CN110257304B - Long-term preservation method of marine bdellovibrio - Google Patents
Long-term preservation method of marine bdellovibrio Download PDFInfo
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Abstract
The invention relates to the technical field of strain preservation, and discloses a long-term preservation method of marine bdellovibrio, which comprises the steps of preparing host strain seawater suspension, preparing marine bdellovibrio seawater suspension and preserving strains. The invention breaks through the traditional concept of storing Bdellovibrio for a long time at low temperature, realizes long-term storage of Bdellovibrio under normal temperature, has simple storage method and good storage effect, and saves the cost of long-term storage.
Description
Technical Field
The invention relates to the technical field of strain preservation, in particular to a long-term preservation method of marine bdellovibrio.
Background
Bdellovibrio organisms, called Bdellovibrio for short, are small bacteria capable of lysing other bacteria. The unique predation characteristic of Bdellovibrio makes the Bdellovibrio strain capable of being used as a 'living antibiotic', has remarkable application value in replacing antibiotics to control harmful bacteria, and has certain application in production practice.
Marine bdellovibrio can crack various gram-negative bacteria, has preferential cracking to the vibrio, and has obvious advantages in controlling the vibrio and diseases caused by the vibrio. However, the Bdellovibrio including marine Bdellovibrio is difficult to simply and effectively preserve for a long time due to the general specialization of the Bdellovibrio, and particularly, the Bdellovibrio is difficult to maintain higher viable bacteria content under normal temperature conditions, so that the application of the Bdellovibrio is severely restricted; on the other hand, the preservation research of the existing Bdellovibrio is mostly focused on Liu Shengzhi vibrio, and the Bdellovibrio is preserved basically by adopting a low-temperature preservation method, so that the preservation effect at normal temperature is not ideal, the preservation cost is high, and the specific preservation condition of the marine Bdellovibrio is still fresh and reported at present. There is therefore an urgent need to develop a simple and effective long-term preservation method for marine bdellovibrio.
Disclosure of Invention
Based on the problems, the invention provides a long-term preservation method of marine bdellovibrio, which is suitable for preserving the strain of Halobacteriovorax at normal temperature, has good preservation effect and is simple in preservation method.
In order to solve the technical problems, the invention provides a long-term preservation method of marine bdellovibrio, which comprises the following steps:
s1: preparation of host bacteria seawater suspension
Preparing a SWYE liquid culture medium, inoculating host bacteria for storing marine Bdellovibrio into the SWYE liquid culture medium, shaking culturing, centrifuging the SWYE liquid culture medium containing the host bacteria after the end of the logarithm period, rinsing with sterile seawater twice to obtain host bacteria culture bacteria, suspending the host bacteria culture bacteria in the sterile seawater to prepare a host bacteria seawater suspension, and regulating the bacterial liquid concentration in the host bacteria seawater suspension to be 10 6 cfu/mL or 10 7 cfu/mL；
S2: preparation of sea Bdellovibrio seawater suspension
The concentration of the prepared bacterial liquid is 5 multiplied by 10 8 Inoculating marine Bdellovibrio into the host bacteria seawater suspension, shaking culturing at 30deg.C until the host bacteria are completely cracked and contain a large amount of Bdellovibrio in the offensive period, centrifuging culture solution under 1000g condition for 10min, collecting supernatant, filtering twice with 0.45 μm filter membrane, centrifuging under 12000g condition for 20min, discarding supernatant, rinsing the centrifuged precipitate with sterile seawater, centrifuging under 12000g condition for 20min, suspending marine Bdellovibrio culture bacteria in sterile seawater to obtain marine Bdellovibrio seawater suspension, and regulating bacterial liquid concentration of marine Bdellovibrio seawater suspension to 10 8 pfu/mL or 3.33X10 7 pfu/mL or 10 7 pfu/mL；
S3: strain preservation
Mixing the sea Bdellovibrio sea water suspension prepared in the step S2 and the host bacteria sea water suspension prepared in the step S1 together in equal volume, filling 4mL of the mixed solution into a 5mL sterile nut tube, and storing the sterile nut tube filled with the mixed solution at 15-37 ℃.
Further, the host bacteria in the step S1 and the step S2 are vibrio alginolyticus.
Further, the marine Bdellovibrio in step S2 is a strain of the genus Halobaeriovirax.
Further, the salinity of the sterile seawater in the step S1 and the step S2 is 25-35, the pH is 7-9, and the sterile seawater is subjected to filtration and sterilization treatment.
Further, the salinity of the sterile seawater in the step S1 and the step S2 is 29.8-30.2, the pH is 7.8-8.2, and the sterile seawater is subjected to filtration and sterilization treatment.
Further, the preservation temperature in the step S3 is 20 ℃, and the bacterial liquid concentration of the selected marine Bdellovibrio sea water suspension is 3.33X10 7 The bacterial liquid concentration of the seawater suspension of the host bacteria selected by pfu/mL is 10 6 cfu/mL。
Further, the preservation temperature in the step S3 is 20-30 ℃, and the bacterial liquid concentration of the selected marine bdellovibrio bacteriovorus seawater suspension is 10 7 The bacterial liquid concentration of the seawater suspension of the host bacteria selected by pfu/mL is 10 7 cfu/mL。
Compared with the prior art, the invention has the beneficial effects that: the invention breaks through the traditional concept of storing Bdellovibrio at low temperature for a long time, realizes long-term storage of Bdellovibrio, in particular to the strain of Halobaeriovirax, under the normal temperature condition, has simple storage method and good storage effect, and saves the cost of long-term storage.
Drawings
FIG. 1 is a block diagram of a preservation method of the present invention;
FIG. 2 is a graph showing the results of the change in the number of Halobaeriovirax strains in example 1 of the present invention;
FIG. 3 is a graph showing the results of the change in the number of Halobactriovorax species in example 2 of the present invention.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
Example 1:
in this example, the strain of Halobactiovirax is selected as the species of Bdellovibrio maritimus to be preserved, the Chinese translation name of Halobactiovirax is halophilic vibrio, and Vibrio parahaemolyticus is selected as the host strain for counting the marine Bdellovibrio maritimus by double-layer plates.
Sterile seawater is prepared, the salinity of the sterile seawater is 25-35, the pH is 7-9, and the sterile seawater is subjected to filtration and sterilization treatment, wherein the salinity of the sterile seawater in the embodiment is 29.8-30.2, and the pH is 7.8-8.2.
SWYE medium was prepared as follows: taking 24g of NaCl and MgCl 7H 2 O 7g、KCl 0.7g、MgCl·6H 2 O5.3 g, distilled water 1L were prepared into a tetra-salt solution having a pH of 7.4-7.8, and then 10g of peptone and 3g of yeast extract were taken, the tetra-salt solution was used to fix the volume to 1L, the pH was adjusted to 7.2-7.4, and the mixture was autoclaved at 121℃for 20 minutes to prepare a culture of the host bacteria of this example.
Preparing seawater double-layer agar culture medium, wherein the bottom layer of the seawater double-layer agar culture medium contains 1.2% agar, the upper layer contains 0.6% agar, respectively preparing seawater with salinity of 20, and autoclaving at 121deg.C for 20min. When in use, the upper layer of the seawater double-layer agar medium is melted and cooled to about 42 ℃ and mixed into the seawater double-layer agar medium with the final content of 1×10 10 cfu/mL of vibrio parahaemolyticus host bacteria cell is used for marine bdellovibrio counting.
A long-term preservation method of marine bdellovibrio, comprising the steps of:
s1: preparation of host bacteria seawater suspension
Preparing a SWYE liquid culture medium, inoculating host bacteria for preserving marine Bdellovibrio into the SWYE liquid culture medium, selecting the vibrio alginolyticus as the host bacteria for preserving marine Bdellovibrio in the embodiment, centrifuging the SWYE liquid culture medium containing the vibrio alginolyticus after shaking culture to the end of the logarithmic phase, rinsing twice with sterile seawater to obtain vibrio alginolyticus culture bacteria, suspending the vibrio alginolyticus culture bacteria in sterile seawater to prepare vibrio alginolyticus seawater suspension, and adjusting the concentration of bacterial liquid in the vibrio alginolyticus seawater suspension to obtain bacterial liquid concentration of 10 6 cfu/mL or 10 7 cfu/mL, 10 in this example 6 cfu/mL of Vibrio alginolyticus seawater suspension marked as A2, 10 7 cfu/mL of the vibrio alginolyticus seawater suspension is marked as A1;
s2: preparation of sea Bdellovibrio seawater suspension
In this embodiment, the marine Bdellovibrio is a strain of Halobaeriovirax genus, and the bacterial liquid concentration is 5×10 according to the preparation method in step S1 8 cfu/mL of a host bacteria seawater suspension for culturing a strain of Halobacterovirax, in this example, vibrio alginolyticus is selected as a host bacteria for culturing Bdellovrax, a strain of Halobacterovirax is inoculated into the above-mentioned Vibrio alginolyticus seawater suspension, when Vibrio alginolyticus is completely lysed and contains a large amount of strain of Halobacterovirax in an attack period, shaking culture solution is taken to centrifuge for 10min under 1000g, supernatant is taken after filtration with a 0.45 μm filter membrane for two times, and centrifuged for 20min under 12000g, supernatant is discarded, precipitate after centrifugation is rinsed with sterile seawater, and centrifuged for 20min under 12000g, then a strain culture solution of Halobacterovirax is suspended in sterile seawater to prepare a strain seawater suspension of Halobacterovirax, and the concentration of strain seawater suspension of Halobacterovirax is adjusted to 10 8 pfu/mL or 3.33X10 7 pfu/mL or 10 7 pfu/mL, 10 in this example 8 The pfu/mL of a seawater suspension of a strain of the genus Halobacterovirax was labeled W1, and 3.33X10 7 The pfu/mL sea water suspension of Halobacterovirax strain is marked as W2, 10 7 The seawater suspension of the strain of Halobacteriovorax of pfu/m is marked as W3;
s3: strain preservation
The bacterial strain seawater suspension of Halobacterovirax prepared in the step S2 and the vibrio alginolyticus seawater suspension prepared in the step S1 are mixed together in equal volume, a combined preservation system of different bacterial solution concentrations of the bacterial strain seawater suspension of Halobacterovirax and the vibrio alginolyticus seawater suspension is shown in table 1, 4mL of the mixed solution is filled into a 5mL sterile nut tube, and the sterile nut tube filled with the mixed solution is preserved at 15-37 ℃, wherein the preservation temperature in the embodiment is 20 ℃.
Table 1, halobacteriovorax, strain preservation System
The method comprises the steps of (1) counting the strains of Halobacteriaceae in a seawater double-layer agar medium before the strains are placed under the condition of 20 ℃ for preservation, then respectively preserving the strains of Halobacteriaceae in the condition of 20 ℃ for 1 month, 3 months, 6 months and 12 months, and respectively counting the strains of Halobacteriaceae preserved for 1 month, 3 months, 6 months and 12 months, wherein the change of the number of the strains of Halobacteriaceae in a combined preservation system of different bacterial liquid concentrations of the bacterial suspension of Halobacteriaceae and the seawater suspension of Vibrio alginolyticus is shown in figure 2, and as can be seen in figure 2, the content of the strains of Halobacteriaceae is still relatively high when the strains of Halobacteriaceae are preserved under the condition of 20 ℃, especially after long-term preservation, and the bacterial content of the strains of Halobacteriaceae in all preservation groups is more than 10 in 6-12 months 5 pfu/mL, wherein the A2W2 group has the best long-term preservation effect, and the content is about 10 6 pfu/mL。
Example 2:
in this example, the strain of Halobactiovirax is selected as the strain of the reserved marine Bdellovibrio, and Vibrio parahaemolyticus is selected as the host strain for counting marine Bdellovibrio by double-layer plates.
Sterile seawater is prepared, the salinity of the sterile seawater is 25-35, the pH is 7-9, and the sterile seawater is subjected to filtration and sterilization treatment, wherein the salinity of the sterile seawater in the embodiment is 29.8-30.2, and the pH is 7.8-8.2.
SWYE medium was prepared as follows: taking 24g of NaCl, 24g of MgCl.7H2O 7g, 0.7g of KCl, 5.3g of MgCl.6H2O and 1L of distilled water to prepare a tetrasalt solution with the pH of 7.4-7.8, then taking 10g of peptone and 3g of yeast extract, fixing the volume of the peptone to 1L by using the tetrasalt solution, adjusting the pH to 7.2-7.4, and sterilizing at 121 ℃ for 20min for later use to culture the host bacteria.
Preparing seawater double-layer agar culture medium, wherein the bottom layer of the seawater double-layer agar culture medium contains 1.2% agar, the upper layer contains 0.6% agar, respectively preparing seawater with salinity of 20, and autoclaving at 121deg.C for 20min. When in use, the upper layer of the seawater double-layer agar culture medium is melted and cooled to about 42 ℃ and mixed with the vibrio parahaemolyticus host bacteria cells with the final content of 1 multiplied by 1010cfu/mL for counting marine bdellovibrio.
A long-term preservation method of marine bdellovibrio, comprising the steps of:
s1: preparation of host bacteria seawater suspension
Preparing a SWYE liquid culture medium, inoculating host bacteria for preserving marine Bdellovibrio into the SWYE liquid culture medium, selecting the vibrio alginolyticus as the host bacteria for preserving marine Bdellovibrio in the embodiment, centrifuging the SWYE liquid culture medium containing the vibrio alginolyticus after shaking culture to the end of the logarithmic phase, rinsing twice with sterile seawater to obtain vibrio alginolyticus culture bacteria, suspending the vibrio alginolyticus culture bacteria in sterile seawater to prepare vibrio alginolyticus seawater suspension, and adjusting the concentration of bacterial liquid in the vibrio alginolyticus seawater suspension to obtain bacterial liquid concentration of 10 6 cfu/mL or 10 7 cfu/mL, 10 in this example 6 cfu/mL of Vibrio alginolyticus seawater suspension marked as A2, 10 7 cfu/mL of the vibrio alginolyticus seawater suspension is marked as A1;
s2: preparation of sea Bdellovibrio seawater suspension
In this example, marine Bdellovibrio was a strain of Halobaeriovirax genus, and the bacterial liquid concentration was 5×10 8 cfu/mL of a host bacteria seawater suspension for culturing a strain of Halobacterovirax, in this example, vibrio alginolyticus is selected as a host bacteria for culturing Bdellovibrio marine, bdellovibrio marine WBX is inoculated into the above-mentioned Bdellovibrio alginolyticus seawater suspension, shaking culture is carried out at 30 ℃ until Vibrio alginolyticus is completely lysed and contains a large amount of strain of Halobacterovirax in an attack period, the culture solution is centrifuged at 1000g for 10min, the supernatant is obtained, vacuum-filtered by a 0.45 μm filter membrane for two times, centrifuged at 12000g for 20min, the supernatant is discarded, the centrifuged precipitate is rinsed by sterile seawater, and centrifuged at 12000g for 20min, then the strain culture solution of Halobacterovirax is suspended in sterile seawater to prepare a seawater suspension of Halobacterovirax, and the strain solution concentration of Halobacterovirax is adjusted to 10 v 8 pfu/mL or 3.33X10 7 pfu/mL or 10 7 pfu/mL, 10 in this example 8 pfu/mL of Halobacteriovorax genusThe seawater suspension of the strain is marked as W1, and 3.33X10 7 The pfu/mL sea water suspension of Halobacterovirax strain is marked as W2, 10 7 The seawater suspension of the strain of Halobacteriovorax of pfu/m is marked as W3;
s3: strain preservation
The bacterial strain seawater suspension of Halobacterovirax prepared in the step S2 and the vibrio alginolyticus seawater suspension prepared in the step S1 are mixed together in equal volume, a combined preservation system of different bacterial solution concentrations of the bacterial strain seawater suspension of Halobacterovirax and the vibrio alginolyticus seawater suspension is shown in a table 2, 4mL of the mixed solution is filled into a 5mL sterile nut tube, and the sterile nut tube filled with the mixed solution is preserved at 15-37 ℃, wherein the preservation temperature in the embodiment is 20-30 ℃.
Table 2, strain-preserving System of Halobacteriovorax genus
Before the Halobacterovirax strain is preserved at 20-30deg.C, it is counted by seawater double-layer agar medium, and then the Halobacterovirax strain is preserved at 20-30deg.C for 1 month, 3 months, 6 months and 12 months respectively, and the strains of Halobactiovrax genus which are preserved for 1 month, 3 months, 6 months and 12 months are respectively counted, and the change of the number of the strains of Halobactiovrax genus in a combined preservation system of different bacterial liquid concentrations of the bacterial strain seawater suspension of Halobactiovrax genus and the bacterial strain seawater suspension of vibrio alginolyticus is shown in figure 3.
As can be seen from FIG. 3, the bacterial count of Halobacteriovorax species is maintained at greater than 10 when the Halobacteriovorax species is stored at 20-30deg.C, particularly after prolonged storage 5 Higher levels of pfu/mL, where the bacterial numbers of Halobactericovorax species of groups A1W2, A1W3 and A2W1 were always maintained at 10 5 pfu/mL or more, the long-term storage effect at 12 months in group A1W3 was relatively good.
The above is an embodiment of the present invention. The foregoing embodiments and the specific parameters of the embodiments are only for clarity of description of the invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and all equivalent structural changes made in the description and drawings of the invention are intended to be included in the scope of the invention.
Claims (5)
1. A method for long-term preservation of marine bdellovibrio, comprising the steps of:
s1: preparation of host bacteria seawater suspension
Preparing a SWYE liquid culture medium, inoculating host bacteria for storing marine Bdellovibrio into the SWYE liquid culture medium, shaking culturing, centrifuging the SWYE liquid culture medium containing the host bacteria after the end of the logarithm period, rinsing with sterile seawater twice to obtain host bacteria culture bacteria, suspending the host bacteria culture bacteria in the sterile seawater to prepare a host bacteria seawater suspension, and regulating the bacterial liquid concentration in the host bacteria seawater suspension to be 10 6 cfu/mL or 10 7 cfu/mL; wherein the host bacteria of the marine Bdellovibrio are all vibrio alginolyticus, and the marine Bdellovibrio is a strain of Halobaeriovirax;
s2: preparation of sea Bdellovibrio seawater suspension
The concentration of the prepared bacterial liquid is 5 multiplied by 10 8 Inoculating marine Bdellovibrio into the host bacteria seawater suspension, shaking culturing at 30deg.C until the host bacteria are completely cracked and contain a large amount of Bdellovibrio in the offensive period, centrifuging culture solution under 1000g condition for 10min, collecting supernatant, filtering twice with 0.45 μm filter membrane, centrifuging under 12000g condition for 20min, discarding supernatant, rinsing the centrifuged precipitate with sterile seawater, centrifuging under 12000g condition for 20min, suspending marine Bdellovibrio culture bacteria in sterile seawater to obtain marine Bdellovibrio seawater suspension, and regulating bacterial liquid concentration of marine Bdellovibrio seawater suspension to 10 8 pfu/mL or 3.33X10 7 pfu/mL or 10 7 pfu/mL；
S3: strain preservation
Mixing the sea Bdellovibrio sea water suspension prepared in the step S2 and the host bacteria sea water suspension prepared in the step S1 together in equal volume, filling 4mL of the mixed solution into a 5mL sterile nut tube, and storing the sterile nut tube filled with the mixed solution at 20-30 ℃.
2. The method for long-term preservation of marine Bdellovibrio according to claim 1, wherein the sterile seawater in step S1 and step S2 has a salinity of 25-35 and a pH of 7-9, and is subjected to filtration and sterilization treatment.
3. The method for long-term preservation of marine Bdellovibrio according to claim 2, wherein the sterile seawater in step S1 and step S2 has a salinity of 29.8-30.2 and a pH of 7.8-8.2, and is subjected to filtration and sterilization treatment.
4. The method for long-term preservation of marine Bdellovibrio according to claim 1, wherein the preservation temperature in step S3 is 20 ℃, and the bacterial liquid concentration of the selected marine Bdellovibrio seawater suspension is 3.33×10 7 The bacterial liquid concentration of the seawater suspension of the host bacteria selected by pfu/mL is 10 6 cfu/mL。
5. The method for long-term preservation of marine Bdellovibrio according to claim 1, wherein the preservation temperature in step S3 is 20-30 ℃, and the bacterial liquid concentration of the selected marine Bdellovibrio seawater suspension is 10 7 The bacterial liquid concentration of the seawater suspension of the host bacteria selected by pfu/mL is 10 7 cfu/mL。
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