CN103146597B - Method for preparing photosynthetic bacteria liquid - Google Patents
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- CN103146597B CN103146597B CN201310036820.1A CN201310036820A CN103146597B CN 103146597 B CN103146597 B CN 103146597B CN 201310036820 A CN201310036820 A CN 201310036820A CN 103146597 B CN103146597 B CN 103146597B
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Abstract
The invention discloses a method for preparing rhodovulum sulfidophilus liquid. The method includes the following steps: (1) a cryopreserved tube containing the rhodovulum sulfidophilus liquid is dissolved and revived, the dissolved bacteria liquid comes to be revived bacteria liquid; (2) on an ultra-clean working platform, by utilization of the revived bacteria liquid, streaking is carried out on a flat plate solid medium, the streaked flat plate solid medium is placed in a constant temperature incubator with the temperature of 30 DEG C for being cultivated for 24-36 hours in a static mode, after single colonies are grown, an inoculating loop is utilized to pick the single colonies on the flat plate solid medium to streak on a slope culture medium, the streaked slope culture medium is placed in the incubator with the temperature of 30 DEG C for being cultivated for 24-36 hours in a static mode, and bacterial colonies are grown; and (3) after the bacterial colonies are grown, by utilization of a rhodovulum sulfidophilus liquid culture medium, the bacterial colonies on the slope culture medium are eluted to be bacterial colony liquid, the bacterial colony liquid is cultivated for the first time so as to obtain first-time culture bacterial liquid, and transfer culture is carried out on the first-time bacterial liquid to obtain transfer culture bacterial liquid. The method for preparing the rhodovulum sulfidophilus liquid has the advantages of being easy in preparation and culture of the culture media, high in the bacteria liquid concentration and good in quality.
Description
Technical field
The present invention relates to a kind of method preparing Bacteria liquid, particularly relate to a kind of method preparing photosynthetic bacteria liquid.
Background technology
Photosynthetic bacterium (photosynthetic bacteria, be called for short PSB), the prokaryotic organism with original luminous energy synthetic system the most ancient on the earth, be under anaerobic carry out photosynthesis and the general name of the bacterioid that do not produce oxygen, this is biological difference with green plants, algae and other photosynthesis.Photosynthetic bacterium is distributed widely in marsh, pond, lake, river, ditch, ocean and soil, is a kind of excellent aquatic environment modifying agent and fodder additives.Current photosynthetic bacteria liquid preparation method is very loaded down with trivial details, the complicated component of substratum, cultivate in special culture medium mostly, and be cultivate under certain pH value and intensity of illumination, the defect of existing preparation method is to prepare in substratum used often to be needed to add certain or some particular matter, as leaven promoting agent, pawpaw extracting solution, Herba Visci extract etc., and complicated component, prepare loaded down with trivial details, and the illumination that in preparation process, most needs are special.
Summary of the invention
The technical problem to be solved in the present invention is: provide a kind of culturing process simply to prepare the method for photosynthetic bacteria liquid.
The technical scheme of technical solution problem of the present invention is:
Prepare a method for thiophilic little red oomycetes bacterium liquid, the steps include:
(1) recovery of bacterial classification
The cryopreservation tube that thiophilic little red oomycetes bacterial classification is housed is carried out the recovery of bacterial classification rapid solution, bacterium liquid after dissolving becomes recovery bacterium liquid, the method of described bacterial classification rapid solution recovery is: the cryopreservation tube that thiophilic little red oomycetes bacterial classification is housed being positioned over immediately temperature is recovery also shake fast in the water-bath of 38 DEG C ~ 40 DEG C, until cryopreservation tube inner icing all dissolves;
(2) line of bacterium liquid and quiescent culture
On Bechtop, rule on Solid media for plates with recovery bacterium liquid, after by the Solid media for plates after line, in 30 DEG C of constant incubators, quiescent culture grows single bacterium colony in 24 ~ 36 hours, rule on slant medium with the single bacterium colony on transfering loop picking Solid media for plates, slant medium after line quiescent culture 24 ~ 36 hours bacterium colonies in 30 DEG C of incubators grow, described Solid media for plates compound method is: by 5 grams of peptones, 1 gram of yeast leaching powder, 0.1 gram of high ferric phosphate, 20 grams of agar, 1000 milliliters are boiled heating of seawater and dissolve, and with the sodium hydroxide solution that concentration is 1mol/L be adjusted to pH be 7.6 substratum lysate be loaded in Erlenmeyer flask, then the Erlenmeyer flask that substratum lysate is housed is placed in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, then the Erlenmeyer flask that substratum lysate is housed is taken out from autoclave sterilizer, substratum lysate in Erlenmeyer flask pours cooled and solidified in the culture dish of sterilizing before curing into can obtain Solid media for plates to room temperature, described inclined-plane solid medium compound method is: by 5 grams of peptones, 1 gram of yeast leaching powder, 0.1 gram of high ferric phosphate, 20 grams of agar, 1000 milliliters are boiled heating of seawater and dissolve, be that 1mol/L sodium hydroxide solution is adjusted to pH and 7.6 becomes substratum lysate and be sub-packed in test tube by concentration, test tube tampon seals, then test tube is vertically put in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, then test tube takes out from autoclave sterilizer, before substratum lysate solidifies, test tube is inclined to the inclined-plane becoming miter angle with horizontal plane, the natural coagulation of substratum lysate can obtain inclined-plane solid medium to room temperature,
(3) bacterium colony wash-out and cultivation:
After bacterium colony grows, with thiophilic little red oomycetes bacterium liquid culture medium the bacterium colony on slant medium eluted and become bacterium colony solution, bacterium colony solution carries out cultivating for the first time and can being cultivated bacterium liquid for the first time, and first cultivation bacterium liquid carries out switching cultivation again can obtain switching cultivation bacterium liquid; Described first cultivation is mixed by bacterium colony solution in rear loading Erlenmeyer flask, and after kraft paper wrapping sealing, be placed in concussion on shaking table and cultivate, the condition of shaking table shaking cultivation is temperature 28 DEG C ~ 35 DEG C, rotating speed 50rpm; Described switching is cultivated as shaking table concussion is cultivated, the condition that shaking table concussion is cultivated is inoculum size 10%, temperature 28 DEG C ~ 35 DEG C, rotating speed 50rpm, or described switching is cultivated as the quiescent culture in constant incubator, the condition of quiescent culture is inoculum size 20%, temperature 28 DEG C ~ 35 DEG C, and not timing every day rocks 3 ~ 6 times; Described photosynthetic bacteria liquid substratum compound method is: take yeast leaching 0.5 gram, powder, take peptone 3 grams to be dissolved in 1 liter and to boil seawater, solution after dissolving loads in Erlenmeyer flask, after kraft paper wrapping sealing, in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, naturally cools to room temperature and can obtain thiophilic little red oomycetes bacterium liquid culture medium after sterilizing.
Technique effect of the present invention is: solid medium preparation of the present invention is simple, and only need yeast to soak powder, peptone, high ferric phosphate, agar and boil seawater preparation, photosynthetic bacteria liquid substratum only needs yeast soak powder, peptone and boil seawater.Cultivation required equipment is simple, only need one can the shaking table incubator of temperature control or constant incubator.The photosynthetic bacteria liquid concentration prepared is high, and quality is good.Therefore, the inventive method there is substratum preparation and culturing process simple, bacterial concentration is high, the measured feature of matter.
Accompanying drawing explanation
1, embodiment of the present invention Solid media for plates diced into sections schematic diagram.
2, the photo when embodiment of the present invention inoculates 10% thiophilic little red oomycetes nutrient solution.
3, the embodiment of the present invention inoculates photo when 10% thiophilic little red oomycetes nutrient solution cultivates the 4th day.
Embodiment
One, the configuration of embodiment of the present invention solid medium
Embodiment of the present invention solid medium adopts 2216E solid medium, and 2216E solid medium is called for short solid medium, is a kind of special culture media cultivating marine bacteria, cultivates for the streak culture of marine bacteria or coating.Embodiment of the present invention solid medium is divided into plate culture medium and slant medium, and the solid medium prepared with culture dish, is called plate culture medium; The solid medium prepared with test tube is slant medium.Composition and the compound method of embodiment of the present invention solid medium are as follows:
The material preparing solid medium prepares: take peptone 5 grams with electronic balance, yeast leaching 1 gram, powder, high ferric phosphate 0.1 gram, 20 grams, agar, and measures and boil 1 liter, seawater.
The preparation of plate culture medium: by the peptone of the above-mentioned amount of taking, yeast leaching powder, high ferric phosphate, agar, and the heating of seawater that boils measured dissolves, the present embodiment Erlenmeyer flask of 2 liters mixes, and heating by electric cooker dissolves, boil 5 minutes, be as the criterion so that content is dissolved completely.Be that the liquid after dissolving is adjusted to pH and 7.6 namely obtains substratum lysate by the sodium hydroxide solution of 1mol/L again by concentration, then this substratum lysate is sub-packed in multiple Erlenmeyer flask, the substratum lysate of packing 100 milliliters in the Erlenmeyer flask that the present embodiment is each 250 milliliters, needs 10 Erlenmeyer flasks altogether.The Erlenmeyer flask that substratum lysate is housed is placed in autoclave sterilizer, temperature 121 DEG C, under the condition of pressure 105kPa, sterilizing is after 25 minutes, Erlenmeyer flask after sterilizing is taken out from autoclave sterilizer, be positioned at the substratum lysate of Erlenmeyer flask before curing, pour this substratum lysate into culture dish through sterilizing respectively, the culture dish of sterilizing in autoclave sterilizer temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa.The diameter of the present embodiment culture dish is 9 centimetres, and 15 milliliters of substratum lysates through sterilizing poured into by each culture dish, treats that substratum lysate cool to room temperature solidifies stored refrigerated in rearmounted 4 DEG C of refrigerators for subsequent use.
The preparation of slant medium: take peptone 5 grams with electronic balance, yeast leaching 1 gram, powder, high ferric phosphate 0.1 gram, 20 grams, agar, and measure and boil seawater 1 liter of heating for dissolving, be that 1mol/L sodium hydroxide solution is adjusted to pH and 7.6 namely obtains substratum lysate by concentration.Substratum lysate is sub-packed in test tube, the present embodiment needs 50 test tubes, each cuvette cartridge 20 milliliters of substratum lysates, test tube tampon seals, vertically put into autoclave sterilizer, temperature 121 DEG C, pressure is sterilizing 25 minutes under the condition of 105kPa, then test tube is taken out from autoclave sterilizer, substratum lysate before curing, test tube is inclined to the inclined-plane becoming miter angle with desktop (horizontal plane), makes stored refrigerated in invisible spectro substratum lysate natural coagulation to the rearmounted 4 DEG C of refrigerators of room temperature for subsequent use.
If preparation 2216E liquid nutrient medium, then do not need to add agar, its composition, sterilizing and making method same as described above.
Two, the configuration of embodiment of the present invention photosynthetic bacteria liquid substratum
Embodiment of the present invention photosynthetic bacteria liquid substratum compound method: take yeast leaching 0.5 gram, powder with electronic balance, peptone 3 grams is dissolved in 1 liter and boils in seawater, it is in the Erlenmeyer flask of 2 liters that culture solution after dissolving loads specification, after kraft paper wrapping sealing Erlenmeyer flask, Erlenmeyer flask after sealing in autoclave sterilizer temperature 121 DEG C, pressure be under the condition of 105kPa sterilizing to naturally cool to room temperature after 25 minutes for subsequent use.
Three, embodiment of the present invention concrete operation method
Embodiment of the present invention concrete operation method is tested with the thiophilic little red oomycetes bacterial classification in photosynthetic bacterium, comprises the recovery of the thiophilic little red oomycetes bacterial classification that laboratory is preserved, first cultivation of cultivating and transfer.Thiophilic little red oomycetes (Rhodovulum sulfidophilus) belongs to bacterium class, α is out of shape Gammaproteobacteria, red bacterium order, red bacterium section, little red oomycetes belongs to (Rhodovulum Hiraishi and Ueda), cell oval to rod, 0.5 ~ 0.9 μm × 0.9 ~ 2.0 μm.One pole is given birth to flagellar movement or is not moved.The capable binary fission of cell, Gram-negative.Amphimicrobian phototrophic bacteria, i.e. all can grow under illumination anaerobism and dark aerobic condition very well.Optimum growh mode utilizes various organic compound to carry out photoheterotrophy growth.When hydrogen sulfide and Sulfothiorine exist, light autotrophy or photoheterotrophic growth can be carried out.Thiophilic little red oomycetes is the type species that little red oomycetes belongs to.Thiophilic little red oomycetes, as the one of photosynthetic bacterium, is widely used in improving water quality, reduces pathogenic micro-organism and bad algae, and as the additive of bait of fishes and shrimps.
1, the recovery of bacterial classification:
The cryopreservation tube that thiophilic little red oomycetes is housed preserved at-80 DEG C is taken out, be positioned over rapid fluid resuscitation in 38 DEG C ~ 40 DEG C water-baths immediately and shake fast, the present embodiment, with hand dynamic, namely holds cryopreservation tube shake wrist, until inner icing all dissolves, need 50 seconds ~ 100 seconds.Cryopreservation tube opened by Bechtop, and Bechtop needs in advance with ultra violet lamp sterilization in 15 minutes.Pick with transfering loop and get thiophilic little red oomycetes bacterium liquid less, ring body by transfering loop stretches in bacterium liquid and picks bacterium liquid, the volume picked is about 5 microlitres, Solid media for plates is rule, during line, as shown in Figure 1, a Solid media for plates is divided into four communities to rule, first community as A district, as the bacterium source region of bacterium to be separated; Second community and the 3rd community, respectively as B district and C district, are the zone of transition of stepwise dilution, and the 4th community, as Ze Shi key area, D district, makes D district occur a large amount of single bacterium colonies purebred use for you to choose.First A district is drawn: Solid media for plates is placed in spirit lamp other (distance spirit lamp 10 centimetres), left hand holds culture dish, left-hand palm is held at the bottom of ware, with thumb and forefinger, ware lid is raised, with at the bottom of ware in miter angle and opening towards spirit lamp, the right hand takes the transfering loop having picked thiophilic little red oomycetes bacterium liquid first at A zoning 3 ~ 4 continuous print parallel lines, lines how much should according to choose bacterium amount number and determine, if choose, bacterium amount is many can many strokes several parallel lines.The present embodiment line length 1 ~ 3 centimetre, neighbouring parallel lines spacing 1 centimetre.The residual bacterium on transfering loop should be burnt immediately, in order to avoid affect the separating effect in each district below because thiophilic little red oomycetes is too much after A ride.Then other district is drawn: the transfering loop after residual for burning-off bacterium is cooled about 1 minute at plate culture medium edge, temperature is cooled to non-scald on hand, and make plate culture medium B district forward the one side of close spirit lamp to, transfering loop takes thiophilic little red oomycetes to B district by A district (bacterium source region), draw the parallel lines of several densifications immediately, the present embodiment B district line length 1 ~ 3 centimetre, neighbouring parallel lines spacing 1 centimetre.Do the line in C district again from B district, namely take C district to from B district by thiophilic little red oomycetes, from the residual bacterium will burnt before B district takes thiophilic little red oomycetes to C district transfering loop.The line in D district is done finally by C district, namely D district is taken to from C district by thiophilic little red oomycetes, at D ride, from the residual bacterium also will burnt before C district takes thiophilic little red oomycetes to D district transfering loop, be sure not again to contact A district, B district when drawing D district, in order to avoid bacterium liquid dense in this twoth district takes D district to, affect the formation of single bacterium colony.Residual bacterium on burning-off transfering loop, be inverted culture dish, be inverted culture dish than just putting culture dish is not easy pollution microbes more, because also have miscellaneous bacteria in air, although in addition at the bottom of ware and ware lid disinfection, but in operation, the appearance of contact still may be polluted, inversion can make the least possible contact substratum of miscellaneous bacteria.Being inverted culture dish also can prevent the substratum moisture in culture dish from evaporating, and makes substratum keep moistening.Be inverted and drip on substratum after also can preventing condensate moisture, cause bacterium colony to spread, cannot count.Solid media for plates after line quiescent culture 24 ~ 36 hours in 30 DEG C of constant incubators.After the Solid media for plates D head of district goes out single bacterium colony, with the single bacterium colony in transfering loop picking Solid media for plates D district, streak culture on slant medium, the present embodiment on slant medium in "the" shape line, the slant medium after line in 30 DEG C of constant incubators quiescent culture after 24 ~ 36 hours bacterium colony grow.
After bacterium colony grows, with photosynthetic Bacteria liquid culture medium, the bacterium colony on inclined-plane solid medium is eluted, elution step is as follows: in Bechtop, with micropipet, 1ml photosynthetic bacteria liquid substratum is put into the test tube cultivated and have the inclined-plane solid medium of thiophilic little red oomycetes bacterium colony, concuss (temperature controls at 15 ~ 30 DEG C) on earthquake device, until the bacterium colony on inclined-plane solid medium all elutes become bacterium liquid.
The bacterium colony that inclined-plane solid medium grows is that single bacterium colony that solid medium is chosen grows, and is the step of an enlarged culturing.
Thiophilic little red oomycetes separation and purification qualification from seawater of the present embodiment, namely Seawater Samples 100 microlitre is fetched from Mouping County, shandong Province sea area, on 2216E solid plate substratum, coating evenly, inversion culture dish is placed in 28 DEG C of constant incubators and cultivates, within 2 ~ 3 days, observe, with transfering loop picking purple bacterium colony, rule on 2216E solid plate substratum again, within 3 ~ 5 days, afterwards visible plate culture medium has loose purple colony growth, the single purple bacterium colony of picking is thiophilic little red oomycetes through molecular biology method qualification.
2, cultivate for the first time
The recover bacterium liquid that elutes and 100ml photosynthetic bacteria liquid substratum of 1ml mixes in rear loading Erlenmeyer flask, and after kraft paper wrapping sealing, be placed in concussion on shaking table and cultivate, shaking table shaking cultivation can make bacterial suspension, and reproduction speed is fast.The condition of shaking table shaking cultivation is temperature 28 DEG C ~ 35 DEG C, rotating speed 50rpm, within 3 days ~ 4 days, can obtain bacterial concentration and reach about 20,000,000,000/milliliter (2 × 10
10/ ml) first cultivation bacterium liquid.
Embodiment of the present invention bacterial concentration measuring method: the above-mentioned bacterium liquid antiseptic sea water eluted from inclined-plane solid medium is diluted successively with 10 times of concentration, namely, 100uL bacterium liquid adds 900uL antiseptic sea water, bacterial concentration is diluted to 1/10 of previous bacterial concentration, dilution like this 10 times, thus be diluted to 10 of original concentration
-10doubly, so finally obtain diluting 10 before rear bacterial concentration is
-10times concentration, finally draws 100uL with the micropipet of 100uL and dilutes 10
-10bacterium liquid is doubly coated with spreading rod on Solid media for plates, be coated on operation in Bechtop, near the flame (spacing 10 centimetres) of spirit lamp, before coating first by spreading rod concentration be 75% alcohol-pickled 30 seconds sterilization, then flash back on spirit lamp flame three times, slightly be coated with after cooling, by the coating of bacterium liquid evenly, during coating, ware lid slightly leaves, ware lid is not lifted down entirely, namely ware lid one end is raised, spreading rod is made to put in substratum coating, be inverted to be put in 30 DEG C of constant incubators after ware lid built by coated culture dish and cultivate, within 3 days, count the single colony number on substratum afterwards, be multiplied by 10 again
10, be the concentration in 1ml original bacteria liquid.Colony diameter 1 ~ 3mm, quantity is 2 ~ 10.
3, switching is cultivated
Inoculum size with 10% will cultivate bacterium liquid and the mixing of photosynthetic bacteria liquid substratum for the first time, such as to cultivate 1 liter of bacterium liquid, namely needing the photosynthetic bacteria liquid substratum Erlenmeyer flask of the first cultured first cultivation bacterium liquid of 100ml and 900ml to mix rear loading specification is in the Erlenmeyer flask of 2 liters, be placed in shaking table (28 DEG C ~ 35 DEG C, rotating speed 50rpm), the present embodiment shaking table adopts full temperature to cultivate shaking table, within 3 days ~ 4 days, can obtain the switching cultivation bacterium liquid that concentration reaches 20,000,000,000/milliliter.
Switching cultivate also can in constant incubator quiescent culture, inoculum size 20% (the photosynthetic bacteria liquid substratum of cultured first cultivation bacterium liquid as first in 200ml and 800ml mixes), temperature 28 DEG C ~ 35 DEG C, not timing every day is rocked several times, the present embodiment rocks 5 ~ 6 times every day, or interval is shaken once in 3 ~ 4 hours, the thiophilic little red oomycetes bacterium liquid that concentration reaches 20,000,000,000/milliliter within 5 days ~ 6 days, also can be obtained.
1, the present embodiment configuration solid medium and photosynthetic bacteria liquid substratum use instrument concrete as follows and reagent:
Electronic balance: Asia-Pacific measuring instrument company limited produces, model DS-200.
Autoclave sterilizer: Shandong Xinhua Ande Medical Articles Co., Ltd. produces, the portable electrothermal pressure steam sterilizer of YXQG02 type.
Glassware: diameter is the culture dish of 9 centimetres; Diameter (external diameter) is the glass test tube of 1.8cm, length 18cm; Specification is respectively the Erlenmeyer flask of 1L and 2L; Specification is respectively the beaker of 500ml and 1L all purchased from Sichuan Shu Niu glassware company.
Yeast leaching powder: Beijing extensive and profound in meaning star biotechnology limited liability company produces, and 250g fills, BR biochemical reagents (Biological reagent biological stain), micro-Huang or off-white powder.
Peptone: Beijing extensive and profound in meaning star biotechnology limited liability company produces, 250g fills, BR biochemical reagents (Biological reagent biological stain), and white is to buff powder.
High ferric phosphate: Tianjin recovery fine chemistry industry institute produces, and 250g fills, greyish white or pale pink powder.
Agar: Beijing health doubly this (chembase) Science and Technology Ltd. is produced, and 500g fills, pale yellow powder.
Sodium hydroxide: Tianjin great Mao chemical reagent factory is produced, analytical pure, 500g fills, white particulate.
Boil seawater: get 1L seawater with the Erlenmeyer flask that specification is 2L and boil 5 minutes cool to room temperature and be and boil seawater.
2, the Other Instruments used by the embodiment of the present invention and reagent:
Transfering loop: Tai Yida bio tech ltd, Beijing produces, long 50 millimeters of bar, the diameter of bar is 0.5 millimeter.
Micropipet: German Ai Bende (Eppendorf) company produces, and range is 100 μ L and 1mL.
Oscillator: its woods Bel instrument manufacturing company limited of Haimen City produces, model VOR76X-5.
Constant incubator: the permanent tech equipment company limited in Shanghai one produces, model DHP-9162.
Bechtop: safe and sound company of Su Jing group produces, model AIR TECH.
Electric heat constant temp. water tank: the permanent tech equipment company limited in Shanghai one produces, model DKB-600B.
Full temperature cultivates shaking table, and Shanghai new talent medicine equipment Manufacturing Co., Ltd produces, model QYC-200.
Antiseptic sea water: will boil seawater temperature 121 DEG C in autoclave sterilizer, sterilizing 25min under pressure 105kPa condition, after sterilizing, cool to room temperature is antiseptic sea water.
According to above-mentioned operation steps, the thiophilic little red oomycetes bacterium liquid cultivated and photosynthetic bacteria liquid substratum mix by the inoculum size with 10% in Erlenmeyer flask, see Fig. 2, then shaking table (28 DEG C ~ 35 DEG C is placed in, rotating speed 50rpm) cultivate, the high-concentration bacterial liquid of 20,000,000,000/milliliter within 4 days, can be obtained, see Fig. 3.
Through test, the inventive method is also applicable to other kinds of photosynthetic bacterium as Crimson rhodospirillum, spherical red pseudomonas, the cultivation of the red pseudomonas of pod membrane.
Claims (1)
1. prepare a method for thiophilic little red oomycetes bacterium liquid, the steps include:
(1) recovery of bacterial classification
The cryopreservation tube that thiophilic little red oomycetes bacterial classification is housed is carried out the recovery of bacterial classification rapid solution, bacterium liquid after dissolving becomes recovery bacterium liquid, the method of described bacterial classification rapid solution recovery is: the cryopreservation tube that thiophilic little red oomycetes bacterial classification is housed being positioned over immediately temperature is recovery also shake fast in the water-bath of 38 DEG C ~ 40 DEG C, until cryopreservation tube inner icing all dissolves;
(2) line of bacterium liquid and quiescent culture
On Bechtop, rule on Solid media for plates with recovery bacterium liquid, after by the Solid media for plates after line, in 30 DEG C of constant incubators, quiescent culture grows single bacterium colony in 24 ~ 36 hours, rule on slant medium with the single bacterium colony on transfering loop picking Solid media for plates, slant medium after line quiescent culture 24 ~ 36 hours bacterium colonies in 30 DEG C of incubators grow, described Solid media for plates compound method is: by 5 grams of peptones, 1 gram of yeast leaching powder, 0.1 gram of high ferric phosphate, 20 grams of agar, 1000 milliliters are boiled heating of seawater and dissolve, and to be adjusted to pH with the sodium hydroxide solution that concentration is 1mol/L be 7.6, substratum lysate is loaded in Erlenmeyer flask, then the Erlenmeyer flask that substratum lysate is housed is placed in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, then the Erlenmeyer flask that substratum lysate is housed is taken out from autoclave sterilizer, substratum lysate in Erlenmeyer flask pours cooled and solidified in the culture dish of sterilizing before curing into can obtain Solid media for plates to room temperature, described slant medium compound method is: by 5 grams of peptones, 1 gram of yeast leaching powder, 0.1 gram of high ferric phosphate, 20 grams of agar, 1000 milliliters are boiled heating of seawater and dissolve, be that to be adjusted to pH be 7.6 to 1mol/L sodium hydroxide solution by concentration, substratum lysate is sub-packed in test tube, test tube tampon seals, then test tube is vertically put in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, then test tube takes out from autoclave sterilizer, before substratum lysate solidifies, test tube is inclined to the inclined-plane becoming miter angle with horizontal plane, the natural coagulation of substratum lysate can obtain slant medium to room temperature,
(3) bacterium colony wash-out and cultivation:
After bacterium colony grows, with thiophilic little red oomycetes bacterium liquid culture medium the bacterium colony on slant medium eluted and become bacterium colony solution, bacterium colony solution carries out cultivating for the first time and can being cultivated bacterium liquid for the first time, and first cultivation bacterium liquid carries out switching cultivation again can obtain switching cultivation bacterium liquid; Described first cultivation is mixed by bacterium colony solution in rear loading Erlenmeyer flask, and after kraft paper wrapping sealing, be placed in concussion on shaking table and cultivate, the condition of shaking table shaking cultivation is temperature 28 DEG C ~ 35 DEG C, rotating speed 50rpm; Described switching is cultivated as shaking table concussion is cultivated, the condition that shaking table concussion is cultivated is inoculum size 10%, temperature 28 DEG C ~ 35 DEG C, rotating speed 50rpm, or described switching is cultivated as the quiescent culture in constant incubator, the condition of quiescent culture is inoculum size 20%, temperature 28 DEG C ~ 35 DEG C, and not timing every day rocks 3 ~ 6 times; Described thiophilic little red oomycetes bacterium liquid culture medium compound method is: take yeast leaching 0.5 gram, powder, take peptone 3 grams to be dissolved in 1 liter and to boil seawater, solution after dissolving loads in Erlenmeyer flask, after kraft paper wrapping sealing, in autoclave sterilizer, temperature 121 DEG C, sterilizing 25 minutes under the condition of pressure 105kPa, naturally cools to room temperature and can obtain thiophilic little red oomycetes bacterium liquid culture medium after sterilizing.
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