CN109055481B - Detection method for attached bacteria on body surface of spindle water fleas - Google Patents
Detection method for attached bacteria on body surface of spindle water fleas Download PDFInfo
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Abstract
The invention discloses a detection method of attached bacteria on the body surface of daphnia fusiforme, which comprises the following steps of pretreatment, desorption and detection: pretreatment: placing the spiny daphnia collected in a target sea area on a sterile filter screen, washing the swordlike daphnia on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen; desorbing: putting the pretreated daphnia pulchella into a centrifugal tube filled with sterile water, adding a desorbent solution, performing ultrasonic treatment and then performing centrifugal treatment, and taking centrifugal supernatant for later use; and (3) detection: and (4) performing bacterial culture on the centrifugal supernatant obtained in the desorption step by using a method for measuring the total number of bacteria in a standard test method for domestic drinking water, and counting the cultured colonies. The detection method provided by the invention has the advantages of simple operation steps and short detection period, so that the desorption efficiency of bacteria attached to the body surface is high, the speed is high, the desorption is more thorough, the consumption of the desorbent is small, the accuracy of the detection method is high, and the detection result is stable and reliable.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a method for detecting bacteria attached to the body surface of daphnia fusiforme.
Technical Field
Spiny daphnia is widely distributed in the ocean and is a main natural bait organism for numerous economic fishes, shrimps and crabs. According to FAO estimates, in the 1999 marine fishery industry, 0.92 million tons of fish were harvested from fish that feed on natural copepods. The daphnia pulchra is widely distributed in coastal areas, Japanese coastal areas, Pacific areas and Indian oceans in China, is one of the dominant species of zooplankton in summer in Zhoushan fishery and adjacent sea areas, and is suitable for being applied to aquaculture due to the characteristics of short life history, wide bait pair and the like. The nutritional value of daphnia fusiformis n-3 highly unsaturated fatty acids (n-3HUPA) are essential fatty acids of seawater larvae and are essential for the normal growth of seawater larvae, especially timnodonic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6 n-3). The lack or deficiency of EPA and DHA affects the growth and development of fish and shrimp larvae, reduces the survival rate, has insufficient vitality and weakens the stress resistance. Seawater fish cannot biologically synthesize linolenic acid (18:3n-3) into EPA and DHA, and the needed EPA and DHA can only be taken in feed. Numerous studies have shown that many coastal copepods are rich in DHA and EPA (accounting for approximately 60% of the fatty acids present) and are able to meet HUFA requirements for larval fish. However, the spiny water fleas collected in the natural sea area carry a plurality of pathogenic bacteria, and the direct feeding of the spiny water fleas is easy to cause the propagation of breeding diseases, so the condition of carrying the bacteria of the spiny water fleas needs to be determined in advance.
At present, the method for detecting bacteria attached to the body surfaces of the invertebrate zooplankton in water at home and abroad mainly adopts the method proposed by Wolman. The method comprises soaking zooplankton in distilled water or normal saline, naturally releasing bacteria attached to zooplankton surface into water to obtain sample with bacteria attached to surface, and optionally using auxiliary means such as hand shaking or shaking table oscillation to increase release efficiency. However, the desorption efficiency of bacteria adhering to the body surfaces of zooplankton is still not high, and the culture medium culture detection is carried out after the bacteria adhering to the body surfaces of the daphnia zooplankton are desorbed by the method, so that the defects that the detection value of the bacteria adhering to the body surfaces of the daphnia zooplankton is low, the fluctuation is large and the like exist. The prior art discloses a method for detecting bacteria attached to the body surface of cyclops zooplankton, such as a Chinese patent with an authorized publication number of CN 103451263B, which comprises the following steps: step 1, taking cyclops in a water sample, and washing the cyclops until no bacteria are detected; step 2, putting the cyclops obtained in the step 1 into a centrifugal tube, and adding a desorbent; step 3, placing the centrifuge tube in the step 2 into a centrifuge for centrifugal treatment; and 4, taking the centrifuged supernatant, carrying out bacterial culture by using a method for measuring the total number of bacteria in a standard test method for drinking water, and counting the cultured colonies. The method has simple operation steps and short detection period, and has obvious detection effect on bacteria attached to the body surfaces of cyclops zooplankton. However, the effect of this method of detection on the desorption of bacteria attached to the surface of the zooplankton such as daphnia is still not ideal.
Disclosure of Invention
One of the purposes of the invention is to provide the detection method for the bacteria attached to the body surfaces of the water fleas, which has the advantages of simple operation steps, short detection period, high desorption efficiency and speed of the bacteria attached to the body surfaces, more thorough desorption, less consumption of the desorbent, high accuracy of the detection method, and stable and reliable detection results.
Aiming at the problems mentioned in the background technology, the invention adopts the technical scheme that:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises pretreatment, desorption and detection, wherein the desorption step is as follows: putting the pretreated daphnia pulcherrima into a 50mL centrifuge tube filled with 25mL of sterile water, adding 0.8-1.2mL of desorbent solution, carrying out ultrasonic treatment for 12-15min under the ultrasonic power of 330-. In the desorption step, the desorption agent, the ultrasonic treatment and the centrifugal treatment are combined to desorb the bacteria attached to the body surfaces of the water fleas, compared with the prior art, the desorption efficiency of the bacteria attached to the body surfaces is high, the speed is high, the desorption is more thorough, the consumption of the desorption agent is less, the detection method is high in accuracy and stable and reliable in detection result, the detection method is simple in operation step and short in detection period, and the detection effect on the bacteria attached to the body surfaces of the water fleas zooplankton is remarkable.
Preferably, the surfactant for desorbent comprises rhamnolipid and tween 80. Bacteria attached to the body surface of the spindle water flea mainly depend on the fact that extracellular polymers are secreted by microorganisms to form a biological membrane which is attached to the body surface of the spindle water flea, reasonable existence of rhamnolipid and tween 80 can generate a gain effect on other components in the desorbent, a hydrophilic membrane outside the biological membrane can be damaged, the spatial three-dimensional structure of the biological membrane is further damaged, disintegration of the bacterial biological membrane is promoted, then the biological membrane falls off and disintegrates by matching with a centrifugal effect, and further, microorganisms in the biological membrane can be completely scattered into the desorbent, so that the accuracy of the detection method is improved.
Further preferably, the surfactant also comprises D-rhamnose, and the mass ratio of rhamnolipid, tween 80 and D-rhamnose in the surfactant is 1:10-12: 0.03-0.05. The mixing combination of rhamnolipid, tween 80 and D-rhamnose in a special proportion in the surfactant obviously reduces the precipitation/adsorption loss of an anionic nonionic surfactant and improves the effective concentration of the surfactant, so that the desorption of the bacteria attached to the body surfaces of the water fleas is synergistically enhanced, the desorption of the bacteria attached to the body surfaces of the spindle water fleas is enhanced, the dosage of the desorbent is obviously reduced, the concentration of the attached bacteria in the solution can be synergistically increased, and the resolution of the attached bacteria is improved.
Preferably, the desorbent further contains a D-amino acid. The D-amino acid can occupy the hydrophilic and hydrophobic point positions of the surface of the bacteria, changes the surface hydrophobicity of the bacteria, and improves the surface hydrophilicity and the Zeta potential absolute value of the bacteria released in the biomembrane, so that the bacteria attached to the surface of the daphnia pulmona can be quickly dissolved in the bacteria under the action of the release agent, and the D-amino acid has no obvious influence on the growth activity of the bacteria, thereby ensuring the stability and the accuracy of the detection result.
Further preferably, the D-amino acid is selected from D-tyrosine, D-leucine, D-isoleucine, D-phenylalanine or D-methionine.
In order to optimize the technical method, the measures taken also include: the desorbent comprises the following components in parts by weight: 0.8 to 1.0 portion of sodium chloride, 0.03 to 0.05 portion of surfactant, 0.1 to 0.25 portion of disodium hydrogen phosphate, 0.2 to 0.4 portion of monopotassium phosphate and 0.01 to 0.02 portion of D-amino acid. The effective components in the desorbent can play a synergistic role, and the thorough and efficient desorption of bacteria attached to the body surfaces of the daphnia fusiformis can be realized under the condition of a small amount of action.
Preferably, the concentration of the desorbent solution is 1.14 to 1.72%.
Preferably, the pretreatment comprises the following specific steps: and (3) placing the spindley daphnia collected in the target sea area on the sterile filter screen, washing the spindley daphnia on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen. Wash spindle water flea that target sea area was collected is the influence of the free bacterium to the attached bacterium detection of spindle water flea body surface in order to avoid the sample in-process water sample, and this preliminary treatment step enables to make free bacterium wash thoroughly, shortens the cleaning time, avoids the too much loss of body surface bacterium in the cleaning process again simultaneously.
Preferably, the detection comprises the following specific steps: and (4) performing bacterial culture on the centrifugal supernatant obtained in the desorption step by using a method for measuring the total number of bacteria in a standard test method for domestic drinking water, and counting the cultured colonies.
Further preferably, the detection of bacteria in the pretreatment and detection steps is performed according to the method for determining the total number of bacteria in GB/T5750.12-2006.
Compared with the prior art, the invention has the advantages that: 1) the detection method has the advantages of simple operation steps, short detection period, obvious detection effect on bacteria attached to the body surfaces of the spiny water flea zooplankton, high accuracy and stable and reliable detection result; 2) the desorption step of the invention ensures that the desorption efficiency of bacteria attached to the body surface is high, the speed is high, the desorption is more thorough, and the dosage of the desorbent is less, thereby ensuring that the detection method of the invention has high accuracy and stable and reliable detection result; 3) the method can realize thorough and efficient desorption of bacteria attached to the body surfaces of the daphnia fusiforme under the condition of less action by utilizing the synergistic effect of all effective components in the desorbent, and can ensure the stability and accuracy of a detection result; 4) the detection method can be used for detecting bacteria in other zooplankton bodies through proper improvement, has wider application range, stronger practicability and higher operability, and is suitable for wide popularization and application.
Detailed Description
The scheme of the invention is further illustrated by the following examples:
example 1:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises pretreatment, desorption and detection, wherein the desorption step is as follows: putting the pretreated daphnia pulchella into a 50mL centrifuge tube filled with 25mL of sterile water, adding 0.8mL of desorbent solution, carrying out ultrasonic treatment for 12min under the ultrasonic power of 330W, then carrying out centrifugal treatment for 2min at 1800rpm and 20 ℃, and taking centrifugal supernatant for later use after the centrifugal treatment. This desorption step adopts desorbent, ultrasonic treatment and centrifugal treatment mode that combines together to carry out the desorption to the attached bacterium of spindle water flea body surface, and prior art for the body surface is attached the bacterium desorption efficiency height, fast, the desorption is more thorough, the desorbent quantity is few, and then makes the detection method degree of accuracy high, the testing result is reliable and stable, and this detection method operating procedure is simple, the detection cycle is short, and is showing to the detection effect of the attached bacterium of spindle water flea class zooplankton body surface.
The above-mentioned surfactant for desorbent comprises rhamnolipid and tween 80. Bacteria attached to the body surface of the spindle water flea mainly depend on the fact that extracellular polymers are secreted by microorganisms to form a biological membrane which is attached to the body surface of the spindle water flea, reasonable existence of rhamnolipid and tween 80 can generate a gain effect on other components in the desorbent, a hydrophilic membrane outside the biological membrane can be damaged, the spatial three-dimensional structure of the biological membrane is further damaged, disintegration of the bacterial biological membrane is promoted, then the biological membrane falls off and disintegrates by matching with a centrifugal effect, and further, microorganisms in the biological membrane can be completely scattered into the desorbent, so that the accuracy of the detection method is improved.
The surfactant also comprises D-rhamnose, and the mass ratio of rhamnolipid, tween 80 and D-rhamnose in the surfactant is 1:10: 0.03. The mixing combination of rhamnolipid, tween 80 and D-rhamnose in a special proportion in the surfactant obviously reduces the precipitation/adsorption loss of an anionic nonionic surfactant and improves the effective concentration of the surfactant, so that the desorption of the bacteria attached to the body surfaces of the water fleas is synergistically enhanced, the desorption of the bacteria attached to the body surfaces of the spindle water fleas is enhanced, the dosage of the desorbent is obviously reduced, the concentration of the attached bacteria in the solution can be synergistically increased, and the resolution of the attached bacteria is improved.
The desorbent also contains D-amino acid. The D-amino acid can occupy the hydrophilic and hydrophobic point positions of the surface of the bacteria, changes the surface hydrophobicity of the bacteria, and improves the surface hydrophilicity and the Zeta potential absolute value of the bacteria released in the biomembrane, so that the bacteria attached to the surface of the daphnia pulmona can be quickly dissolved in the bacteria under the action of the release agent, and the D-amino acid has no obvious influence on the growth activity of the bacteria, thereby ensuring the stability and the accuracy of the detection result.
Wherein the D-amino acid is selected from D-tyrosine, D-leucine, D-isoleucine, D-phenylalanine or D-methionine.
The desorbent comprises the following components in parts by weight: 0.8 part of sodium chloride, 0.03 part of surfactant, 0.1 part of disodium hydrogen phosphate, 0.2 part of monopotassium phosphate and 0.01 part of D-tyrosine. The effective components in the desorbent can play a synergistic role, and the thorough and efficient desorption of bacteria attached to the body surfaces of the daphnia fusiformis can be realized under the condition of a small amount of action.
Wherein the concentration of the desorbent solution is 1.14%.
The pretreatment comprises the following specific steps: and (3) placing the spindley daphnia collected in the target sea area on the sterile filter screen, washing the spindley daphnia on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen. Wash spindle water flea that target sea area was collected is the influence of the free bacterium to the attached bacterium detection of spindle water flea body surface in order to avoid the sample in-process water sample, and this preliminary treatment step enables to make free bacterium wash thoroughly, shortens the cleaning time, avoids the too much loss of body surface bacterium in the cleaning process again simultaneously.
The detection method comprises the following specific steps: the centrifugal supernatant obtained in the desorption step was subjected to bacterial culture by the method for measuring the total number of bacteria in the standard test method for drinking water, and the number of colonies after the culture was counted, as shown in Table 1.
Wherein, the bacteria detection in the pretreatment and detection steps is carried out according to the method for measuring the total number of bacteria in GB/T5750.12-2006.
Example 2:
a detection method for attached bacteria on the body surface of spindle water fleas comprises pretreatment, desorption and detection, and specifically comprises the following steps:
1) the pretreatment comprises the following specific steps: collecting spindle water fleas collected in a target sea area, placing the spindle water fleas on a sterile filter screen, washing the spindle water fleas on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen;
2) desorbing: putting the pretreated daphnia pulchella into a 50mL centrifuge tube filled with 25mL of sterile water, adding 1.0mL of a desorbent solution with the concentration of 1.45%, carrying out ultrasonic treatment for 14min under the ultrasonic power of 350W, then carrying out centrifugal treatment for 3min at 2000rpm and 25 ℃, and taking a centrifugal supernatant for later use after the centrifugal treatment;
3) the detection method comprises the following specific steps: the centrifugal supernatant obtained in the desorption step was subjected to bacterial culture by the method for measuring the total number of bacteria in the standard test method for drinking water, and the number of colonies after the culture was counted, as shown in Table 1.
Wherein the desorbent comprises the following components in parts by weight: 0.9 part of sodium chloride, 0.04 part of surfactant, 0.18 part of disodium hydrogen phosphate, 0.3 part of monopotassium phosphate and 0.015 part of D-amino acid. The mass ratio of rhamnolipid, tween 80 and D-rhamnose in the surfactant is 1:11: 0.04; the D-amino acid is D-tyrosine and D-leucine with the mass ratio of 1: 0.25.
Wherein, the bacteria detection in the pretreatment and detection steps is carried out according to the method for measuring the total number of bacteria in GB/T5750.12-2006.
Example 3:
a detection method for attached bacteria on the body surface of spindle water fleas comprises pretreatment, desorption and detection, and specifically comprises the following steps:
1) the pretreatment comprises the following specific steps: collecting spindle water fleas collected in a target sea area, placing the spindle water fleas on a sterile filter screen, washing the spindle water fleas on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen;
2) desorbing: putting the pretreated daphnia pulchella into a 50mL centrifuge tube filled with 25mL of sterile water, adding 1.2mL of a desorbent solution with the concentration of 1.72%, carrying out ultrasonic treatment for 15min under the ultrasonic power of 380W, then carrying out centrifugal treatment for 5min at 2300rpm and 30 ℃, and taking a centrifugal supernatant for later use after the centrifugal treatment;
3) the detection method comprises the following specific steps: the centrifugal supernatant obtained in the desorption step was subjected to bacterial culture by the method for measuring the total number of bacteria in the standard test method for drinking water, and the number of colonies after the culture was counted, as shown in Table 1.
The desorbent comprises the following components in parts by weight: 1.0 part of sodium chloride, 0.05 part of surfactant, 0.25 part of disodium hydrogen phosphate, 0.4 part of monopotassium phosphate and 0.02 part of D-amino acid. The mass ratio of rhamnolipid, tween 80 and D-rhamnose in the surfactant is 1:12: 0.05; the D-amino acid is D-phenylalanine.
Wherein, the bacteria detection in the pretreatment and detection steps is carried out according to the method for measuring the total number of bacteria in GB/T5750.12-2006.
Comparative example 1:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises the following components in parts by weight in the desorption step: 0.9 part of sodium chloride, 0.04 part of surfactant, 0.18 part of disodium hydrogen phosphate, 0.3 part of monopotassium phosphate and 0.015 part of D-amino acid. The mass ratio of the Tween 80 to the D-rhamnose in the surfactant is 12: 0.04; the D-amino acid is D-tyrosine and D-leucine with the mass ratio of 1: 0.25. The rest of the steps are completely consistent with those of example 2, and the detection results are shown in Table 1.
Comparative example 2:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises the following components in parts by weight in the desorption step: 0.9 part of sodium chloride, 0.04 part of surfactant, 0.18 part of disodium hydrogen phosphate, 0.3 part of monopotassium phosphate and 0.015 part of D-amino acid. The mass ratio of rhamnolipid to D-rhamnose in the surfactant is 12: 0.04; the D-amino acid is D-tyrosine and D-leucine with the mass ratio of 1: 0.25. The rest of the steps are completely consistent with those of example 2, and the detection results are shown in Table 1.
Comparative example 3:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises the following components in parts by weight in the desorption step: 0.9 part of sodium chloride, 0.04 part of surfactant, 0.18 part of disodium hydrogen phosphate, 0.3 part of monopotassium phosphate and 0.015 part of D-amino acid. The mass ratio of rhamnolipid to tween 80 in the surfactant is 1: 11; the D-amino acid is D-tyrosine and D-leucine with the mass ratio of 1: 0.25. The rest of the steps are completely consistent with those of example 2, and the detection results are shown in Table 1.
Comparative example 4:
a detection method for attached bacteria on the body surface of daphnia fusiforme comprises the following components in parts by weight in the desorption step: 0.9 part of sodium chloride, 0.04 part of surfactant, 0.18 part of disodium hydrogen phosphate and 0.3 part of monopotassium phosphate. The mass ratio of rhamnolipid, tween 80 and D-rhamnose in the surfactant is 1:11: 0.04. The rest of the steps are completely consistent with those of example 2, and the detection results are shown in Table 1.
TABLE 1 detection results of bacteria attached to the body surface of Spodoptera
Comparing the data of Table 1, it can be seen that the total amount of bacteria attached to the surface of the daphnia fusiformis measured by the method of the present invention is significantly greater than the test method of the comparative example. The detection result of the invention is stable, the data obtained by 4 times of parallel tests are relatively close, and the difference of each detection result is more than 30 CFU. Meanwhile, the invention can play a synergistic role among all effective components in the desorbent, and can realize thorough and efficient desorption of bacteria attached to the body surfaces of the spindle water fleas under the condition of a small amount of action.
The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A detection method for attached bacteria on the body surface of spindle water fleas comprises pretreatment, desorption and detection, and is characterized in that:
the pretreatment steps are as follows: collecting spindle water fleas collected in a target sea area, placing the spindle water fleas on a sterile filter screen, washing the spindle water fleas on the sterile filter screen by using sterile ultrapure water until bacteria cannot be detected in washing water flowing out of the filter screen;
the desorption step is as follows: putting the pretreated daphnia pulmona into a 50mL centrifuge tube filled with 25mL of sterile water, adding 0.8-1.2mL of desorbent solution with the concentration of 1.14-1.72%, performing ultrasonic treatment for 12-15min under the ultrasonic power of 330-380W, then performing centrifugal treatment for 2-5min at the temperature of 20-30 ℃ at the speed of 1800-2300rpm, and taking the centrifugal supernatant for later use after the centrifugation is finished; the desorbent comprises the following components in parts by weight: 0.8-1.0 part of sodium chloride, 0.03-0.05 part of surfactant, 0.1-0.25 part of disodium hydrogen phosphate, 0.2-0.4 part of monopotassium phosphate and 0.01-0.02 part of D-amino acid, wherein the surfactant comprises rhamnolipid, tween 80 and D-rhamnose in a mass ratio of 1:10-12: 0.03-0.05;
the detection steps are as follows: and (4) performing bacterial culture on the centrifugal supernatant obtained in the desorption step by using a method for measuring the total number of bacteria in a standard test method for domestic drinking water, and counting the cultured colonies.
2. The method for detecting the bacteria attached to the body surfaces of the spindle water fleas according to claim 1, which is characterized in that: the D-amino acid is selected from D-tyrosine, D-leucine, D-isoleucine, D-phenylalanine or D-methionine.
3. The method for detecting the bacteria attached to the body surfaces of the spindle water fleas according to claim 1, which is characterized in that: the bacteria detection is carried out according to the method for measuring the total number of bacteria in GB/T5750.12-2006.
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