆₂₀, thus obtain the absolute inhibiting rate of compound to thalli growth;

3rd, 7,11 are classified as control group (80 μ L sterilized waters, 80 μ L high power liquid nutrient mediums, 40 μ L work bacterium liquid), characterize same culture conditions hypothallus normal growth amount;

4th, 5,6 and 8,9,10 row are respectively 12 compound concentration gradient, and each concentration gradient 3 is parallel.

Described toxic reaction, namely application of sample carries out mixing 1000r/min speed concussion 1min after terminating, and to make system in 96 orifice plates even, then 96 orifice plates being put into isothermal vibration incubator, is 37 DEG C in temperature, and incubator rotating speed is cultivate 12h under 180r/min condition.

Toxicological Characterization in described step (4), specifically comprises the following steps: step (3) sample takes out after cultivating 12h, detects its optical density value (OD by microplate reader ₆₂₀), compound to the toxicity size of subtilis with compound to subtilis absorb light OD ₆₂₀inhibiting rate characterize, be calculated as follows:

Inhibiting rate (%)=[1-(sample OD ₆₂₀-blank OD ₆₂₀)/(contrast OD ₆₂₀-blank OD ₆₂₀)] x100% formula (1)

Wherein, OD is contrasted ₆₂₀refer to the blank OD before and after sample ₆₂₀mean value.

The method that the present invention sets up is the basic skills of subtilis toxotest, is applicable to various pollutent and measures the toxic action of subtilis.

The present invention compared with prior art, has the following advantages and beneficial effect:

1. enzyme-linked immunosorbent assay is due to simple to operate, highly sensitive, and the features such as high specificity widely use at environmental area.Microplate reader is as the conventional instrument of enzyme-linked immunoassay, and measuring principle is based on material material absorbing spectrum and visible ray colorimetric basis, simple, convenient, and reduce the error that in experimental implementation, artificial reading causes, experimental repeatability is good; Sample detection speed is fast, once can detect multiple sample, saves the operating time; Experimental result is directly stored in computer, and Data Processing in Experiment is convenient;

2. use 96 hole microwell plates to measure compound to the toxicity of subtilis, practical sample trace, saves the consumption of experiment material and experiment reagent, reduces experimental cost., directly measure after cultivating, environment is little to sample interference, not easily by living contaminants meanwhile.

Accompanying drawing explanation

Fig. 1 is the schema of the embodiment of the present invention.

Fig. 2 is that the embodiment of the present invention 96 hole microwell plate application of sample arranges schematic diagram.

Fig. 3 is that pyrocatechol and m-nitrophenol are to the chronic toxicity effect figure of subtilis.Wherein: (1) for pyrocatechol is to the chronic toxicity effect of subtilis, EC ₅₀value is 396mg/L (adopting spectrophotometric determination result in document for being 437mg/L); (2) for m-nitrophenol is to the chronic toxicity effect of subtilis, EC ₅₀value is 440mg/L (adopting ultraviolet-visible spectrophotometer measurement result to be 430mg/L in document).

Fig. 4 measures the chronic toxicity effect result figure of 5 kinds of sulfa antibiotics to subtilis for applying institute's establishment method, wherein: (1), for sulfapyridine (SPY) is to the chronic toxicity effect figure of subtilis, EC50 value is 51.5nmol/L;

(2) for cistosulfa (SCP) is to the chronic toxicity effect figure of subtilis, EC50 value is 6.78nmol/L;

(3) for sulfamethoxazole (SMX) is to the chronic toxicity effect figure of subtilis, EC50 value is 57.3nmol/L;

(4) for sulfanilamide (SN) Sulfafurazole (SSZ) is to the chronic toxicity effect figure of subtilis, EC50 value is 9.57nmol/L;

(5) for sulphormethoxine (SFD) is to the chronic toxicity effect figure of subtilis, EC50 value is 33.1nmol/L.

Embodiment

The present invention is further illustrated below in conjunction with embodiment.

Embodiment 1

Use 96 hole microwell plates to measure an experimental technique for bacillus subtilis mushroom toxin, it comprises the following steps (as shown in Figure 1):

(1) bacterial classification is recovered and is gone down to posterity, and specifically comprises the following steps:

Bacterial classification recovery and going down to posterity is carried out on solid slant culture base;

Solid slant culture based formulas: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g,

NaCl5.0g, distilled water 1.0L, agar 15.0g

Solid medium 1mol/LNaOH adjusts pH to 7.0.

Dissolved by subtilis lyophilized powder (purchased from culture presevation management committee of Microbe Inst., Chinese Academy of Sciences), being inoculated into, inclined-plane goes down to posterity obtains the bacterial strain F of stable growth for 3 times afterwards ₃, be placed in 4 DEG C of Refrigerator stores for subsequent use;

(2) the bacterium liquid that works is cultivated and balance, specifically comprises the following steps:

(2a) the bacterium liquid that works is cultivated

F will be passaged to ₃bacterial strain transfering loop be inoculated in 5mL liquid nutrient medium, be 37 DEG C in temperature, under incubator rotating speed 180r/min condition cultivate 6 ~ 8h, using the work bacterium liquid as toxotest;

Liquid culture based formulas: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g,

NaCl5.0g, distilled water 1.0L;

Liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0.

(2b) the bacterium liquid that works balances

According to 100,000/ amount of dilution removing step (1) work bacterium liquid in sterilized water, controlling work bacterium liquid bacterial count after dilution is 10 ³individual/mL, magnetic stirrer 40min, stablize to make bacterium liquid.

(3) toxicity of compound reaction, specifically comprises the following steps:

(as shown in Figure 2, W represents sterilized water, namely makes a circle outside 96 orifice plates and adds 200 μ L sterilized waters respectively to prevent fringing effect to arrange 96 orifice plate Loading sequence; B represents blank group, namely adds 120 μ L sterilized waters, 80 μ L high power liquid nutrient mediums, to record substratum substrate OD under same culture conditions ₆₂₀, thus obtain the absolute inhibiting rate of compound to thalli growth; Ctrl represents control group, namely adds 80 respectively _μl sterilized water, 80 μ L high power liquid nutrient mediums, 40 μ L work bacterium liquid, characterize same culture conditions hypothallus normal growth amount; C ₁-C ₁₂be respectively 12 compound concentration gradient, each concentration gradient 3 is parallel.), controlling every hole overall accumulated amount of application of sample is 200 μ L, namely 80 μ L test-compounds are (according to the compound different concns gradient of preparation compound mother liquid concentration and setting, the compound solution of different volumes is added in orifice plate, 80 μ L are supplemented to sterilized water less than 80 μ L), the balance bacterium liquid of 80 μ L high power liquid nutrient mediums and 40 μ L steps (2b).

96 orifice plate Loading sequence, namely outer making a circle adds 200 μ L sterilized waters to prevent orifice plate rims effect, and second is classified as blank group (120 μ L sterilized waters, 80 μ L high power liquid nutrient mediums), to record substratum substrate OD under same culture conditions ₆₂₀, thus obtain the absolute inhibiting rate of compound to thalli growth; 3,7,11 are classified as control group (80 μ L sterilized waters, 80 μ L high power liquid nutrient mediums, 40 μ L work bacterium liquid), characterize same culture conditions hypothallus normal growth amount; 4,5,6 and 8,9,10 row are respectively 12 compound concentration gradient, and each concentration gradient 3 is parallel.

High power substratum (2.5 times) is for ensureing normal nutrition amount needed for thalli growth in chronic toxicity (12h) process.

High power liquid culture based formulas: extractum carnis 12.5g, peptone 25.0g, yeast extract 12.5g, glucose 12.5g,

NaCl12.5g, distilled water 1.0L;

High power liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0.

Toxic reaction, namely application of sample terminates rear mixing elf 1000r/min speed concussion 1min, and to make system in 96 orifice plates even, then 96 orifice plates being put into isothermal vibration incubator, is 37 DEG C in temperature, and incubator rotating speed is cultivate 12h under 180r/min condition.

(4) compound is to the Toxicological Characterization of subtilis, specifically comprises the following steps:

Step (3) sample takes out after cultivating 12h, and microplate reader detects its optical density value (OD ₆₂₀), compound to the toxicity size of subtilis with compound to subtilis absorb light OD ₆₂₀inhibiting rate characterize, be calculated as follows:

Inhibiting rate (%)=[1-(sample OD ₆₂₀-blank OD ₆₂₀)/(contrast OD ₆₂₀-blank OD ₆₂₀)] x100% formula (1)

Wherein, OD is contrasted ₆₂₀refer to the blank OD before and after sample ₆₂₀mean value.

Embodiment 2:

The method utilizing embodiment 1 to provide is to pyrocatechol and ask the toxotest of nitrophenols to subtilis

Document [1] adopts microcalorimetry to record the half-inhibition concentration (EC of pyrocatechol to subtilis ₅₀) value is 437mg/L, document [3] adopts Nephelometric Determination to ask the half-inhibition concentration (EC of nitrophenols to subtilis ₅₀) value is 430mg/L.96 orifice plates adopting this invention to set up measure pyrocatechol respectively, ask that nitrophenols is to subtilis dose-effect curve, draw pyrocatechol and ask the chronic toxicity EC of nitrophenols to subtilis ₅₀value is respectively 396mg/L, 440mg/L (as shown in Fig. 3 (1) and Fig. 3 (2)).

Embodiment 3:

The method utilizing embodiment 1 to provide to test sulfa antibiotics to the toxic action of subtilis (as shown in Fig. 4 (1) ~ (5), wherein, Fig. 4 (1) is for SPY is to subtilis 12h toxicity dose-effect curve; Fig. 4 (2) is for SCP is to subtilis 12h toxicity dose-effect curve; Fig. 4 (3) is for SMX is to subtilis 12h toxicity dose-effect curve; Fig. 4 (4) is for SSZ is to subtilis 12h toxicity dose-effect curve; Fig. 4 (5) is for SFD is to subtilis 12h toxicity dose-effect curve).

Table 1 experiment microbiotic

Be called for short	Chinese	Chemical formula	No. CAS	Molecular weight	Purity (%)
						SPY	Sulfapyridine	C 11H 11N 3O 2S	144-83-2	249.30	≥99.0
SMX	Sulfamethoxazole	C 10H 11N 3O 3S	723-46-6	253.30	≥99.0
						SSZ	Sulfanilamide (SN) Sulfafurazole	C 11H 13N 3O 3S	127-69-5	267.31	≥99.0
SCP	Cistosulfa	C 10H 9ClN 4O 2S	80-32-0	284.72	≥99.0
						SFD	Sulphormethoxine	C 12H 14N 4O 4S	2447-57-6	310.33	≥99.0

Experiment records the half-inhibition concentration EC of 5 kinds of sulfa antibiotics to the chronic toxicity (12h) of subtilis ₅₀all in 100nmol/L scope, toxicity size is roughly from high to low: SMX > SPY > SFD > SSZ > SCP.

Above-mentioned is can understand for ease of those skilled in the art and apply this model to the description of example.Person skilled in the art obviously easily can make various amendment to these embodiments, and one principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to embodiment here, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.

Reference

1.HuilunChen,JunYao,FeiWang,etal.Toxicityofthreephenoliccompoundsandtheirmixturesonthegram-positivebacteriaBacillussubtilisintheaquaticenvironment.ScienceoftheTotalEnvironment.2010,408(5):1043-1049

2. Li Shuying, Su Yali, Zhou Yuanqing etc., heavy metal stress cultivates the impact on 2 kinds of growths curve of bacteria. Agriculture of Anhui science, 2011,39 (1): 443-446

3. the scape body river in Jiangsu Province which flows into the Huangpu River of Shanghai, Xu Jingbo, oil of mirbane is analyzed intestinal bacteria and the growth inhibiting structure effect of subtilis. Jilin University's journal, 2004,42 (1): 130-134

4. a treasured is apt to, and Wang Jun, Chen Jinping etc., garlic juice is to the inhibiting research of subtilis. northwest Botany Gazette,

2009,29(6)：1269-1275

5. Wang Yu Na, Geng Huimin, Li Chunge, 4 kinds of microbiotic are to the external associating inhibition test of subtilis. Henan Agricultural Sciences, 2012,41 (8): 177-179

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Claims (8)

1. measure a method for bacillus subtilis mushroom toxin, it is characterized in that: it comprises the following steps:

(1) bacterial classification is recovered and is gone down to posterity;

(2) work the cultivation of bacterium liquid and balance;

(3) the balance bacterium liquid of compound to step (2) carries out toxic reaction test;

(4) after toxic reaction, test samples OD620 carries out Toxicological Characterization.

2. method according to claim 1, it is characterized in that: the recovery of bacterial classification in described step (1) to be carried out with going down to posterity on solid slant culture base, dissolved by subtilis lyophilized powder, being inoculated into, inclined-plane goes down to posterity obtains the bacterial strain F of stable growth for 3 times afterwards ₃, be placed in 4 DEG C of Refrigerator stores for subsequent use.

3. method according to claim 2, is characterized in that: described solid slant culture based formulas is: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g, NaCl5.0g, distilled water 1.0L, agar 15.0g; Described solid medium 1mol/LNaOH adjusts pH to 7.0.

4. method according to claim 1, is characterized in that: the work bacterium liquid in described step (2) is cultivated and balance, comprises the following steps:

(2a) training bacterium liquid

F will be passaged to ₃bacterial strain transfering loop be inoculated in 5mL liquid nutrient medium, be 37 DEG C in temperature, under incubator rotating speed 180r/min condition cultivate 6 ~ 8h, using the work bacterium liquid as toxotest;

Wherein, liquid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, yeast extract 5.0g, glucose 5.0g, NaCl5.0g, distilled water 1.0L; Described liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0;

(2b) balancing work bacterium liquid

According to 100,000/ the work bacterium liquid of amount of dilution removing step (2a) in sterilized water, controlling work bacterium liquid bacterial count after dilution is 10 ³individual/mL, magnetic stirrer 40min, stablize to make bacterium liquid.

5. method according to claim 1, is characterized in that: the toxicity of compound reaction in described step (3) is carried out in 96 hole microwell plates, comprises the following steps:

Arrange 96 orifice plate Loading sequence, controlling every hole overall accumulated amount of application of sample is 200 μ L, comprising the balance bacterium liquid of 80 μ L test compounds, 80 μ L high power liquid nutrient mediums and 40 μ L steps (2b);

Wherein, high power liquid culture based formulas is: extractum carnis 12.5g, peptone 25.0g, yeast extract 12.5g, glucose 12.5g, NaCl12.5g, distilled water 1.0L; High power liquid nutrient medium 1mol/LNaOH adjusts pH to 7.0.

6. method according to claim 5, is characterized in that: the Loading sequence of described 96 orifice plates is:

Outer the making a circle of 96 orifice plates adds 200 μ L sterilized waters to prevent orifice plate rims effect,

2nd is classified as blank group comprises 120 μ L sterilized waters and 80 μ L high power liquid nutrient mediums, to record substratum substrate OD under same culture conditions ₆₂₀, thus obtain the absolute inhibiting rate of compound to thalli growth;

3rd, 7,11 are classified as control group comprises 80 μ L sterilized waters, 80 μ L high power liquid nutrient mediums and 40 μ L work bacterium liquid, characterizes same culture conditions hypothallus normal growth amount;

4th, 5,6 and 8,9,10 row are respectively 12 compound concentration gradient, and each concentration gradient 3 is parallel.

7. method according to claim 1, it is characterized in that: described toxic reaction is: application of sample terminates rear 1000r/min speed mixing 1min, to make system even, then 96 orifice plates are put into isothermal vibration incubator, be 37 DEG C in temperature, incubator rotating speed is cultivate 12h under 180r/min condition.

8. method according to claim 1, is characterized in that: the Toxicological Characterization in described step (4), comprises the following steps: take out after the sample of step (3) is cultivated 12h, detect its optical density value OD by microplate reader ₆₂₀, compound to the toxicity size of subtilis with compound to subtilis absorb light OD ₆₂₀inhibiting rate characterize, be calculated as follows:

Inhibiting rate (%)=[1-(sample OD ₆₂₀-blank OD ₆₂₀)/(contrast OD ₆₂₀-blank OD ₆₂₀)] x100%

Wherein, OD is contrasted ₆₂₀refer to the blank OD before and after sample ₆₂₀mean value.