CN108034691B - Method for accurately counting abundance of microorganisms in invertebrate haemolymph - Google Patents
Method for accurately counting abundance of microorganisms in invertebrate haemolymph Download PDFInfo
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- CN108034691B CN108034691B CN201711249372.8A CN201711249372A CN108034691B CN 108034691 B CN108034691 B CN 108034691B CN 201711249372 A CN201711249372 A CN 201711249372A CN 108034691 B CN108034691 B CN 108034691B
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Abstract
The invention discloses a method for accurately counting the abundance of microorganisms in invertebrate haemolymph. The method comprises the following steps: sterilizing the blood-collecting part with 75% ethanol, extracting hemolymph with 1 mL syringe, and mixing hemolymph with ACD anticoagulant at volume ratio of 1: 1. Filtering with 5 μm filter membrane to remove blood cells in haemolymph, and filtering the obtained filtrate with 0.2 μm filter membrane. By SYBR®Green I fluorescent dye solution stains a filter membrane with the aperture of 0.2 mu m, and the filter membrane is observed on a fluorescent microscope after being filmed. And (4) counting the number of microorganisms in each visual field, and finally calculating the abundance of the microorganisms in the haemolymph according to a formula. The method can accurately count all microorganisms including culturable microorganisms and uncultured microorganisms in hemolymph of invertebrates such as scylla paramamosain, litopenaeus vannamei and ostrea portulacea, the counting result is about 2 orders of magnitude higher than that of a flat plate bacterial colony counting method, and the coefficient of variation is obviously lower than that of the flat plate bacterial colony counting method. The method has the advantages of high counting accuracy, strong anti-interference capability, quick result output, wide application and the like.
Description
Technical Field
The invention relates to a method for counting the abundance of microorganisms, in particular to a method for accurately counting the abundance of microorganisms in invertebrate haemolymph.
Background
Scylla paramamosain (Scylla paramamosainBlue crab for short) is a wide-salt and wide-temperature ocean economic shell movementThe crab meat is a dominant species of the blue crab in China, and has large individual, sweet meat flavor and high nutritional and economic values. The blue crabs are mainly distributed in the coastal waters of Guangdong, Fujian, Zhejiang, Guangxi and Hainan, are one of the most important marine culture crabs in China (Linqi et al, 2007), currently, the annual output of the blue crabs exceeds 14 million tons, the annual output value is 100 hundred million, and the culture output of marine fishes and marine crustaceans is the second highest in China (Wangui Cheng et al, 2016).
Litopenaeus vannamei (A) and (B)Litopenaeus vannamei) Latin America native to the Pacific west has thin carapace and the shrimp body is grayish or bluish. The litopenaeus vannamei has wide feeding habits and strong environmental adaptability, and can survive in an environment with the temperature of 13-40 ℃ and the salinity of 2-34 (Xu Fang, 2008). The litopenaeus vannamei is the shrimp with the largest breeding yield in China, and the breeding yield of litopenaeus vannamei in 2015 accounts for 85.85 percent of the total yield of the litopenaeus vannamei in the current year, and reaches 162.524 ten thousand tons (2016 < fishery statistics yearbook >).
Oyster root of Portugal oyster (Crassostrea angulata) Belongs to the phylum of mollusca (Mollusoa) and class Bivalvia, is mainly cultivated in the south of China and favors high-temperature and high-salt environment (forest cleaning, 2013). The oyster meat has delicious taste and rich nutrition, and is the economic shellfish with the largest yield in China and even the world at present. The fishery statistics yearbook of 2016 shows that the annual output of oysters is 457.33 ten thousand tons, and the annual output of the seashells is the first in the marine shellfish culture output of China.
In recent years, diseases (viruses, bacteria and parasites) frequently burst in the breeding process of crabs, shrimps, oysters and the like, the sustainable development of the breeding industry is seriously hindered, and the yield is reduced by over 60 percent every year and the annual economic loss is billions of yuan only in the breeding of blue crabs in Shantou city of Guangdong province. Once the disease occurs, the treatment effect of the medicament aiming at the specific pathogenic microorganism is not obvious, and the ideal disease prevention and control effect can be achieved by maintaining the steady state of the micro-ecosystem in the animal body. Therefore, in recent years, research on symbiotic microorganisms in animals has become a popular content for preventing and controlling the occurrence of diseases. There is evidence for the ubiquitous presence of microorganisms in hemolymph of healthy individuals of marine invertebrates such as crabs, shrimps, oysters and the like (Wang et al, 2015). Tubiash et al counted delicious swimming crabs by a dilution culture counting method in 1975Callinectes sapidus) The microbial abundance in haemolymph is about 1.9X 103MPN/mL. Thereafter, the researchers counted Litopenaeus vannamei successively by using a plate colony counting method (Litopenaeus vannamei，101-103CFU/mL), marsupenaeus japonicus (C.A.)Marsupenaeus japonicus，101-103CFU/mL), Penaeus monodon (Penaeus monodon，~102CFU/mL) and Pacific oyster (Crassostrea gigas，101-102CFU/mL) of invertebrates (Brandin et al 1985; scott et al, 1986; olafsen et al, 1993; Gomez-Gil et al, 1998). In addition, the following characteristics are found in the previous detection of the relation between the disease and the microorganism of the blue crab by the applicant: firstly, some of the symptoms of the disease crabs are that the hemolymph is not coagulated, is milk white or yellow, and contains a large number of pathogenic microorganisms (vibrio parahaemolyticus, aeromonas hydrophila and blood egg dinoflagellate; Li et al, 2008; xiaoean et al, 2010; Li et al, 2012). Secondly, microorganisms are ubiquitous in the hemolymph of healthy blue crabs. Finally, the abundance of haemolymph microorganisms in moribund blue crabs is significantly higher by about 2 orders of magnitude than that of healthy individuals. Therefore, counting the abundance of all microorganisms in invertebrate haemolymph is an important means to assess their health status.
All microorganisms in hemolymph include culturable microorganisms and uncultured microorganisms. At present, the method for counting the abundance of haemolymph microorganisms is based on a pure culture technology of microorganisms, namely, cultures the microorganisms which can be cultured in haemolymph by utilizing 2216E, LB, TCBS and other culture media under the conditions of specific temperature, pH, aerobic or anaerobic condition, and counts after the microorganisms grow out to be visible to the naked eye. While the abundance of uncultured microorganisms in haemolymph has not been known to date, studies have speculated that the number of uncultured microorganisms accounts for up to 99% of the total number of microorganisms in the environment (Amann et al, 1995). This is probably due to the fact that the external culture environment (temperature, pH, salinity, pressure, etc.), nutrient conditions (carbon source, nitrogen source, etc.), etc. are not suitable, and cannot grow on a specific medium, or they grow extremely slowly without forming colonies visible to the naked eye on the medium.
The fluorescence microscopic counting method is a method in which a fluorescent dye and a specific substance (nucleic acid, protein, or the like) in a biological cell are bonded to each other, and specific fluorescence (green, red, or the like) can be observed under a fluorescence microscope in a certain excitation light wavelength range, and then cells containing fluorescence are directly counted. The method is developed in the last 30 years, overcomes the defects of the traditional counting method based on culturable microorganisms, and can be used for quickly and accurately counting all microbial cells including culturable and non-culturable microbial cells in a plurality of complex environment samples such as water, soil, sediments and the like. Of course, direct counting of microorganisms in environmental samples using fluorescence microscopy requires in most cases a certain pre-treatment step of the sample to remove impurities (such as mainly mineral impurities in soils and sediments, and small-particle organic matter, etc.) that interfere with the detection. Two commonly used pretreatment methods for isolating microorganisms from environmental samples are currently available: density gradient centrifugation and chemical separation. The density gradient centrifugal separation method is based on the principle that microbial cells and impurities have different densities, and is characterized by adding a sample into one or more density gradient media (such as sodium polytungstate and Nycodenz; Morono et al, 2013) to carry out centrifugal sedimentation, distributing the microbial cells to a specific position in the gradient under a certain centrifugal force, and distributing the impurities to other positions in the gradient, thereby achieving the purpose of separation. The chemical separation method mainly adopts detergent (such as detergent with the formula of 100mM EDTA, 100mM sodium pyrophosphate and 1% (v/v) Tween 80) to destroy the close adsorption of the microorganisms and the surface of the impurities, so as to separate the microorganisms and the impurities.
However, the direct use of this method for counting microorganisms in invertebrate hemolymph presents the following problems: firstly, the blood cells and the microbial cells have no essential difference in chemical components and density, so that the blood cells and the microbial cells can be dyed by common fluorescent dyes (comprising SYBR Green I) on the market, and the microbial cells cannot be separated by a density gradient centrifugal separation method; secondly, in the haemolymph of healthy individuals, the microbial cells are mostly in a single free state, and no significant microbes aggregated into clusters or bound to blood cells are found, so that no chemical separation method is required; thirdly, the number of the blood cells in the haemolymph of the invertebrate is about one hundred times of that of the microorganism cells, and the volume of the blood cells is much larger than that of the microorganism (the diameter of the blood cells is 6-15 μm, and the diameter of the microorganism is 1-2 μm), so that some microorganism cells are covered by the blood cells when the microorganism cells are observed by a microscope; finally, under a fluorescence microscope, the fluorescence intensity of blood cells is much higher than that of microbial cells, making the microbial cells difficult to observe. Therefore, if the microorganisms in haemolymph are to be counted under a microscope clearly, it is necessary to eliminate the interference of the blood cells with the observation and then to identify the microorganisms therein with a fluorescent dye.
To date, no report has been made on the statistics of all microorganisms in invertebrate hemolymph using a method that does not rely on "pure culture techniques for microorganisms".
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a method for accurately counting the abundance of microorganisms in invertebrate hemolymph based on a filtering principle and fluorescence microscopy, wherein the method for accurately counting the abundance of all microorganisms including culturable microorganisms and uncultured microorganisms in invertebrate hemolymph comprises the following steps:
(1) preparing ACD anticoagulant, adjusting pH to acidity, sterilizing with high pressure steam, and storing at low temperature;
(2) sterilizing invertebrates, and repeating for multiple times;
(3) collecting hemolymph from invertebrate suitable part, and rapidly mixing with ACD anticoagulant;
(4) filtering with a filter membrane with the aperture of 5 mu m, and collecting the filtrate;
(5) filtering with a filter membrane with the aperture of 0.2 mu m, and transferring the filter membrane into a clean culture dish;
(6) shading to drop a proper amount of fluorescent dye solution into the filter membrane for dyeing, and dropping a proper amount of glycerol solution after removing the fluorescent dye solution to prevent fluorescent quenching;
(7) counting by fluorescence microscope observation, and calculating the abundance of haemolymph microorganisms according to the following formula:
the main function of step (3) is to prevent blood coagulation. The most important component in blood that affects detection is blood cells. This method is particularly notable where the blood sample needs to be injected into the ACD anticoagulant immediately after the hemolymph is sampled, to prevent blood coagulation. Blood contains a blood coagulation component, and once blood coagulates, it cannot be detected.
Different from the detection of microorganisms in water samples and soil, invertebrate haemolymph contains a large amount of blood cells, the blood cells and the microorganism cells have no essential difference in chemical components and density, and the separation of the blood cells and the microorganism cells is difficult by a conventional pretreatment method, so that the problem of restricting the application of a fluorescence microscopic counting method is solved.
Through consideration and research, the great difference between the body types of blood cells and microorganisms becomes a separate breakthrough. The diameter of the blood cells is generally 6-15 μm, the diameter of the microorganisms is 1-2 μm, and the selected filter membrane has too large aperture, so that a large amount of blood cells can enter the filtrate after passing through the filter membrane, and the purpose of removing the blood cells cannot be achieved; the filter membrane has an excessively small aperture, so that part of microbial cells are trapped on the filter membrane, the loss of the microbial cells is caused, and the counting accuracy is further influenced. After many times of investigation, the blood cells are filtered and removed by a 5-micron filter membrane. All blood cells were filtered through a 5 μm filter, and the microorganisms in the blood were in the filtered filtrate. This filtrate containing the microorganisms is then filtered through a 0.2 μm filter, and since microorganisms are generally larger than 0.2 μm in diameter, these microorganisms are retained on the 0.2 μm filter and can be counted under a microscope after fluorescent staining. The purpose of the 5 μm filter was to filter out blood cells and the purpose of the 0.2 μm filter was to retain the microorganisms for visual counting. In this way, the hemolymph cells can be filtered to remove substances (including blood cells, hemocyanin, etc.) other than microorganisms, which interfere with microscopic observation and counting.
Preferably, the ACD anticoagulant is formulated as 450mM sodium chloride, 100mM glucose, 26mM citric acid, 30mM sodium citrate, adjusted to pH 4.6, sterilized with steam at 121 ℃ for 20min under a pressure of 1.05kg/cm2, and stored at 4 ℃.
Preferably, the invertebrate comprises one or more of scylla paramamosain, litopenaeus vannamei or ostrea viticola.
Preferably, the above invertebrate is used for hemolymph extraction at the following sites: the Scylla paramamosain is the podocarpus membrane of the second and third steps, the Litopenaeus vannamei is the abdominal blood sinus, and the portugal oyster is the adductor muscle. The parts are positioned on the body surface of the animal, contain a large amount of hemolymph and are easy to extract.
Preferably, the ACD anticoagulant is mixed in an amount to hemolymph volume ratio of 1: 1. At this time, the dilution factor was 1.
Preferably, the filtrate collected above is 200. mu.L, i.e., the loading is 200. mu.L.
Preferably, the fluorescent dye solution is SYBR Green I fluorescent dye solution, and is prepared as follows: 1:40v/v SYBR®Green I was dissolved in 1 × Tris-EDTA buffer.
Preferably, the glycerol solution is a 10% volume fraction glycerol solution.
Preferably, the fluorescent dye is added in an amount of 0.2. mu.L/mm2Dyeing time is 20min, and the dripping amount of glycerol solution is 0.05 mu L/mm2。
Preferably, the above-mentioned fluorescence microscope is used for observation and counting at 1000-fold fluorescence microscope, and 200 fields are counted for each filter.
The invention has the following advantages:
(1) the counting accuracy is high. The conventional methods based on pure culture techniques of microorganisms (such as plate colony counting method and dilution culture counting method) can only count culturable microorganisms in invertebrate hemolymph, and the invention can be used for directly counting all microorganisms including culturable microorganisms and uncultured microorganisms in invertebrate hemolymph more accurately. The method of the invention has about 2 orders of magnitude higher abundance of culturable microorganisms than the method of plate colony counting, and the coefficient of variation of the method of the invention is obviously lower than that of the plate colony counting.
(2) The anti-interference capability is strong. The blood cell diameter of the haemolymph is about 6-15 μm, and its abundance is greater than 106hemocytes/mL (FIGS. 1a, c, e). The diameter of the microorganism in the haemolymph is about 1-2 μm, and the abundance is less than 106cells/mL (FIG. 1b, d, f). The invention can effectively remove the interference of blood cells on the microbial cell counting by filtering a filter membrane with the aperture of 5 mu m (figure 1). The microorganisms which can be counted by the invention include all microorganisms in invertebrate hemolymph which can penetrate through a 5-micron filter membrane and be trapped by a 0.2-micron filter membrane, including culturable microorganisms and non-culturable microorganisms, including bacteria, archaea, fungi and the like.
(3) The result is quick. The method based on the pure culture technology of the microorganisms can obtain macroscopic colonies only within 24 hours, but the counting result can be obtained within 2 hours by applying the method, so that the time for counting the abundance of the haemolymph microorganisms is greatly shortened. Accurate statistics of the abundance of all microorganisms in invertebrate haemolymph is an important means to assess their health status.
(4) Can better grasp and control the steady state of the micro-ecosystem in the animal body. The method has higher counting accuracy and can better grasp and master the steady state of the microecosystem in the animal body. The conventional counting method may have a situation of misjudgment of the steady state due to low precision, which causes unnecessary loss to production and construction.
(5) The application is wide. The method can be applied to scientific research aspects such as symbiotic microbiome of invertebrates and the like, and can also be applied to daily disease detection work of breeding enterprises.
Drawings
FIG. 1 is a fluorescence microscope photograph of hemocytes and microorganisms before and after hemolymph of healthy Scylla paramamosain, Litopenaeus vannamei and Crassostrea vitis processed by the method of the present invention; wherein, the pictures a, c and e are respectively the hemolymph microscope photos of the scylla paramamosain, the litopenaeus vannamei and the portulaca oleracea, and the pictures b, d and f are respectively the microscope photos of the hemolymph of the three animals after being filtered by a filter membrane with the aperture of 5 mu m by using the method of the invention; in the figure, A is a microorganism, B is a blood cell, and the scale is 10 μm;
FIG. 2 is a graph showing the comparison of the method of the present invention with a plate colony counting method to count the abundance of hemolymph microorganisms of healthy individuals of Scylla paramamosain, Litopenaeus vannamei and Crassostrea viticola, respectively; each dot represents an animal, the black horizontal line represents the median, the asterisk represents statistical significance (man-whitney U-test, P < 0.05);
FIG. 3 is a graph showing the comparison of the method of the present invention with a plate colony counting method for counting the abundance of hemolymph microorganisms of dying blue crabs; each dot represents an animal, the black horizontal line represents the median, the asterisk represents statistical significance (man-whitney U-test, P < 0.05);
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1: the method is adopted to count the abundance of hemolymph microorganisms of healthy individuals of scylla paramamosain, litopenaeus vannamei and portuguese oyster
Selecting 6 scylla paramamosain (100.0 g/crab), 6 litopenaeus vannamei (7.5 g/crab) groups and 6 ostrea portugalis (39.7 g/oyster) groups from the same culture facility, wherein the blood extracted from 20 shrimps is mixed to serve as 1 group, and each individual is required to be healthy and active, have healthy appendages and have no attachments on the body surface. The method is adopted to count the abundance of the haemolymph microorganisms.
The method comprises the following operation steps:
(1) preparing ACD anticoagulant: the anticoagulant is prepared from sodium chloride 450mM, glucose 100mM, citric acid 26mM, and sodium citrate 30mM by adjusting pH to 4.6, sterilizing with high pressure steam, and storing at 4 deg.C.
(2) Animal disinfection: the whole body of the animal is wiped dry by using absorbent paper, then the surface of the blood-taking part is sprayed by using 75% ethanol, and the absorbent paper wipes off dirt attached to the surface of the blood-taking part. The above steps were repeated three times.
(3) Blood lymph sampling: a1 mL syringe rinsed with ACD anticoagulant is used for drawing hemolymph, and then the hemolymph is transferred into a centrifuge tube containing anticoagulant, and finally the volume ratio of the hemolymph to the anticoagulant is ensured to be 1: 1. The blood drawing part of the scylla paramamosain is the plantar ganglion membrane of the second step and the third step, the blood drawing part of the litopenaeus vannamei is the abdominal blood sinus of the scylla paramamosain, and the blood drawing part of the portuguese oyster is the adductor muscle of the portuguese concha.
(4) Removing blood cells: a mixture of 200. mu.L hemolymph and anticoagulant was filtered through a filter (Millipore, SX 0002500) containing a 5 μm pore size filter (Millipore, TMTP 02500) and the filtrate was collected in another sterile 1.5 mL centrifuge tube.
The number of blood cells in invertebrate haemolymph is about one hundred times that of microbial cells, the volume of the blood cells is several times that of the microbes (blood cell diameter 6-15 μm, microbe diameter 1-2 μm), and they can be used by common fluorescent dyes (including SYBR) on the market®Green I) staining, rendering it impossible to count directly under the microscope. As shown in fig. 1, before filtration, the microbial cells are covered by huge volume of blood cells and are difficult to see; after filtration, a large number of microorganisms, but in a small volume, are exposed, making it possible to observe the counts.
(5) Obtaining haemolymph microorganisms: the filtrate obtained in step (5) was filtered with a filter (Millipore, SX 0002500) containing a 0.2 μm pore size filter (Millipore, GTBP 02500), and then the 0.2 μm filter was transferred to a clean petri dish.
(6) Microbial staining and flaking: and (3) dripping 100 mu L of SYBR Green I fluorescent dye liquor (1: 40v/v SYBR Green I dissolved in 1 x Tris-EDTA buffer) into the filter membrane obtained in the step (6), and dyeing for 20min in a dark place. The staining solution was removed, the filter was placed on a glass slide, 25 μ L of 10% v/v glycerol solution was added dropwise to prevent fluorescence quenching, and the slide was covered.
(7) Counting by a fluorescence microscope: the slides were transferred to a 1000-fold fluorescence microscope (Nikon, ECLIPSE 90i) and observed with a blue filter, where the microbial cells were green-emitting. Each film was counted for 200 fields. Finally, the abundance of haemolymph microorganisms was calculated according to the following formula:
the total time spent: for 2 hours.
Comparative example 1: counting the abundance of hemolymph microorganisms of healthy individuals of Scylla paramamosain, Litopenaeus vannamei and Crassostrea viticola by using a plate colony counting method
The same scylla paramamosain 6 (-100.0 g/crab), litopenaeus vannamei 6 (-7.5 g/shrimp, 1 group of blood extracted from 20 shrimps is mixed, and oyster 6 (39.7 g/shrimp) are selected, and each individual is required to be healthy and active, have healthy appendages and have no attachments on the body surface. The difference from the embodiment 1 is that: and (4) counting the abundance of the haemolymph microorganisms by adopting a plate colony counting method.
The operation steps of the plate colony counting method are as follows:
(1) the animal sterilization and hemolymph sampling procedures were the same as in example 1.
(2) Preparing 2216E solid flat plates: 2216E solid culture medium formula, 5 g/L peptone, 1 g/L yeast extract, 0.1 g/L ferric chloride, 20 g/L sodium chloride, 15 g/L agar, adding 1L distilled water for dissolving, adjusting pH to 7.6, autoclaving, and pouring into sterile plate when the culture medium is cooled to about 45 deg.C.
(3) Coating: the 100 mu L of mixed solution of hemolymph and anticoagulant is absorbed by a pipette and inoculated on a 2216E solid plate, the bacteria solution is evenly coated on the plate by a sterile glass scraper, and the plate is inversely cultured in a 30 ℃ incubator.
(4) Counting: after 24 hours of plate culture, the number of colonies on each plate was counted, and the abundance of total culturable bacteria (CFU/mL) = the number of colonies on the plate × 10 × 2 (ensuring 30-300 colonies in one plate) was calculated using the following formula.
The total time spent: 1-2 days.
Comparing the method of the invention with a plate colony counting method to respectively count the abundance of hemolymph microorganisms of healthy scylla paramamosain, litopenaeus vannamei and portuguese oyster
The results of comparing example 1 with comparative example 1 are as follows:
as shown in fig. 2: the average value of the abundance of the haemolymph microorganisms of healthy individuals of the scylla paramamosain, the litopenaeus vannamei and the ostrea portulacea counted by the method is 2.6 multiplied by 104 cells/mL、1.3×105 cells/mL、1.7×105cells/mL, the coefficient of variation is respectively 16%, 11% and 7%; the average value of the abundance of hemolymph culturable bacteria of healthy individuals of Scylla paramamosain, Litopenaeus vannamei and Crassostrea viticola calculated by using a plate colony counting method is 6.6 multiplied by 102 CFU/mL、5.1×103 CFU/mL、4.5×102 CFU/mL, coefficient of variation is 92%, 66%, 58% respectively. Wherein the variation coefficient of the Scylla paramamosain experimental group is up to 92%, and the haemolymph microorganism abundance of healthy individuals counted by a plate colony counting method is from 55 CFU/mL to 1.7 multiplied by 103 CFU/mL, there is a significant error.
Compared with the comparative example 1, the result shows that the method can be applied to the abundance count of the haemolymph microorganisms of the scylla paramamosain, the litopenaeus vannamei and the ostrea portulaca, and compared with a flat plate bacterial colony counting method, the method has the advantages of more accurate abundance count of the microorganisms in the haemolymph, smaller variation coefficient and shorter total consumption time.
Example 2: the method of the invention counts the abundance of the dying scylla paramamosain hemolymph microorganisms
Selecting 6 dying scylla paramamosain from the same culture facility as the embodiment 1 (-100.0 g/crab), and requiring that each individual is in an expiring state, and only the mouth breathes and the foot hardly moves. The method is adopted to count the abundance of the haemolymph microorganisms, and the specific operation steps refer to example 1.
Comparative example 2: counting the abundance of the dying scylla paramamosain hemolymph microorganisms by adopting a flat plate colony counting method
The same dying pauperus scholaris 6 (-100.0 g/pauperus scholaris) as the same as the embodiment 2 is selected, each individual is required to be in an expiring state, and only the mouth breathes and the foot hardly moves. The difference from the embodiment 2 is that: and (4) counting the abundance of the haemolymph microorganisms by adopting a plate colony counting method. The specific procedure was as in comparative example 1.
Comparing the method of the invention with the plate colony counting method to respectively count the abundance of the haemolymph microorganisms of the dying Scylla paramamosain
The results of comparing example 2 with comparative example 2 are as follows:
as shown in fig. 3: the average value of the abundance of the moribund scylla paramamosain hemolymph microorganisms counted by the method is 3.6 multiplied by 106cells/mL, 2.6X 10 abundance of haemolymph microorganisms over healthy individuals4cells/mL (see example 1) is about 138 times higher; the average value of the abundance of the moribund scylla paramamosain hemolymph microorganisms counted by using a plate colony counting method is 3.9 multiplied by 103 CFU/mL, 6.6X 10 abundance of haemolymph microorganisms over healthy individuals2 CFU/mL (see comparative example 1) was about 6 times higher.
However, the abundance of haemolymph microorganisms in individual healthy individuals was as high as 1.7X 10 by plate colony counting3 CFU/mL, average of haemolymph microbial abundance from dying individual 3.9X 103 The CFU/mL is close, which can cause the occurrence of the event that healthy blue crabs are mistaken for diseased blue crabs or the diseased blue crabs are mistaken for healthy blue crabs in the breeding production process.
The comparison between the example 2 and the comparative example 2 shows that compared with a flat plate colony counting method, the method has higher detection sensitivity on the abundance of the haemolymph microorganisms of the dying scylla paramamosain and is less prone to misjudgment.
While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
Claims (10)
1. A method for accurately counting the abundance of microorganisms in invertebrate hemolymph comprising the steps of:
(1) preparing ACD anticoagulant, adjusting pH to 4.6, sterilizing with high pressure steam, and storing at low temperature;
(2) sterilizing invertebrates, and repeating for multiple times;
(3) collecting hemolymph from invertebrate suitable part, and rapidly mixing with ACD anticoagulant;
(4) filtering with a filter membrane with the aperture of 5 mu m, and collecting the filtrate;
(5) filtering with a filter membrane with the aperture of 0.2 mu m, and transferring the filter membrane into a clean culture dish;
(6) dropping a fluorescent dye solution into the filter membrane for dyeing when the light is avoided, and dropping a glycerol solution after the fluorescent dye solution is removed;
(7) counting by fluorescence microscope observation, and calculating the abundance of haemolymph microorganisms according to the following formula:
2. the method for accurately counting the abundance of microorganisms in invertebrate hemolymph according to claim 1, wherein step (1) comprises formulating the ACD anticoagulant with 450mM NaCl, 100mM glucose, 26mM citrate, 30mM sodium citrate, adjusted pH 4.6 at 1.05kg/cm2Sterilizing with 121 deg.C steam under pressure for 20min, and storing at 4 deg.C.
3. The method of claim 1, wherein the invertebrate in step (3) comprises one or more of scylla paramamosain, litopenaeus vannamei or ostrea portulacea.
4. The method for accurately counting the abundance of microorganisms in invertebrate hemolymph according to claim 1 or 3, wherein the invertebrate hemolymph is drawn at the following positions: the Scylla paramamosain is the podocarpus membrane of the second and third steps, the Litopenaeus vannamei is the abdominal blood sinus, and the Ostrea portulaca is the adductor muscle.
5. The method for accurately counting the abundance of microorganisms in invertebrate hemolymph according to claim 1, wherein the amount of ACD anticoagulant to hemolymph in step (3) is in a volume ratio of 1: 1.
6. The method of claim 1, wherein the collection filtrate of step (4) is 200 μ L.
7. The method of claim 1, wherein the fluorescent staining solution of step (6) is a fluorescent staining solution
Green I fluorescent dye solution is prepared as follows: 1:40v/v
Green I was dissolved in 1 × Tris-EDTA buffer.
8. The method for accurately counting the abundance of microorganisms in invertebrate hemolymph according to claim 1, wherein the glycerol solution of step (6) is a 10% volume fraction glycerol solution.
9. The method for accurately counting the abundance of microorganisms in invertebrate hemolymph as claimed in claim 1, wherein the amount of fluorescent dye added in step (6) is 0.2 μ L/mm2Dyeing time is 20min, and the dripping amount of glycerol solution is 0.05 mu L/mm2。
10. The method for accurately counting the abundance of microorganisms in invertebrate hemolymph as claimed in claim 1, wherein step (7) uses a 1000-fold fluorescence microscope to count 200 fields per filter.
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