CN104673877B - One mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method - Google Patents
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Abstract
The present invention relates to a mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method, this method is improved to traditional drug sensitive experiment method, change the pH value this point of culture medium using the metabolite in its growth course, invention new method, with the pH value of pH meter detection mycoplasma culture, minimum inhibitory concentration of the Chinese medicine to mycoplasma is judged by the height of pH value, and pass through the processing to condition of culture, the improvement of test tube size substitutes 96 orifice plates, in pH meter the data obtained, decline stage data first are more than or equal to () test tube data difference to 1 test tube, drug concentration in the previous test tube of the test tube, as minimum inhibitory concentration.The relatively conventional method operation of the present invention is more simple, convenient, data processing method is coordinated by accurate pH meter, make judgement of the deeper Chinese medicine of color to the minimum inhibitory concentration of mycoplasma more convenient, directly perceived, accurate, this method be particularly suitable for use in the deeper Chinese medicine of color mycoplasma MIC detection.
Description
Technical field
The present invention relates to a mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method.
Background technology
Mycoplasma is a kind of more special microorganism, and solid culture is highly difficult, even if growth, its bacterium colony also will be by
Putting microscope can just see, and because the liquid culture of mycoplasma is generation that is fully transparent, leaning in its growth course
Thank to the pH value that product changes fluid nutrient medium, and the growth feelings of mycoplasma are judged by the color change of indicator in culture medium
Condition.The judgement of its Chinese medicine drug sensitive experiment result is relatively difficult, typically using following methods：Microdilution cultivation：96 hole cells
In culture plate, decoction is first passed into a times dilution with fresh fluid nutrient medium, totally 10 dilution factors, then the bacterium of equivalent is added toward each hole
Liquid, while positive, negative control is set, 37 DEG C of cultures, until color change occurs for Positive control wells.With minimum no color change
The drug concentration in hole is set to minimal inhibitory concentration.But because the color and luster of some Chinese herbs decoctions is deeper, such as cordate houttuynia is dilute in medicine
When degree of releasing is relatively low, culture medium is caught color, often influence to visually observe, it is difficult to judge the color change of indicator, may
Experimental result is set error occur.The deeper Chinese medicine of color, addition activated carbon is adsorbed some wherein, can reduce color
Depth, but suspection can adsorb a part of active ingredient, so as to influence experimental result.Color is deep to need accurate Mlc again
When, somebody takes：To the Mlc of suspection, a certain amount of culture is taken out, is added in fresh fluid nutrient medium
Again the regular hour is cultivated, sees if there is mycoplasma growth, to judge the fungistatic effect of this concentration of this concentration.The method is time-consuming
Arduously.The MIC of chicken virus mycoplasma is detected there have been this problem, and chicken virus mycoplasma is the disease for causing fowl chronic respiratory disease
One of substance, cause great economic loss to aviculture and influence product quality.In order to improve therapeutic effect and solve clinical
The residual problem of Western medicine medicine, in-vitro screening Chinese medicine is as medicine.But because the color and luster of traditional Chinese medicine liquid is relatively deep and chicken virus mycoplasma
Growth characteristic, traditional drug sensitive experiment are difficult critical point of the accurate observation to detection liquid discoloration.
The content of the invention
The technical problem to be solved in the present invention is how to overcome the drawbacks described above of prior art, to traditional drug sensitive experiment side
Method is improved, and is changed the pH value this point of culture medium using the metabolite in its growth course, invention new method, is used pH meter
The pH value of mycoplasma culture is detected, minimum inhibitory concentration of the Chinese medicine to mycoplasma is judged by the height of pH value, there is provided one
Mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method.
In order to solve the above technical problems, this mycoplasma Chinese medicine minimum inhibitory concentration new detecting method comprises the following steps：
Step 1)：Target Chinese medicine is taken, the target Chinese medicine is gone out by decocting, concentrating the decoction for 0.5g/mL, high pressure
Bacterium；Mycoplasma culture to be checked is recovered, and prepares fluid nutrient medium；
Step 2)：To take 12 length be 7cm, the teat glass that radius is 1.5cm, after sterilizing number consecutively for " (-), 1.,
2., 3., 4., 5., 6., 7., 8., 9., 10., (+) ", be put on suitable rack for test tube；
Step 3)：The 2mL steps 1) liquid medium is added into (-) test tube；To 1., 2., 3., 4., 5., 6., 7.,
8., 9., 10., in (+) 11 test tubes respectively plus the 1mL steps 1) liquid medium；
Step 4)：The 1mL steps 1) decoction is added into 1. test tube, 1. will be moved with liquid medium test tube herb liquid
Liquid device fully mixes, and then passs and is diluted to again and 10. manages；
Step 5)：To 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 11 test tubes, it is each to add 0.8mL liquid
The mixed liquor of nutrient solution and 0.2mL bacterium solutions；
Step 6)：12 test tubes are stoppered into plug, cultivates 7~15 days and cultivates in 37 DEG C of insulating boxs under no light condition；
The pH value of culture in each pipe is determined by pH meter；
Step 7)：12 test tubes that will be handled through step 6), by accurate pH meter come determine (-), 1.~10., in (+) pipe
The pH value of culture, take record data successively；
1.~10. test tube data be the pH variation tendencies fallen after rising, find in its decline stage data, first with
(-) test tube data difference is more than or equal to -1 test tube, the drug concentration in the previous test tube of the test tube, as minimum antibacterial dense
Degree.
As further explanation, the mycoplasma culture method for resuscitation to be checked described in step 1) is the mycoplasma that will be frozen
After strain melts, by certain mass than 1:9 ratios are added in fresh nutrient solution, in 37 DEG C of cultures, you can recovery mycoplasma.
As further explanation, the fluid nutrient medium described in step 1) is improvement FregShi fluid nutrient mediums, and its raw material is matched somebody with somebody
Fang Wei, NaCl -5.0g, KCl -0.4g, MgSO4.7H2O—0.2g、Na2HPO4.12H2O—1.6g、KH2PO4- 0.2g, Portugal
Grape sugar -10g, milk protein hydrolysate -5g, dusty yeast -5g, 1 ﹪ cysteine solutions -10mL, 2 ﹪ arginine solutions -
Pink solution-the 1.0mL of 20mL, Swine serum -100mL, 1 ﹪, 100,000 units/mL penicillin -10mL, 1 ﹪ acetic acid supports solution -
10mL；Above-mentioned each composition is weighed, 1000mL is added water to and adjusts pH value to 7.6~7.8,0.22um apertures with 1.0 ﹪ mol/L NaOH
Sieving, degerming, packing, 20 DEG C of preservations of ﹣.
As further explanation, step 4) is described to pass a times dilution concrete operations and is, by the liquid medium in 1. test tube and
After nutrient solution is fully mixed with pipettor, take 1mL to move into 2. test tube, and mixed with the liquid medium in 2. test tube, then from 2.
Take 1mL to move into 3. test tube in test tube, by that analogy to 10. test tube, finally take 1mL to discard from taking in 10. test tube mixing liquid.
As specific optimization, the step 1) Chinese medicine is rheum officinale, balloonflower root, the coptis.
As specific optimization, the step 1) mycoplasma is chicken virus mycoplasma.
As specific optimization, (-) described in step 4), 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., (+) 12 test tubes
In drug concentration for respectively 0,125mg/mL, 62.5mg/mL, 31.25mg/mL, 15.625mg/mL, 7.8125mg/mL,
3.9063mg/mL、1.9531mg/mL、0.9766mg/mL、0.4883mg/mL、0.2441mg/mL、0。
An of the invention mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method, relatively conventional method operation is more simple,
It is convenient, data processing method is coordinated by accurate pH meter, makes the deeper Chinese medicine of color sentencing to the minimum inhibitory concentration of mycoplasma
Disconnected more convenient, directly perceived, accurate, this method is particularly suitable for use in the mycoplasma MIC detections of the deeper Chinese medicine of color.
Brief description of the drawings
A mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method of the invention is described further below in conjunction with the accompanying drawings：
Fig. 1 is the experimental result schematic diagram of the embodiment 1 of this mycoplasma Chinese medicine minimum inhibitory concentration new detecting method；
Fig. 2 is the experimental result schematic diagram of the embodiment 2 of this mycoplasma Chinese medicine minimum inhibitory concentration new detecting method；
Fig. 3 is the experimental result schematic diagram of the embodiment 3 of this mycoplasma Chinese medicine minimum inhibitory concentration new detecting method.
Embodiment
Embodiment 1：
Step 1)：Rheum officinale is taken, by rheum officinale by decocting, concentrating the decoction for 0.5g/mL, autoclaving；It is former to chicken poison branch
Body is recovered, and prepares fluid nutrient medium；The chicken virus mycoplasma method for resuscitation is to melt the chicken virus mycoplasma frozen
Afterwards, by certain mass than 1:9 ratios are added in fresh nutrient solution, in 37 DEG C of cultures, you can recovery mycoplasma.Described liquid
Culture medium is improvement FregShi fluid nutrient mediums, and its composition of raw materials is NaCl -5.0g, KCl -0.4g, MgSO4.7H2O—
0.2g、Na2HPO4.12H2O—1.6g、KH2PO4- 0.2g, glucose -10g, milk protein hydrolysate -5g, dusty yeast -5g,
1 ﹪ cysteine solutions -10mL, 2 ﹪ arginine solutions -20mL, Swine serum -100mL, the pink solution -1.0mL of 1 ﹪, 10
Ten thousand units/mL penicillin -10mL, 1 ﹪ acetic acid support solution -10mL；Above-mentioned each composition is weighed, adds water to 1000mL with 1.0 ﹪
Mol/L NaOH adjusts pH value to the sieving of 7.6~7.8,0.22um apertures, degerming, packing, 20 DEG C of preservations of ﹣.
Step 2)：To take 12 length be 7cm, the teat glass that radius is 1.5cm, after sterilizing number consecutively for " (-), 1.,
2., 3., 4., 5., 6., 7., 8., 9., 10., (+) ", be put on suitable rack for test tube；
Step 3)：The 2mL steps 1) liquid medium is added into (-) test tube；To 1., 2., 3., 4., 5., 6., 7.,
8., 9., 10., in (+) 11 test tubes respectively plus the 1mL steps 1) liquid medium；
Step 4)：The 1mL steps 1) decoction is added into 1. test tube, 1. will be moved with liquid medium test tube herb liquid
Liquid device fully mixes, and then passs and is diluted to again and 10. manages；It is described to pass times a dilution concrete operations and be, by the Liquid Culture in 1. test tube
After liquid and nutrient solution are fully mixed with pipettor, take 1mL to move into 2. test tube, and mixed with the liquid medium in 2. test tube, then
Take 1mL to move into 3. test tube from 2. test tube, by that analogy to 10. test tube, finally take 1mL to discard from taking in 10. test tube mixing liquid；
Step 5)：To 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 11 test tubes, it is each to add 0.8mL liquid
The mixed liquor of nutrient solution and 0.2mL bacterium solutions；So far, (-), 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., (+) 12
Drug concentration in test tube is respectively 0,125mg/mL, 62.5mg/mL, 31.25mg/mL, 15.625mg/mL, 7.8125mg/
mL、3.9063mg/mL、1.9531mg/mL、0.9766mg/mL、0.4883mg/mL、0.2441mg/mL、0。
Step 6)：12 test tubes are stoppered into plug, cultivates 7~15 days and cultivates in 37 DEG C of insulating boxs under no light condition；
The pH value of culture in each pipe is determined by pH meter；As a result it is as shown in Figure 1.
Step 7)：12 test tubes that will be handled through step 6), by accurate pH meter come determine (-), 1.~10., in (+) pipe
The pH value of culture, take record data successively；
1.~10. test tube data be the pH variation tendencies fallen after rising, find in its decline stage data, first with
(-) test tube data difference is more than or equal to -1 test tube, the drug concentration in the previous test tube of the test tube, as minimum antibacterial dense
Degree.
Embodiment 2：
Step 1)：The coptis is taken, by the coptis by decocting, concentrating the decoction for 0.5g/mL, autoclaving；It is former to chicken poison branch
Body is recovered, and prepares fluid nutrient medium；The chicken virus mycoplasma method for resuscitation is to melt the chicken virus mycoplasma frozen
Afterwards, by certain mass than 1:9 ratios are added in fresh nutrient solution, in 37 DEG C of cultures, you can recovery mycoplasma.Described liquid
Culture medium is improvement FregShi fluid nutrient mediums, and its composition of raw materials is NaCl -5.0g, KCl -0.4g, MgSO4.7H2O—
0.2g、Na2HPO4.12H2O—1.6g、KH2PO4- 0.2g, glucose -10g, milk protein hydrolysate -5g, dusty yeast -5g,
1 ﹪ cysteine solutions -10mL, 2 ﹪ arginine solutions -20mL, Swine serum -100mL, the pink solution -1.0mL of 1 ﹪, 10
Ten thousand units/mL penicillin -10mL, 1 ﹪ acetic acid support solution -10mL；Above-mentioned each composition is weighed, adds water to 1000mL with 1.0 ﹪
Mol/L NaOH adjusts pH value to the sieving of 7.6~7.8,0.22um apertures, degerming, packing, 20 DEG C of preservations of ﹣.
Step 2)：To take 12 length be 7cm, the teat glass that radius is 1.5cm, after sterilizing number consecutively for " (-), 1.,
2., 3., 4., 5., 6., 7., 8., 9., 10., (+) ", be put on suitable rack for test tube；
Step 3)：The 2mL steps 1) liquid medium is added into (-) test tube；To 1., 2., 3., 4., 5., 6., 7.,
8., 9., 10., in (+) 11 test tubes respectively plus the 1mL steps 1) liquid medium；
Step 4)：The 1mL steps 1) decoction is added into 1. test tube, 1. will be moved with liquid medium test tube herb liquid
Liquid device fully mixes, and then passs and is diluted to again and 10. manages；It is described to pass times a dilution concrete operations and be, by the Liquid Culture in 1. test tube
After liquid and nutrient solution are fully mixed with pipettor, take 1mL to move into 2. test tube, and mixed with the liquid medium in 2. test tube, then
Take 1mL to move into 3. test tube from 2. test tube, by that analogy to 10. test tube, finally take 1mL to discard from taking in 10. test tube mixing liquid；
Step 5)：To 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 11 test tubes, it is each to add 0.8mL liquid
The mixed liquor of nutrient solution and 0.2mL bacterium solutions；So far, (-), 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., (+) 12
Drug concentration in test tube is respectively 0,125mg/mL, 62.5mg/mL, 31.25mg/mL, 15.625mg/mL, 7.8125mg/
mL、3.9063mg/mL、1.9531mg/mL、0.9766mg/mL、0.4883mg/mL、0.2441mg/mL、0。
Step 6)：12 test tubes are stoppered into plug, cultivates 7~15 days and cultivates in 37 DEG C of insulating boxs under no light condition；
The pH value of culture in each pipe is determined by pH meter；As a result it is as shown in Figure 2.
Step 7)：12 test tubes that will be handled through step 6), by accurate pH meter come determine (-), 1.~10., in (+) pipe
The pH value of culture, take record data successively；
1.~10. test tube data be the pH variation tendencies fallen after rising, find in its decline stage data, first with
(-) test tube data difference is more than or equal to -1 test tube, the drug concentration in the previous test tube of the test tube, as minimum antibacterial dense
Degree.
Embodiment 3：Step 1)：Balloonflower root is taken, by balloonflower root by decocting, concentrating the decoction for 0.5g/mL, autoclaving；It is right
Chicken virus mycoplasma is recovered, and prepares fluid nutrient medium；The chicken virus mycoplasma method for resuscitation is, the chicken poison branch frozen is former
After body melts, by certain mass than 1:9 ratios are added in fresh nutrient solution, in 37 DEG C of cultures, you can recovery mycoplasma.It is described
Fluid nutrient medium be improvement FregShi fluid nutrient mediums, its composition of raw materials is, NaCl -5.0g, KCl -0.4g,
MgSO4.7H2O—0.2g、Na2HPO4.12H2O—1.6g、KH2PO4- 0.2g, glucose -10g, milk protein hydrolysate -5g,
Dusty yeast -5g, 1 ﹪ cysteine solutions -10mL, 2 ﹪ arginine solutions -20mL, Swine serum -100mL, 1 ﹪ are pink molten
Liquid -1.0mL, 100,000 units/mL penicillin -10mL, 1 ﹪ acetic acid support solution -10mL；Above-mentioned each composition is weighed, is added water to
1000mL adjusts pH value to the sieving of 7.6~7.8,0.22um apertures, degerming, packing, 20 DEG C of preservations of ﹣ with 1.0 ﹪ mol/L NaOH.
Step 2)：To take 12 length be 7cm, the teat glass that radius is 1.5cm, after sterilizing number consecutively for " (-), 1.,
2., 3., 4., 5., 6., 7., 8., 9., 10., (+) ", be put on suitable rack for test tube；
Step 3)：The 2mL steps 1) liquid medium is added into (-) test tube；To 1., 2., 3., 4., 5., 6., 7.,
8., 9., 10., in (+) 11 test tubes respectively plus the 1mL steps 1) liquid medium；
Step 4)：The 1mL steps 1) decoction is added into 1. test tube, 1. will be moved with liquid medium test tube herb liquid
Liquid device fully mixes, and then passs and is diluted to again and 10. manages；It is described to pass times a dilution concrete operations and be, by the Liquid Culture in 1. test tube
After liquid and nutrient solution are fully mixed with pipettor, take 1mL to move into 2. test tube, and mixed with the liquid medium in 2. test tube, then
Take 1mL to move into 3. test tube from 2. test tube, by that analogy to 10. test tube, finally take 1mL to discard from taking in 10. test tube mixing liquid；
Step 5)：To 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 11 test tubes, it is each to add 0.8mL liquid
The mixed liquor of nutrient solution and 0.2mL bacterium solutions；So far, (-), 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., (+) 12
Drug concentration in test tube is respectively 0,125mg/mL, 62.5mg/mL, 31.25mg/mL, 15.625mg/mL, 7.8125mg/
mL、3.9063mg/mL、1.9531mg/mL、0.9766mg/mL、0.4883mg/mL、0.2441mg/mL、0。
Step 6)：12 test tubes are stoppered into plug, cultivates 7~15 days and cultivates in 37 DEG C of insulating boxs under no light condition；
The pH value of culture in each pipe is determined by pH meter；As a result it is as shown in Figure 3.
Step 7)：12 test tubes that will be handled through step 6), by accurate pH meter come determine (-), 1.~10., in (+) pipe
The pH value of culture, take record data successively；
1.~10. test tube data be the pH variation tendencies fallen after rising, find in its decline stage data, first with
(-) test tube data difference is more than or equal to -1 test tube, the drug concentration in the previous test tube of the test tube, as minimum antibacterial dense
Degree.
Upper table is the data obtained of embodiment 1~3, understands that rheum officinale MIC is 3.9063mg/mL, balloonflower root MIC according to this method
It is 0.4883mg/mL for 3.9063mg/mL, coptis MIC.The accuracy of the data result is verified in testing again.
It can be that professional and technical personnel in the field realize or used that above-mentioned embodiment, which is intended to illustrate the present invention, to above-mentioned
Embodiment is modified and will be apparent for those skilled in the art, therefore the present invention includes but is not limited to
Above-mentioned embodiment, it is any to meet the claims or specification description, meet with principles disclosed herein and novelty,
The method of inventive features, technique, product, each fall within protection scope of the present invention.
Claims (1)
1. a mycoplasma species Chinese medicine minimum inhibitory concentration new detecting method, it is characterized in that：This method comprises the following steps：
Step 1)：Target Chinese medicine is taken, by the target Chinese medicine by decocting, concentrating the decoction for 0.5g/mL, autoclaving；It is right
Mycoplasma culture to be checked is recovered, and prepares fluid nutrient medium；Described mycoplasma culture method for resuscitation to be checked is to incite somebody to action
After the mycoplasma strain frozen melts, by certain mass than 1:9 ratios are added in fresh nutrient solution, are cultivated in 37 DEG C, you can
Recovery mycoplasma；Described fluid nutrient medium is improvement FregShi fluid nutrient mediums, and its composition of raw materials is, NaCl -5.0g,
KCl—0.4g、MgSO4·7H2O—0.2g、Na2HPO4·12H2O—1.6g、KH2PO4- 0.2g, glucose -10g, newborn egg
White hydrolysate -5g, dusty yeast -5g, 1% cysteine solution -10mL, 2 ﹪ arginine solutions -20mL, Swine serum -
Pink solution-the 1.0mL of 100mL, 1 ﹪, 100,000 units/mL penicillin -10mL, 1 ﹪ acetic acid support solution -10mL；Weigh above-mentioned
Each composition, add water to 1000mL with 1.0 ﹪ mol/L NaOH adjust pH value to the sieving of 7.6~7.8,0.22um apertures, it is degerming, point
Dress, 20 DEG C of preservations of ﹣；The Chinese medicine is rheum officinale, balloonflower root, the coptis；Mycoplasma is chicken virus mycoplasma；
Step 2)：To take 12 length be 7cm, the teat glass that radius is 1.5cm, after sterilizing number consecutively for " (-), 1., 2.,
3., 4., 5., 6., 7., 8., 9., 10., (+) ", be put on suitable rack for test tube；
Step 3)：The 2mL steps 1) liquid medium is added into (-) test tube；To 1., 2., 3., 4., 5., 6., 7., 8.,
9., 10., in (+) 11 test tubes respectively plus the 1mL steps 1) liquid medium；
Step 4)：The 1mL steps 1) decoction is added into 1. test tube, will 1. test tube herb liquid and liquid medium pipettor
Fully mix, then pass and be diluted to the again and 10. manage；It is described to pass times a dilution concrete operations and be, by the liquid medium in 1. test tube and
After nutrient solution is fully mixed with pipettor, take 1mL to move into 2. test tube, and mixed with the liquid medium in 2. test tube, then from 2.
Take 1mL to move into 3. test tube in test tube, by that analogy to 10. test tube, finally take 1mL to discard from taking in 10. test tube mixing liquid；It is described
(-), drug concentration 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 12 test tubes are respectively 0,125mg/mL,
62.5mg/mL、31.25mg/mL、15.625mg/mL、7.8125mg/mL、3.9063mg/mL、1.9531mg/mL、
0.9766mg/mL、0.4883mg/mL、0.2441mg/mL、0；
Step 5)：To 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., in (+) 11 test tubes, it is each to add 0.8mL Liquid Cultures
The mixed liquor of liquid and 0.2mL bacterium solutions；
Step 6)：12 test tubes are stoppered into plug, cultivates 7~15 days and cultivates in 37 DEG C of insulating boxs under no light condition；Pass through
PH meter determines the pH value of culture in each pipe；
Step 7)：Will through step 6) handle 12 test tubes, by accurate pH meter come determine (-), 1.~10., (+) pipe in cultivate
The pH value of thing, take record data successively；
1.~10. test tube data be the pH variation tendencies fallen after rising, find in its decline stage data, first with (-) examination
Pipe data difference is more than or equal to -1 test tube, the drug concentration in the previous test tube of the test tube, as minimum inhibitory concentration.
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Inventor after: Yang Mingcai Inventor after: Zhang Zhongyu Inventor after: Xu Bin Inventor after: Yang Mingjuan Inventor after: Sui Zhaofeng Inventor after: Han Xiutong Inventor after: Xu Jianyi Inventor after: Chi Lingzhi Inventor after: Cui Jian Inventor after: Zhang Jing Inventor before: Yang Mingcai Inventor before: Zhang Zhongyu Inventor before: Xu Bin Inventor before: Yang Mingjuan Inventor before: Sui Zhaofeng Inventor before: Xu Jianyi Inventor before: Chi Lingzhi Inventor before: Cui Jian Inventor before: Song Huizhen Inventor before: Zhang Jing |
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Granted publication date: 20171124 Termination date: 20180319 |