CN106085833A - Microorganism metering device and metering method thereof in water - Google Patents

Microorganism metering device and metering method thereof in water Download PDF

Info

Publication number
CN106085833A
CN106085833A CN201610376306.6A CN201610376306A CN106085833A CN 106085833 A CN106085833 A CN 106085833A CN 201610376306 A CN201610376306 A CN 201610376306A CN 106085833 A CN106085833 A CN 106085833A
Authority
CN
China
Prior art keywords
microorganism
water
sampling rod
absorbent resin
super absorbent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610376306.6A
Other languages
Chinese (zh)
Other versions
CN106085833B (en
Inventor
周应科
侯金亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
North China University of Science and Technology
Original Assignee
North China University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by North China University of Science and Technology filed Critical North China University of Science and Technology
Priority to CN201610376306.6A priority Critical patent/CN106085833B/en
Publication of CN106085833A publication Critical patent/CN106085833A/en
Application granted granted Critical
Publication of CN106085833B publication Critical patent/CN106085833B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/48707Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48735Investigating suspensions of cells, e.g. measuring microbe concentration
    • G01N2015/1024

Abstract

The invention discloses microorganism metering device and metering method thereof in water, including supply port, guidance panel, groove, load position, microorganism metering is held, lower vessel portion divides, upper vessel portion divides, thin film, measure solution, agitator, measure electrode, negative electrode, anelectrode, microorganism-collecting terminal, sampling rod, anti-tampering cap, super absorbent resin, magnet, rotor, motor, supply port one end is connected to negative electrode and anelectrode, when the other end connects power source, negative polarization and positive polarization is there is respectively in the microorganism measured in liquid on negative electrode and anelectrode, regulated microorganism-collecting voltage and microorganism metered voltage alternately by guidance panel simultaneously;Microorganism metering device makes microorganism polarize by applied voltage, making microorganism be fallen in microorganism-collecting terminal by zigzag electrode to measure, micro organism quantity directly displays out by digital display panel, simple to operate, being susceptible to external interference, accuracy is high.

Description

Microorganism metering device and metering method thereof in water
Technical field
The present invention relates to microorganism metering field, more particularly, it relates to microorganism metering device and metering side thereof in water Method.
Background technology
Water is Source of life, is the natural surroundings that is widely present of microorganism, all contains a number of in various natural waters Microorganism.The metering of microorganism in water, is ensureing the safety of water source and is controlling to have in infectious disease extremely important meaning, be also simultaneously The important indicator of evaluating water quality situation.
In water, the metering of microorganism, has colony counting method, will testing sample after suitably dilution, microorganism therein Fully dispersed one-tenth individual cells, takes a certain amount of diluted sample liquid and is applied on flat board, through cultivating, numerous by each single cells grown Growing and form macroscopic bacterium colony, should represent in raw sample one of i.e. one single bacterium colony is unicellular, adds up clump count, according to Its extension rate and sampling inoculum concentration can converse in sample containing bacterium number.Secondly also have a kind of filter membrane method, will appropriate well Filter put into by the filter membrane in footpath, filtered sample, owing to microorganism is retained on the surface of the film by the effect of filter membrane, and microorganism in sample Growth inhibitor can remove with aseptic water washing filter after filtration, then, filter membrane is placed in culture medium cultivation, nutrient Being swapped by the micropore of filter membrane with metabolite, the bacterium colony turned out on filter membrane surface can count, and and sample size phase Close.
The shortcoming of above method is: the longest, and complex operation is vulnerable to environmental pollution, and the result obtained differs and fixes Really, extension rate is the most, and accuracy is the poorest, it is impossible to be accurately obtained microorganism particular number.
Summary of the invention
It is an object of the invention to provide microorganism metering device and metering method thereof in water, to solve above-mentioned background technology The problem of middle proposition.
For achieving the above object, the present invention provides following technical scheme: microorganism metering device and metering method thereof in water, Including supply port, guidance panel, digital display panel, groove and loading position, described bottom portion of groove includes motor, turns Son and magnet, be used for placing microorganism measuring container, and described microorganism measuring container is divided into upper vessel portion to divide by thin film and holds Device lower part, wherein upper vessel portion demultiplexing is in placing measurement solution, and by diaphragm seal, lower vessel portion divides and includes agitator and survey Amount electrode, measures solution and includes that pure water, thin film include aluminium foil, measures electrode and includes microorganism acquisition terminal, negative electrode and just Electrode, described sampling rod includes push-pull device at fixed, protection water sample cover, prevents the water sample that super absorbent resin collects by extraneous ring The impact in border, described sampling rod one end is surrounded by super absorbent resin, is wherein surrounded by one end of super absorbent resin for gathering Water sample, inserts in microorganism measuring container, and the cap aperture of anti-tampering cap does not has one end of super absorbent resin through sampling rod, places In loading position, cover microorganism measuring container completely;
Supply port one end is connected to negative electrode and anelectrode, when the other end connects power source, and the microorganism measured in liquid exists There is negative polarization and positive polarization on negative electrode and anelectrode respectively, regulated microorganism-collecting voltage and micro-by guidance panel simultaneously Alternately, wherein, described microorganism-collecting voltage is 2800 KHz, 12V to biometric voltage, described microorganism metering Voltage is 900 KHz, 1V.
Preferably, microorganism measuring container is made up of polythene material.
Preferably, negative electrode and positive electricity extremely zigzag, sawtooth is uneven and is staggeredly placed.
The metering method of microorganism metering device in described water, its operating procedure is:
Step one, microorganism measuring container is put in groove;
Step 2, utilize push-pull device at fixed to push out the super absorbent resin of sampling rod, from the water containing microorganism, gather water Sample, then utilizes push-pull device at fixed to be withdrawn into by the super absorbent resin containing water sample in protection water sample cover, prevents the water collected Sample is affected by external environment;
Step 3, putting in microorganism measuring container by the sampling rod being surrounded by super absorbent resin one end, sampling rod inserts micro-life In thing measuring container, protection water sample cover (23) sequentially passes through ground floor aluminum foil film, measures solution, second layer aluminum foil film, surveys Amount solution uses pure water, and after second layer aluminum foil film is the worse for wear, pure water part from container is flowed into lower vessel portion and divides, Measure electrode to be flooded completely;
Step 4, one end of super absorbent resin is not had to pass from sampling rod the cap aperture of anti-tampering cap;
Step 5, covers measurement container completely by the anti-tampering cap through sampling rod, is placed in loading position;
Step 6, the push-pull device at fixed utilized in sampling rod, push out the super absorbent resin containing water sample, by sampling rod with cap Hole is fulcrum, stirs pure water, makes the water sample on super absorbent resin fully mix with pure water;
Step 7, connect with the mains, guidance panel is adjusted to microorganism-collecting voltage 2800 KHz, 12V and microorganism simultaneously Metered voltage 900 KHz, 1V, the motor rotor driven of bottom portion of groove rotates, and the magnet concomitant rotation on rotor, magnet carries again Agitator in dynamic microorganism measuring container is sufficiently stirred for, and makes microorganism fall on zigzag electrode, and microorganism is at applied voltage Under the influence of there is positive polarization and negative polarization;
The direct capacitance that in step 8, water, microorganism metering device and metering method thereof are capable of detecting when between anelectrode and negative electrode By rate of change, amount and static capacity rate of change, can show that microorganism falls microorganism-collecting eventually from anelectrode and negative electrode The quantity of end, and show in digital display panel.
Compared with prior art, the invention has the beneficial effects as follows: this microorganism metering device makes micro-life by applied voltage Thing polarizes, and microorganism is fallen in microorganism-collecting terminal by zigzag electrode and measured, and micro organism quantity is aobvious by numeral Show that panel directly displays out, simple to operate, it is susceptible to external interference, accuracy is high.
Accompanying drawing explanation
Fig. 1 is microorganism metering device and metering method schematic diagram thereof in water of the present invention;
Fig. 2 is that microorganism measuring container of the present invention is positioned over groove schematic diagram;
Fig. 3 is that the anti-tampering cap of the present invention is positioned over loading position schematic diagram;
Fig. 4 is the microorganism measuring container schematic diagram of the present invention;
Fig. 5 is the measurement electrode schematic diagram of the present invention;
Fig. 6 is that the anelectrode of the zigzag measurement electrode of the present invention is staggeredly placed and enlarged diagram at A1 with negative electrode;
Fig. 7 is the microorganism metering device operation chart of the present invention;
Fig. 8 is the sampling rod structure schematic diagram of the present invention;
In figure: 1, supply port, 2, guidance panel, 4, groove, 5, load position, 6, microorganism metering hold, 7, lower vessel portion divide, 8, upper vessel portion divides, 9, thin film, 10, measure solution, 11, agitator, 12, measure electrode, 13, negative electrode, 14, anelectrode, 15, Microorganism-collecting terminal, 16, sampling rod, 17, anti-tampering cap, 18, super absorbent resin, 19, magnet, 20, rotor, 21, electronic Machine.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Based on Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under not making creative work premise Embodiment, broadly falls into the scope of protection of the invention.
In conjunction with accompanying drawing, the present invention provides a kind of technical scheme: microorganism metering device and metering method thereof in water, including supplying Electricity port 1, guidance panel 2, digital display panel 3, groove 4 and loading position 5, include motor 21, rotor 20 bottom groove 4 With magnet 19, being used for placing microorganism measuring container 6, microorganism measuring container 6 is divided into upper vessel portion to divide 7 and container by thin film 9 Lower part 8, wherein upper vessel portion divides 7 for placing measurement molten 10, and is sealed by thin film 9, and lower vessel portion divides 8 to include agitator 11 With measurement electrode 12, measure solution 10 and include that pure water, thin film 9 include aluminium foil, measure electrode 12 and include microorganism acquisition terminal 15, negative electrode 13 and anelectrode 14, sampling rod 16 includes push-pull device at fixed 22, protection water sample cover 23, prevents super absorbent resin 18 The water sample collected is affected by external environment, and sampling rod 16 one end is surrounded by super absorbent resin 18, is wherein surrounded by high-hydroscopicity One end of resin 18 is used for gathering water sample, inserts in microorganism measuring container 6, and the cap aperture of anti-tampering cap 17 does not has through sampling rod 16 There is one end of super absorbent resin, be positioned over loading position 5, cover microorganism measuring container 6 completely;
Supply port one end is connected to negative electrode 13 and anelectrode 14, when the other end connects power source, measures the micro-life in liquid There is negative polarization and positive polarization respectively in thing on negative electrode 13 and anelectrode 14, is regulated microorganism by guidance panel 2 simultaneously and receive Collecting voltage and microorganism metered voltage are alternately.
Microorganism measuring container 6 is made up of polythene material, and negative electrode 13 and anelectrode 14 are zigzag, and sawtooth is concavo-convex not Putting down and be staggeredly placed, microorganism-collecting voltage is 2800 KHz, 12V, and microorganism metered voltage is 900 KHz, 1V.
Embodiment:
Step one, microorganism measuring container 6 is put in groove 4;
Step 2, push-pull device at fixed 22 is utilized to push out, from the water containing microorganism the super absorbent resin 18 of sampling rod 16 Gather water sample, then utilize push-pull device at fixed 22 to be withdrawn into by the super absorbent resin 18 containing water sample in protection water sample cover 23, anti- The water sample only collected is affected by external environment;
Step 3, putting in microorganism measuring container 6 by the sampling rod 16 being surrounded by super absorbent resin 18 one end, sampling rod 16 is inserted In a subtle way in biometric container 6, protection water sample cover (23) sequentially passes through ground floor aluminum foil film 9, measures solution 10, second layer aluminum Paper tinsel thin film 9, measures solution 10 and uses pure water, and after second layer aluminum foil film 9 is the worse for wear, pure water part 7 from container flows Enter and divide 8 to lower vessel portion, measure electrode 12 and flooded completely;
Step 4, one end of super absorbent resin 18 is not had to pass from sampling rod 16 cap aperture of anti-tampering cap 17;
Step 5, covers measurement container completely by the anti-tampering cap 17 through sampling rod 16, is placed in loading position 5;
Step 6, the push-pull device at fixed 22 utilized in sampling rod 16, push out the super absorbent resin 18 containing water sample, will sampling Rod 16, with cap aperture as fulcrum, stirs pure water, makes the water sample on super absorbent resin 18 fully mix with pure water;
Step 7, connect with the mains, guidance panel 2 is adjusted to microorganism-collecting voltage 2800 KHz, 12V and microorganism simultaneously Metered voltage 900 KHz, 1V, motor 21 rotor driven bottom groove 4 rotates 20, and the magnet 19 on rotor 20 turns therewith Dynamic, magnet 19 drives again the agitator 11 in microorganism measuring container 6 to be sufficiently stirred for, and makes microorganism fall on zigzag electrode, Positive polarization and negative polarization is there is under the influence of microorganism is the most alive;
What in step 8, water, microorganism metering device and metering method thereof were capable of detecting when between anelectrode 14 and negative electrode 13 is quiet By rate of change, capacitance and static capacity rate of change, can show that microorganism falls micro-life from anelectrode 14 and negative electrode 13 The quantity of thing collection terminal 15, and show in digital display panel 3.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and In addition equivalent or change are conceived in improvement, all should contain within the scope of the present invention.

Claims (4)

1. microorganism metering device in water, including supply port (1), guidance panel (2), digital display panel (3), groove (4) With loading position (5), it is characterised in that: described groove (4) bottom includes motor (21), rotor (20) and magnet (19), For placing microorganism measuring container (6), described microorganism measuring container (6) by thin film (9) be divided into upper vessel portion divide (7) and Lower vessel portion divides (8), and wherein upper vessel portion divides (7) to be used for placing measurement solution (10), and is sealed by thin film (9), lower vessel portion Dividing (8) include agitator (11) and measure electrode (12), described measurement solution (10) includes pure water, described thin film (9) Including aluminium foil, described measurement electrode (12) includes microorganism acquisition terminal (15), negative electrode (13) and anelectrode (14), described Sampling rod (16) include push-pull device at fixed (22), protection water sample cover (23), prevent the water sample that super absorbent resin (18) collects Being affected by external environment, described sampling rod (16) one end is surrounded by super absorbent resin (18), is wherein surrounded by high-hydroscopicity tree One end of fat (18) is used for gathering water sample, inserts in microorganism measuring container (6), and the cap aperture of anti-tampering cap (17) passes sampling rod (16) there is no one end of super absorbent resin, be positioned over loading position (5), cover microorganism measuring container (6) completely;
Described supply port one end is connected to negative electrode (13) and anelectrode (14), when the other end connects power source, measures liquid There is negative polarization and positive polarization respectively in the microorganism in body on negative electrode (13) and anelectrode (14), by guidance panel (2) Alternately, wherein, described microorganism-collecting voltage is for regulation microorganism-collecting voltage and microorganism metered voltage simultaneously 2800 KHz, 12V, described microorganism metered voltage is 900 KHz, 1V.
Microorganism metering device in water the most according to claim 1, is characterized in that: described microorganism measuring container (6) It is made up of polythene material.
Microorganism metering device in water the most according to claim 1, is characterized in that: described negative electrode (13) and anelectrode (14) being zigzag, sawtooth is uneven and is staggeredly placed.
4., according to the metering method of microorganism metering device in the water one of claim 1-3 Suo Shu, its operating procedure is:
Step one, microorganism measuring container (6) is put in groove (4);
Step 2, push-pull device at fixed (22) is utilized to push out, from containing microorganism the super absorbent resin (18) of sampling rod (16) Water in gather water sample, then utilize push-pull device at fixed (22) that the super absorbent resin (18) containing water sample is withdrawn into protection water sample Cover (23) is inner, prevents the water sample collected from being affected by external environment;
Step 3, the sampling rod (16) being surrounded by super absorbent resin (18) one end is put in microorganism measuring container (6), sampling Rod (16) inserts in microorganism measuring container (6), and protection water sample cover (23) sequentially passes through ground floor aluminum foil film (9), measures molten Liquid (10), second layer aluminum foil film (9), described measurement solution (10) uses pure water, when second layer aluminum foil film (9) is the worse for wear After, pure water part (7) from container is flowed into lower vessel portion and divides (8), measures electrode (12) and is flooded completely;
Step 4, one end of super absorbent resin (18) is not had to pass from sampling rod (16) cap aperture of anti-tampering cap (17);
Step 5, the anti-tampering cap (17) through sampling rod (16) is covered measurement container completely, be placed in loading position (5);
Step 6, the push-pull device at fixed (22) utilized in sampling rod (16), push out the super absorbent resin (18) containing water sample, By sampling rod (16) with cap aperture as fulcrum, stir pure water, make the water sample on super absorbent resin (18) fully and pure water mixes Close;
Step 7, connect with the mains, guidance panel (2) is adjusted to microorganism-collecting voltage 2800 KHz, 12V and micro-life simultaneously Thing metered voltage 900 KHz, 1V, motor (21) rotor driven of groove (4) bottom rotates (20), the magnet on rotor (20) (19) concomitant rotation, magnet (19) drives again the agitator (11) in microorganism measuring container (6) to be sufficiently stirred for, makes microorganism fall On zigzag electrode, under the influence of microorganism is the most alive, there is positive polarization and negative polarization;
Step 8, the static capacity detected between anelectrode (14) and negative electrode (13) and static capacity rate of change, pass through Rate of change can show that microorganism falls the quantity of microorganism-collecting terminal (15) from anelectrode (14) and negative electrode (13), and Display in digital display panel (3).
CN201610376306.6A 2016-05-31 2016-05-31 Microorganism metering device and its metering method in water Expired - Fee Related CN106085833B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610376306.6A CN106085833B (en) 2016-05-31 2016-05-31 Microorganism metering device and its metering method in water

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610376306.6A CN106085833B (en) 2016-05-31 2016-05-31 Microorganism metering device and its metering method in water

Publications (2)

Publication Number Publication Date
CN106085833A true CN106085833A (en) 2016-11-09
CN106085833B CN106085833B (en) 2018-03-13

Family

ID=57230365

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610376306.6A Expired - Fee Related CN106085833B (en) 2016-05-31 2016-05-31 Microorganism metering device and its metering method in water

Country Status (1)

Country Link
CN (1) CN106085833B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621483A (en) * 2021-09-10 2021-11-09 谢爱军 Microorganism evenly distributed processing apparatus for microorganism culture

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1072799C (en) * 1995-10-18 2001-10-10 中国科学院生物物理研究所 Sensitive method for quickly testing microbe pollution by electroluminescence and its use
CN1537224A (en) * 2001-07-30 2004-10-13 ���»���ϵͳ��ʽ���� Microorganism-collecting chip, microorganism-collecting kit, method of quantifying microorgaisms, specimen for confirming normal state of microorganism-quantifying apparatus
US20110183371A1 (en) * 2008-06-27 2011-07-28 Hideyuki Noda Microbe-collecting carrier cartridge, carrier treating apparatus, and method of measuring microbes
CN104232478A (en) * 2014-09-24 2014-12-24 昆山超强光电设备有限公司 Bacteria collector
CN105143435A (en) * 2013-02-28 2015-12-09 远程医疗零距离公司 Diagnostic device for evaluating microbial content of a sample
CN105540763A (en) * 2005-10-06 2016-05-04 派克逖克斯公司 Electrochemical ion exchange treatment of fluids

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1072799C (en) * 1995-10-18 2001-10-10 中国科学院生物物理研究所 Sensitive method for quickly testing microbe pollution by electroluminescence and its use
CN1537224A (en) * 2001-07-30 2004-10-13 ���»���ϵͳ��ʽ���� Microorganism-collecting chip, microorganism-collecting kit, method of quantifying microorgaisms, specimen for confirming normal state of microorganism-quantifying apparatus
CN105540763A (en) * 2005-10-06 2016-05-04 派克逖克斯公司 Electrochemical ion exchange treatment of fluids
US20110183371A1 (en) * 2008-06-27 2011-07-28 Hideyuki Noda Microbe-collecting carrier cartridge, carrier treating apparatus, and method of measuring microbes
CN105143435A (en) * 2013-02-28 2015-12-09 远程医疗零距离公司 Diagnostic device for evaluating microbial content of a sample
CN104232478A (en) * 2014-09-24 2014-12-24 昆山超强光电设备有限公司 Bacteria collector

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621483A (en) * 2021-09-10 2021-11-09 谢爱军 Microorganism evenly distributed processing apparatus for microorganism culture

Also Published As

Publication number Publication date
CN106085833B (en) 2018-03-13

Similar Documents

Publication Publication Date Title
CN105137021B (en) Reservoir multi-parameter water quality vertical dynamic data collecting method
CN103335955B (en) A kind of on-line water quality monitoring method and device
CN105158023B (en) A kind of autocontrol classified filtering water body recovery technology harvester
CN104807862A (en) Method and device for detecting dissolved oxygen in water
CN206096107U (en) Water quality detecting system based on singlechip
CN203535000U (en) Portable detector for detection of heavy metal in water environment
CN106085833A (en) Microorganism metering device and metering method thereof in water
CN103207266B (en) Sediment oxygen demand rate determination device based on electromagnetic dynamic and using method
CN105784419A (en) Device and method for sampling suspended matters based on LabVIEW upper computer
CN209471000U (en) Wave erosion experimental rig
CN105004781A (en) Dopamine detecting method based on paper-base electrochemistry device
CN109337813A (en) Suitable for biological tissue's culture and the system and method for real-time monitoring
CN101923037A (en) Method for screening neoplasm metastasis resistant medicine based on fluorescent image and application thereof
CN203178192U (en) Dual-disc excrement analysis instrument
CN206725453U (en) A kind of P in soil H value detection devices
CN109900589A (en) For drawing the measurement device and its application method of characteristic curve of soil moisture
CN203310763U (en) Rapid grading instrument for silkworm fresh cocoons
JPH10260178A (en) Measuring device for oxygen consumption of sea bottom
CN207650198U (en) A kind of water quality detecting device
CN202974689U (en) Specimen collection device
CN206570327U (en) A kind of air microbe detector
CN205262421U (en) Stereometry device of insect
CN207259606U (en) A kind of thimerosal making apparatus with water outlet control function
CN208902487U (en) The electrophoresis tank of gradient pH value seawater can be prepared
CN111948275A (en) Dissolved oxygen detection device based on micro-fluidic chip

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20180313

Termination date: 20200531

CF01 Termination of patent right due to non-payment of annual fee