CN108034691A - A kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph - Google Patents
A kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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Abstract
The invention discloses a kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph.Step：With 75% ethanol to taking blood position to carry out disinfection, hemolymph is extracted with 1 mL syringes, by hemolymph with 1:1 volume ratio is mixed with ACD anti-coagulants.The haemocyte in hemolymph is removed with 5 μm of aperture membrane filtrations, the filtrate of acquisition is refiltered into 0.2 μm of aperture filter membrane.Use SYBR®GreenI fluorescence dye liquor dyes 0.2 μm of aperture filter membrane, then to being observed after its film-making on fluorescence microscope.The micro organism quantity in each visual field is counted, the abundance of microorganism in hemolymph is finally calculated according to formula.The method of the present invention can the invertebrate hemolymph such as accurate counting Scylla paramamosain, litopenaeus vannamei and Portuguese oyster include all microorganisms including Bacterial diversity and Anticipated transient without scram, its count results about 2 orders of magnitude higher than the method for plate culture count, its coefficient of variation are also significantly lower than the method for plate culture count.The method of the present invention has the advantages that counting accuracy height, strong antijamming capability, goes out that result is fast, is widely used.
Description
Technical field
The present invention relates to the method for enumeration of micro organisms abundance, specifically a kind of accurate counting invertebrate hemolymph
The method of the abundance of middle microorganism.
Background technology
Scylla paramamosain（Scylla paramamosain, abbreviation mud crab）It is the eurythermal marine economy crustacean of wide salt,
It is the sociales that China mud crab belongs to, its individual is big, meat flavour is sweet and refreshing, and nutrition is high with economic value.Mud crab be distributed mainly on Guangdong,
Fujian, Zhejiang, Guangxi and Hainan littoral sea, are one of most important sea-farming crab classes in China（Lin Qi etc., 2007）, at present
Mud crab annual output is cured 10,000,000,000 more than 140,000 tons, annual value of production, is occupied in China's seawater fish and seawater crustacean aquaculture yield
Second（Wang Guizhong etc., 2016）.
Litopenaeus vannamei（Litopenaeus vannamei）Originate in the Latin America of Pacific Ocean west bank, its crust compared with
Thin, shrimp body is in cinerous or cyanic colours.Litopenaeus vannamei feeding habits are wide, strong environmental adaptability, can be 13 ~ 40 DEG C in temperature, salinity
Survive in the environment of 2-34（Xu Guofang, 2008）.Litopenaeus vannamei is the shrimps of China's cultured output maximum, China in 2015
Litopenaeus vannamei cultured output accounts for the 85.85% of prawn total output then, reaches 162.524 ten thousand tons（2016《Fisheries statistics
Yearbook》）.
Portuguese oyster（Crassostrea angulata）Belong to Mollusca（Mollusoa）, Bivalvia
（Bivalvia）, main cultivation is in south China, hobby high temperature and high salt environment（Lin Qing, 2013）.Oyster meat delicious, nutrition are rich
Richness, is the economic shellfish of current China or even world wide production maximum.2016《Fisheries statistics yearbook》It has been shown that, oyster annual output
For 457.33 ten thousand tons, rank first place in China's marine shellfish cultured output.
Break out disease again and again in the breeding process such as crab, shrimp, oyster in recent years（Virus, bacterium and parasite）, serious resistance
Hindered the sustainable development of aquaculture industry, the only blue crab cultivation of the Shantou City, Guangdong Province therefore annual underproduction more than 60%, year economic damage
Lose more than one hundred million members.Once disease occurs, for the pharmaceutical treatment effect and unobvious of specific pathogen microorganism, and by maintaining animal
The stable state of internal microecosystem tends to reach ideal disease control effect.Thus in recent years, to common in animal body
The research of raw microorganism becomes the hot content of prevention and control disease generation.Evidence suggests in oceans such as crab, shrimp, oysters without ridge
Generally existing microorganism in the hemolymph of the healthy individuals of Vertebrate（Wang etc., 2015）.Tubiash et al. is most early in 1975
Delicious leisurely and carefree crab has been counted with dilution culture counting method（Callinectes sapidus）Bacterial abundance in hemolymph is about
1.9×103MPN/mL.Hereafter, researcher has counted litopenaeus vannamei using the method for plate culture count in succession（Litopenaeus vannamei, 101-103CFU/mL）, Marsupenaeus japonicus（Marsupenaeus japonicus, 101-103CFU/mL）, spot
Save prawn（Penaeus monodon, ~ 102CFU/mL）And Pacific oyster（Crassostrea gigas, 101-102 CFU/
mL）Deng the abundance of the hemolymph microorganism of invertebrate（Brandin etc. 1985；Scott etc., 1986；Olafsen etc.,
1993；Gomez-Gil etc., 1998）.Found in addition, being directed in applicant in detection of the mud crab disease with microorganism relation early period
Following features：First, the Symptoms of the sick crab in part do not solidify for hemolymph, are creamy white or yellow, and wherein containing a large amount of
Pathogenic microorganism（Vibrio parahaemolytious, Aeromonas hydrophila and blood ovum whirlpool whipworm；Li et al., 2008；Xia little An etc., 2010；Li et al.,
2012）.Secondly, the generally existing microorganism in the hemolymph of healthy mud crab.Finally, the hemolymph microorganism in dying mud crab
Abundance about 2 orders of magnitude more significantly high than healthy individuals.Therefore, the abundance of all microorganisms in invertebrate hemolymph is counted
It is a kind of important means for assessing its health status.
All microorganisms include educable microorganism and unculturable microorganism in hemolymph.Hemolymph is counted at present
The method of bacterial abundance is all based on the pure culture technigne of microorganism, i.e., using culture mediums such as 2216E, LB, TCBS, specific
Temperature, pH, educable microorganism in hemolymph is cultivated under aerobic or anaerobic condition, it is macroscopic to treat that it grows
Counted again after bacterium colony.And the abundance of the microorganism for not being cultured out in those hemolymphs is unknown so far, there is research to push away
The accounting for surveying the quantity of those microorganisms for not being cultured out microbial count in the environment is up to 99%（Amann etc.,
1995）.This is likely due to external culture environment（Temperature, pH, salinity, pressure etc.）, and nutritional condition（Carbon source, nitrogen source
Deng）Deng being not suitable for, it can not be grown on specific culture medium, or its growth is extremely slow, naked eyes are formed not on culture medium
Visible bacterium colony.
Fluorescent microscope counting method is a kind of predetermined substance using in fluorescent dye and biological cell（Nucleic acid or albumen etc.）
It is combined with each other, can observes special fluorescence under fluorescence microscope in the range of certain excitation wavelength（It is green or red
Color etc.）, then to method that wherein cell containing fluorescence is directly counted.This method is most to grow up in the past 30 years
, it overcomes traditional method of counting based on Bacterial diversity, can be used for numerous to water body, soil, deposit etc.
The educable and not educable all microbial cells that include in complex environment sample are fast and accurately counted.
Certainly, the microorganism in environmental sample is directly counted using Fluorescent microscope counting method, is in most cases needed to sample
Product carry out certain pre-treatment step to remove the impurity of Interference Detection（Such as in soils and sediments be mainly dirt,
And little particle organic matter etc.）.The preprocess method of the currently used separate microorganism from environmental sample has two kinds：Density level bands
Spend centrifugal separation and fractionation.Density gradient centrifugation is different with the density of impurity based on microbial cell
Principle, one or more density gradient medias are added by sample（Such as poly- sodium tungstate and Nycodenz；Morono etc., 2013）In
Centrifugal sedimentation is carried out, specific location microbial cell being assigned under certain centrifugal force in gradient, and impurity is assigned to
Other positions in gradient, so as to reach separated purpose.Fractionation mainly uses detergent（Such as, it is formulated as 100
MM EDTA, 100 mM sodium pyrophosphates, the detergent of 1% (v/v) Tween 80）Destroy the close of microorganism and contaminant surface
Suction-operated, microorganism and impurity are separated.
However, there are problems with for microorganism this method being directly used in counting invertebrate hemolymph：First,
Haemocyte and microbial cell are without essential difference in chemical composition and density, therefore they can be by common on the market
Fluorescent dyeing（Including SYBR Green I）, and density gradient centrifugation can not separate microorganism cell；Secondly,
In the hemolymph of healthy individuals, microbial cell is in single free state more, do not find significantly to assemble it is agglomerating or with
The microorganism that haemocyte combines, therefore fractionation need not be used；Again, haemocyte in invertebrate hemolymph
Quantity is 100 times of microbial cell or so, and the volume of haemocyte is also more much larger than microorganism（Haemocyte diameter 6-15
μm, 1-2 μm of microorganism diameter）, cause some microbial cells to be covered in by haemocyte when carrying out microscopic；Finally,
Under fluorescence microscope, the fluorescence intensity ratio microbial cell of haemocyte is much higher, and it is difficult to be observed to cause microbial cell.
Therefore, if count the microorganism in hemolymph under the microscope with will be clear that, interference of the haemocyte to observation must be just eliminated,
Then microorganism therein is identified with fluorescent dye.
Up to the present, it is there is not yet any in relation to using the method independent of " pure culture technigne of microorganism ", coming
Count the report of the method for all microorganisms in invertebrate hemolymph.
The content of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, based on work principle of filter and fluorescence microscopy, there is provided Yi Zhongzhun
The method for really counting the abundance of microorganism in invertebrate hemolymph, accurate counting invertebrate hemolymph includes can
The abundance of all microorganisms including culture microorganism and Anticipated transient without scram, comprises the following steps：
(1) ACD anti-coagulants is prepared, pH is adjusted as acidity, is preserved after high pressure steam sterilization in low temperature；
(2) invertebrate sterilizes, repeatedly；
(3) hemolymph is extracted in invertebrate proper site, is mixed with ACD anti-coagulants rapidly；
(4) with 5 μm of aperture membrane filtrations are contained, filtrate is collected；
(5) with 0.2 μm of aperture membrane filtration is contained, filter membrane is transferred in clean culture dish；
(6) lucifuge is added dropwise appropriate fluorescence dye liquor to filter membrane and dyes, and qs glycerin solution is added dropwise after removing fluorescence dye liquor, to prevent
Fluorescent quenching；
(7) fluorescence microscope counts, and the abundance of hemolymph microorganism is calculated according to equation below：
Step（3）Main function be to prevent blood clotting.The most important component that detection is influenced in blood is haemocyte.
This method it is especially noted that place be hemolymph sampling after need that blood sample is injected into ACD anti-coagulants immediately, prevent
Blood clotting.Contain hemostatic composition in blood, blood can not be detected once solidification.
It is different from the detection of water sample and Soil Microorganism, substantial amounts of haemocyte, blood are contained in invertebrate hemolymph
Cell and microbial cell are difficult to be separated in chemical composition and density without essential difference, conventional preprocess method, into
In order to restrict the problem of Fluorescent microscope counting method application.
By thinking deeply and studying, haemocyte becomes separated breach with the greatest differences on microorganism build.Haemocyte
General diameter is at 6-15 μm, and a diameter of 1-2 μm of microorganism, the filter sizes of selection are excessive, it will cause a large amount of haemocytes to lead to
Enter filtrate after crossing this filter membrane, be unable to reach the purpose for removing haemocyte；Filter sizes are too small, it will make part microbial cell
Retention causes the loss of microbial cell on this filter membrane, and then influences the accuracy counted.Repeatedly grope by us, most
5 μm of membrane filtrations are selected to remove haemocyte afterwards.First fall all haemocytes in blood with 5 μm of membrane filtrations, at this time in blood
Microorganism filtrate all after filtration in.Then this filtrate for containing microorganism is filtered into 0.2 μm of filter membrane, due to micro- life
The diameter of thing is generally more than 0.2 μm, therefore these microorganisms are all trapped within 0.2 μm of filter membrane, can after fluorescent staining
To count under the microscope.The purpose of 5 μm of filter membranes is that filtering removes haemocyte, and the purpose of 0.2 μm of filter membrane is retention microorganism
It is observed counting.Filter like this, can in blood lymphocyte in addition to microorganism other interference microscopes observation and
The material of counting（Including haemocyte, hemocyanin etc.）All filtering is removed.
Preferably, above-mentioned preparation ACD anti-freezing agent prescriptions are 450 mM of sodium chloride, 100 mM of glucose, 26 mM of citric acid,
30 mM of sodium citrate, it is 4.6 to adjust pH, under 1.05kg/cm2 pressure with after 20 min of steam sterilizing of 121oC in 4 DEG C
Preserve.
Preferably, above-mentioned invertebrate includes the one or more of Scylla paramamosain, litopenaeus vannamei or Portuguese oyster.
Preferably, the position of above-mentioned invertebrate extraction hemolymph is as follows：Scylla paramamosain is its second and third step base pitch
At film, litopenaeus vannamei is its abdomen blood sinus, and Portuguese oyster position is its closed shell flesh.These positions are located at animal body surface, and contain
Substantial amounts of hemolymph, is easily extracted.
Preferably, the amount and hemolymph volume ratio 1 of above-mentioned ACD anti-coagulants mixing:1.At this time, extension rate is 1 times.
Preferably, it is 200 μ L that filtrate is collected in above-mentioned collection, i.e., applied sample amount is 200 μ L.
Preferably, above-mentioned fluorescence dye liquor is SYBR Green I fluorescence dye liquors, is formulated as follows：1:40 v/v SYBR®
Green I are dissolved in 1 × Tris-EDTA buffer.
Preferably, above-mentioned glycerite is the glycerite of volume fraction 10%.
Preferably, above-mentioned fluorescence dye liquor dripping quantity of stating is 0.2 μ L/mm2, dyeing time is 20 min, and glycerite is added dropwise
Measure as 0.05 μ L/mm2。
Preferably, above-mentioned state uses 1000 times of fluorescence microscopes when fluorescence microscope counts, and every filter membrane counts 200
A visual field.
The invention has the advantages that：
（1）Counting accuracy is high.The common method based on microorganism pure culture technigne（Such as the method for plate culture count and dilution
Number method is surveyed in culture）The Bacterial diversity in the hemolymph of invertebrate can only be counted, can be more accurately using the present invention
Include all microorganisms including Bacterial diversity and Anticipated transient without scram to invertebrate hemolymph directly to be counted
Number.The method of the present invention about 2 orders of magnitude higher than the Bacterial diversity abundance that the method for plate culture count obtains, and the method for the present invention
The coefficient of variation be significantly lower than the method for plate culture count.
（2）Strong antijamming capability.For haemocyte diameter in hemolymph at 6-15 μm or so, its abundance is more than 106
hemocytes/mL（Fig. 1 a, c, e）.And the microorganism diameter in hemolymph is at 1-2 μm or so, its abundance is less than 106
cells/mL（Fig. 1 b, d, f）.The present invention can effectively remove haemocyte to microbial cell meter by filtering 5 μm of aperture filter membranes
Several interference（Fig. 1）.The microorganism that the present invention can count includes that all 5 μ can be perforated through in invertebrate hemolymph
M filter membranes and by the microorganism of 0.2 μm of membrane retention, including educable microorganism and not educable microorganism, have bacterium,
Ancient bacterium, fungi etc..
（3）It is fast to go out result.It is visible that the method for pure culture technigne based on microorganism at least needs 24 h to obtain naked eyes
Bacterium colony, and count results can be obtained in 2 h using the present invention, substantially reduce the abundance that counts hemolymph microorganism when
Between.The abundance of all microorganisms is a kind of important means for assessing its health status in accurate statistics invertebrate hemolymph.
（4）The stable state with controlling microecosystem in animal body can preferably be held.The method of the present invention counting accuracy is more
Height, preferably can hold and control the stable state of microecosystem in animal body.And traditional counting method can because precision is not high and
Situation about judging steady state error is likely to occur, production and construction are caused with unnecessary loss.
（5）It is widely used.In terms of can either being applied to the scientific researches such as the symbiotic microorganism group of invertebrate, also can
Daily Defect inspection applied to breeding enterprise works.
Brief description of the drawings
Fig. 1 handles healthy Scylla paramamosain, litopenaeus vannamei and the hemolymph of Portuguese oyster respectively for the method for the present invention
Forward and backward haemocyte and the fluorescent microscopy images of microorganism；Wherein, figure a, c, e be respectively Scylla paramamosain, litopenaeus vannamei,
The hemolymph microphotograph of Portuguese oyster, figure b, d, f are to be divided the hemolymph of above-mentioned three kinds of animals using the method for the present invention
Microphotograph that Guo Lv be after 5 μm of aperture filter membranes；A is microorganism in figure, and B is haemocyte, and scale is 10 μm；
To count Scylla paramamosain, litopenaeus vannamei and Portugal respectively with the method for plate culture count male to compare the method for the present invention by Fig. 2
The abundance of the hemolymph microorganism of the healthy individuals of oyster；Each point represents an animal, and the horizontal line of black represents median, asterisk
Represent statistically significant（Mann Whitney U test, P<0.05）；
Fig. 3 is to compare the abundance that the method for the present invention counts dying mud crab hemolymph microorganism with the method for plate culture count；Each
Point represents an animal, and the horizontal line of black represents median, and asterisk represents statistically significant（Mann Whitney U test, P<
0.05）；
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
It is described in detail on step ground.
Embodiment 1：Using the method for the present invention statistics Scylla paramamosain, litopenaeus vannamei and the healthy individuals of Portuguese oyster
The abundance of hemolymph microorganism
Choose the Scylla paramamosain 6 from same breeding facility（~ 100.0 g/ are only）, 6 groups of litopenaeus vannamei（~ 7.5 g/, with
1 group is used as after the blood mixing of 20 shrimp extractions）, Portuguese oyster 6（39.7 g/ is only）, it is desirable to every individual health is active, attached
Limb perfects, body surface is without attachment.Using the abundance of the method for the present invention statistics hemolymph microorganism.
The method of the present invention operating procedure is：
（1）Prepare ACD anti-coagulants：Anti-freezing agent prescription is 450 mM of sodium chloride, 100 mM of glucose, 26 mM of citric acid, lemon
Sour 30 mM of sodium, pH are adjusted to 4.6,4 DEG C are stored in after high pressure steam sterilization.
（2）Animal sterilizes：First animal whole body is dried with blotting paper, the surface at blood position is then taken with 75% ethanol spray,
Blotting paper cleans the dirt of its surface attachment.Repeat the above steps three times.
（3）Hemolymph samples：Hemolymph is extracted with 1 mL syringes of ACD anti-coagulants rinses, transfers it to and contains
Have in the centrifuge tube of anti-coagulants, it is final to ensure that hemolymph and anti-coagulants volume ratio are 1:1.The blood drawing position of Scylla paramamosain for its
2nd, at three step coxacorias, the blood drawing position of litopenaeus vannamei is its abdomen blood sinus, and the blood drawing position of Portuguese oyster is its closed shell
Flesh.
（4）Remove haemocyte：With containing 5 μm of aperture filter membranes（Millipore, TMTP02500）Filter
（Millipore, SX0002500）The mixture of 200 μ L hemolymphs and anti-coagulants is filtered, is centrifuged with 1.5 mL of another sterilizing
Pipe collects above-mentioned filtrate.
The quantity of haemocyte in invertebrate hemolymph is 100 times of microbial cell or so, its volume is also micro-
The several times of biology are big（6-15 μm of haemocyte diameter, 1-2 μm of microorganism diameter）, and they can be by common on the market
Fluorescent dye（Including SYBR®Green I）Dyeing, causes it can not directly be observed counting under the microscope.Such as Fig. 1
Shown, before filtering, microbial cell is covered by the haemocyte of bulky, it is difficult to is seen clearly；After filtering, numerous but volume
Small microorganism just exposes out so that observation counting is possibly realized.
（5）Obtain hemolymph microorganism：With containing 0.2 μm of aperture filter membrane（Millipore, GTBP02500）Filter
（Millipore, SX0002500）Filtration step（5）The filtrate of acquisition, is then transferred to clean culture dish by 0.2 μm of filter membrane
In.
（6）Microbial staining, film-making：To step（6）The filter membrane of acquisition instills 100 μ L SYBR Green I fluorescence dye
Liquid（1:40 v/v SYBR Green I are dissolved in 1 × Tris-EDTA buffer）, 20 min of lucifuge dyeing.Remove dye liquor,
Filter membrane is put on glass slide, instills 25 μ L, 10% v/v glycerites to prevent fluorescent quenching, covered.
(7) fluorescence microscope counts：Glass slide is transferred to 1000 times of fluorescence microscopes (Nikon, ECLIPSE 90i),
It is observed with blue color filter, microbial cell is in fluorescence microscope emitted green light.Every film counts 200 visuals field.Most
Afterwards, the abundance of hemolymph microorganism is calculated according to equation below：
It is total to spend the time：2 it is small when.
Comparative example 1：Using the method for plate culture count statistics Scylla paramamosain, litopenaeus vannamei and the health of Portuguese oyster
The abundance of the hemolymph microorganism of body
Choose Scylla paramamosain same as Example 16（~ 100.0 g/ are only）, 6 groups of litopenaeus vannamei（~ 7.5 g/, with 20
1 group is used as after the blood mixing of shrimp extraction）, Portuguese oyster 6（39.7 g/ is only）, it is desirable to every individual health is active, appendage
Sound, body surface is without attachment.Difference from Example 1 is：Using the method for plate culture count statistics hemolymph microorganism
Abundance.
The method of plate culture count operating procedure is：
（1）Animal sterilizes and hemolymph sampling procedure is the same as embodiment 1.
（2）Prepare 2216E solid plates：2216E solid culture based formulas, 5 g/L peptones, 1 g/L yeast extracts, 0.1
G/L ferric trichlorides, 20 g/L sodium chloride, 15 g/L agar, add the dissolving of 1L distilled water, and pH is adjusted to 7.6, and autoclaving, waits to train
Foster base is poured into aseptic flat board when being cooled to 45 DEG C or so.
（3）Coating：The mixed liquor that 100 μ L hemolymphs and anti-coagulants are drawn with liquid-transfering gun is inoculated in 2216E solid plates
On, bacterium solution is coated with tablet uniformly with sterile glass scraper plate, tablet is inverted culture in 30 DEG C of insulating boxs.
（4）Count：After 24 h of tablet culture, the clump count on each tablet is counted, being calculated with equation below can always cultivate
The abundance of bacterium, can always cultivate the abundance of bacterium（CFU/mL）Clump count × 10 × 2 on=tablet（Ensure have in a tablet
30-300 bacterium colony）.
It is total to spend the time：1-2 days.
Compare the method for the present invention and count healthy Scylla paramamosain, litopenaeus vannamei and Portugal respectively with the method for plate culture count
The abundance of the hemolymph microorganism of oyster
Embodiment 1 and 1 comparing result of comparative example are as follows：
As shown in Figure 2：Scylla paramamosain, litopenaeus vannamei and the healthy individuals of Portuguese oyster are counted using the method for the present invention
The average value of the abundance of hemolymph microorganism is respectively 2.6 × 104 cells/mL、1.3×105 cells/mL、1.7×105
Cells/mL, the coefficient of variation are respectively 16%, 11%, 7%；Scylla paramamosain, litopenaeus vannamei are counted using the method for plate culture count
The average value that bacterium abundance can be cultivated with the hemolymph of the healthy individuals of Portuguese oyster is respectively 6.6 × 102 CFU/mL、5.1×
103 CFU/mL、4.5×102 CFU/mL, the coefficient of variation are respectively 92%, 66%, 58%.Wherein, the variation of Scylla paramamosain experimental group
Coefficient is up to 92%, the hemolymph bacterial abundances of the healthy individuals counted with the method for plate culture count from 55 CFU/mL to
1.7×103 CFU/mL, there are great error.
Embodiment 1 is contrasted with comparative example 1, is as a result illustrated, the method for the present invention can apply to count Scylla paramamosain, vannamei boone
The abundance of the hemolymph microorganism of prawn and Portuguese oyster, compared with the method for plate culture count, the method for the present invention counts blood strangury
The abundance of the microorganism of Palestine and China is more accurate, coefficient of variation smaller, total time-consuming are shorter.
Embodiment 2：The method of the present invention counts the abundance of dying Scylla paramamosain hemolymph microorganism
Choose from 1 same breeding facility of embodiment, dying Scylla paramamosain 6（~ 100.0 g/ are only）, it is desirable to every
Body is in state on the point of dying, and only mouth is breathing, and step is almost motionless.The micro- life of hemolymph is counted using the method for the present invention
The abundance of thing, concrete operation step is with reference to embodiment 1.
Comparative example 2：The abundance of dying Scylla paramamosain hemolymph microorganism is counted using the method for plate culture count
Choose dying Scylla paramamosain 6 same as Example 2（~ 100.0 g/ are only）, it is desirable to every individual is in feeble breathing one
The state of breath, only mouth are breathing, and step is almost motionless.Difference from Example 2 is：Using the method for plate culture count
Count the abundance of hemolymph microorganism.Concrete operation step is with reference to comparative example 1.
Compare the method for the present invention counted respectively with the method for plate culture count dying Scylla paramamosain hemolymph microorganism it is rich
Degree
Embodiment 2 and 2 comparing result of comparative example are as follows：
As shown in Figure 3：The average value that the abundance of dying Scylla paramamosain hemolymph microorganism is counted using the method for the present invention is
3.6×106Cells/mL, than healthy individuals hemolymph bacterial abundance 2.6 × 104cells/mL（See embodiment 1）It is high about
138 times；Counted using the method for plate culture count the average value of the abundance of dying Scylla paramamosain hemolymph microorganism for 3.9 ×
103 CFU/mL, than healthy individuals hemolymph bacterial abundance 6.6 × 102 CFU/mL（See comparative example 1）It is higher by about 6 times.
However, the hemolymph bacterial abundance that indivedual healthy individuals are counted using the method for plate culture count is up to 1.7
×103 CFU/mL, the average value 3.9 × 10 with dying individual hemolymph bacterial abundance3 CFU/mL is approached, this is being cultivated
It may cause healthy mud crab being mistakenly considered morbidity mud crab in production process, or the mud crab that will fall ill is mistakenly considered the event of healthy mud crab and goes out
It is existing.
Embodiment 2 is contrasted with comparative example 2, is as a result illustrated, compared with the method for plate culture count, the method for the present invention is to dying
The detection sensitivity higher of the abundance of the hemolymph microorganism of Scylla paramamosain, it is more difficult to judge by accident.
The above disclosed power for being only a kind of preferred embodiment of the present invention, the present invention cannot being limited with this certainly
Sharp scope, therefore equivalent variations made according to the claims of the present invention, are still within the scope of the present invention.
Claims (10)
1. a kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph, it is characterised in that including following step
Suddenly：
(1) ACD anti-coagulants is prepared, pH to 4.6 is adjusted, is preserved after high pressure steam sterilization in low temperature；
(2) invertebrate sterilizes, repeatedly；
(3) hemolymph is extracted in invertebrate proper site, is mixed with ACD anti-coagulants rapidly；
(4) with 5 μm of aperture membrane filtrations are contained, filtrate is collected；
(5) with 0.2 μm of aperture membrane filtration is contained, filter membrane is transferred in clean culture dish；
(6) dyeing of fluorescence dye liquor is added dropwise to filter membrane in lucifuge, and glycerite is added dropwise after removing fluorescence dye liquor；
(7) fluorescence microscope counts, and the abundance of hemolymph microorganism is calculated according to equation below：
2. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（1）The preparation ACD anti-freezing agent prescriptions are 450 mM of sodium chloride, 100 mM of glucose, 26 mM of citric acid, lemon
Sour 30 mM of sodium, it is 4.6 to adjust pH, in 1.05kg/cm2With 121 under pressureoPreserved after 20 min of steam sterilizing of C in 4 DEG C.
3. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（3）The invertebrate includes the one or more of Scylla paramamosain, litopenaeus vannamei or Portuguese oyster.
4. according to the method for the abundance of microorganism in the accurate counting invertebrate hemolymph of claim 1 or 3, its feature
It is, the position that the invertebrate extracts hemolymph is as follows：Scylla paramamosain is all to receive at its second and third step coxacoria
Shore prawn is its abdomen blood sinus, and Portuguese oyster position is its closed shell flesh.
5. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（3）The amount of the ACD anti-coagulants and the amount of hemolymph are volume ratio 1:1.
6. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（4）It is 200 μ L that filtrate is collected in the collection.
7. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（6）The fluorescence dye liquor is SYBR Green I fluorescence dye liquors, is formulated as follows：1:40 v/v SYBR® Green
I is dissolved in 1 × Tris-EDTA buffer.
8. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（6）The glycerite is the glycerite of volume fraction 10%.
9. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（6）The fluorescence dye liquor dripping quantity is 0.2 μ L/mm2, dyeing time is 20 min, and glycerite dripping quantity is
0.05 μL/mm2。
10. according to claim 1 in accurate counting invertebrate hemolymph the abundance of microorganism method, its feature exists
In step（7）The fluorescence microscope uses 1000 times of fluorescence microscopes when counting, every filter membrane counts 200 visuals field.
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