CN103409356A - Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof - Google Patents
Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof Download PDFInfo
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Abstract
The invention discloses a borrelia BSK storage liquid culture medium and a single colony separating and purifying method and application thereof. The borrelia BSK storage liquid culture medium comprises CMRL-1066, bovine serum albumin V, new peptone, hydroxyethyl piperazine ethanesulfonic acid, sodium citrate, glucose, yeast powder, sodium bicarbonate, sodium pyruvate, acetyl glucosamine, rabbit serum, sodium hydroxide, and the like. The single colony separating and purifying method comprises the following steps of: preparing the BSK storage liquid culture medium, preparing sepharose gel, sterilizing at high pressure, gradually melting, uniformly mixing with the BSK storage liquid culture medium in proportion, and pouring to prepare a lower solid culture medium; mixing a borrelia burgdorferi bacterium suspension diluted in a gradient way and the BSK storage liquid culture medium, pouring into an upper culture medium so that a macroscopic single colony appears; raising the single colony by using a toothpick, inspecting by using a dark-field microscope to observe a representative burgdorferi bacterium strain. The method disclosed by the invention has the advantages of easiness and fastness for operation and clarity in result. According to the invention, the representative burgdorferi bacterium strain is uniform in genetic background without heterogeneity, suitable for subsequent scientific research and used as a standard strain.
Description
Technical field
The present invention relates to the medical microbiology field, be specifically related to a kind of burgdorferi BSK stock solution substratum, also relate to the method for utilizing this stock solution substratum separation and purification burgdorferi list bacterium colony, the method that also relates to this separation and purification in preparing microbiology scientific research, clinical diagnosis single bacterium colony and the purposes in pure growth.
Background technology
Single bacterium colony separates and the microorganism pure culture technigne is one of the most common most important technology in microbiology scientific research, medical treatment, industrial production.The microorganisms cultures that contains more than one is called mixed culture, if all cells all comes from a parental cell in a bacterium colony, this bacterium colony is called pure culture so.When carrying out strain identification, industrial production and scientific research, microorganism used is general all to be required as pure culture.Because the different bacterial classification in multiple source may be arranged in mixed culture, may cause the heterogeneity of microorganisms cultures, show as the unstable of biological character, to industrial production, bring loss, and may cause the mistake of clinical diagnosis.Separate a kind of microbiological technique method that contains single bacterium and be called single bacterium colony separation, the process that is obtained pure culture by single bacterium colony is called separation and purification, and the single bacterium colony of common microorganism separates and the method for pure culture has following several.
1, spread plate method
At first the microorganism suspension is carried out to the dilution of gradient, getting a certain amount of diluent is placed on the aseptic nutrient agar plate solidified, then with aseptic glass slicker, diluent is coated on media surface equably, incubator is inverted to cultivate and can be obtained single bacterium colony.Get single bacterium colony and make suspension, repeat above-mentioned steps for several times, just can obtain pure growth.
2, plate streak
The method of the simplest, the most common separate microorganism list bacterium colony is plate streak.With aseptic transfering loop, get culture a little is rule on flat board.When transfering loop moves backward on media surface, bacterium liquid on transfering loop dilutes gradually, and its purpose is all that bacterium is disperseed to open better and better, and individual cells finally is scattered here and there on the line of being drawn, the single bacterium colony of each self-forming after cultivating, will can obtain purebred bacterium after single bacterium colony culture transferring propagation.The method is also that the single bacterium colony of microorganism separates modal method.
3, enrichment culture method
By creating specific growth conditions, as temperature, special nutrition composition, carbon source, nitrogenous source, pH etc., under these conditions, needed microorganism can be at war with other microorganisms effectively, aspect energy for growth considerably beyond other microorganisms.Under identical substratum and culture condition, through repeatedly repeating culture transferring, eliminate other microorganism, the bacterial strain of last enrichment is easy to grow single bacterium colony on solid medium.
Burgdorferi (Borrelia) is gram negative bacterium, and this microorganism belonging to genus has 5-10 thin and irregular spiral, and motion is active, easy coloring, and micro-aerobic, thermophilic is the 28-37 degree, artificial culture is more difficult.This genus bacterium comprises a plurality of mankind's pathogenic bacterium, as causes the Borrelia burgdoyferi (Borrelia burgdorferi) of Lyme disease, is propagated to the mankind by tick.The infection of Borrelia burgdoyferi is influential to a plurality of systems of human body, and through different clinical stages, long-term dysfunction can occur affected organ; Cause the borrelia obermeyri of mankind's typhinia, according to the difference of typhinia communication media insect, can be divided into two classes.One is LBRF, or claims epidemic relapsing fever, and its pathogenic agent is borrelia obermeyri (Borrelia recurrentis).Another is tick-borne relapsing fever, claims again the region typhinia, 15 kinds of its pathogenic agent as many as, such as the logical burgdorferi (Borrelia duttonii) of Du, Hermes burgdorferi (Borrelia hermsii) etc.The popular typhinia in Asia and China is mainly borrelia persica (Borrelia persica) and borrelia latyschewii (Borrelia latyschewii).
Burgdorferi has special genome structure feature, and the wire that its genome is differed in size by karyomit(e) and about 20 5-56kb of the approximately 910kb of a wire or the plasmid of ring-type are formed.Some plasmids are unsettled in the subculture in vitro separately culturing process, however be but maintain in the host animal body have infectious necessary.Heterogeneity in heredity can also derive from same clone's derivative, i.e. offspring after plasmid loss, these are plasmid-encoded important phenotypic characteristic, such as antigenic drift and NAD biosynthesizing cofactor etc.Therefore, single bacterium colony separation and the microorganism pure culture technigne of burgdorferi seem even more important in strain identification, somatotype, clinical diagnosis and the scientific research in this field.Because the clone after plasmid loss or generation homologous recombination probably impacts last evaluation, diagnosis, result for the treatment of.
Yet single bacterium colony of burgdorferi separates and pure culture but can not be carried out according to conventional method, be at first because this strain growth speed is slow.The speed of burgdorferi multiplication needs 5 hours at the soonest under the vitro culture optimal conditions, therefore, from single bacterium colony of a Hemapoiesis until obtain pure growth, often needed for two above times in week, if spread plate method is routinely carried out single bacterium colony with plate streak, separate, how in culture cycle, to keep dull and stereotyped humidity is a challenge.Borrelia burgdoyferi is obligatory parasitism at nature in life cycle, and metabolic capacity is limited, can only be at the uncertain BSK(Stoenner-Kelly Medium of nutritive ingredient complexity, crucial) grow in substratum.The BSK medium component of those uncertain keys, limited the auxotrophic evaluation of Borrelia burgdoyferi as the crude extract of rabbit anteserum, bovine serum albumin etc., and this just makes enrichment culture method separate single bacterium colony and obtains pure culture and hardly may.What is more important, burgdorferi are a kind of not aerobic bacteriums, and conventional spread plate method and plate streak carry out single bacterium colony while separating, and planar surface exposes fully, is unfavorable for the growth of burgdorferi.These all make conventional microbiology list bacterium colony separation method be difficult in the research, diagnosis of the burgdorferi of application.
What single bacterium colony of burgdorferi separated employing at present is limiting dilution assay, be about to want the spirochete separated mixed culture to dilute with substratum, then put into respectively 96 orifice plates, after cultivating a week, if be less than 12 holes, the burgdorferi cell is arranged, can think that the culture in each hole unicellularly copies generation by one, obtain thus pure culture and realize separation and the purifying of burgdorferi.
Summary of the invention
The objective of the invention is to be to provide cultivation that a kind of burgdorferi BSK stock solution substratum, this substratum be applicable to the burgdorferi bacterium and separate, Purification and Characterization.And preparation method's Simple fast, can be separated to the mono-clonal bacterium colony that genetic background is single.
Another object of the present invention is to be to provide a kind of burgdorferi list bacterium colony separation purification method, and the method is simple to operate can directly see single bacterium colony fast on solid plate, directly perceived and simple to operate, is applicable to promoting the use of.
Last purpose of the present invention is to be to provide the application of a kind of burgdorferi separation purification method in preparing pure growth, utilize the acquisition pure growth that the method can be easily and fast, and the genetic background homogeneous is without heterogeneity, be applicable to follow-up scientific research and as the standard substance bacterial strain.
In order to achieve the above object, the present invention takes following technical measures:
A kind of BSK stock solution substratum, it is made by the raw material of following weight part:
A kind of BSK stock solution substratum, it makes (preferable range) by the raw material of following weight part:
A kind of burgdorferi BSK stock solution substratum, it makes (optimum value) by the raw material of following weight part:
A kind of BSK stock solution substratum, it is made by the raw material of following weight part:
Said components is added in distilled water in order one by one, and room temperature (20-30 ℃) stirs to dissolving fully, adds next component after a upper component is dissolved fully again, finally adjusts pH constant volume.
A kind of burgdorferi list bacterium colony separation purification method, its step is as follows:
(1) sepharose preparation: with low melting-point agarose configuration 5%(mass volume ratio) sepharose, the sterilizing 20-40 minute of 121 ℃ of high pressure (103.4 kPas), be placed in room temperature (20-30 ℃) standby.
(2) preparation of bottom solid medium: regulate water-bath temperature to 55 degree, by 5% sepharose as for wherein melting gradually.Then the ratio that is 1:1 according to volume ratio mixes with BSK stock solution substratum.Be poured onto culture dish, the culture dish that diameter is 10 centimetres, each needs 10 milliliters of bottom solid mediums.The standing 12-24 hour of room temperature, until substratum solidify fully, stand-by in 4 degree Refrigerator stores after surperficial evaporated condensation water.
(3) burgdorferi bacterial strain to be separated is cultured to logarithmic phase with BSK-H conventional liq substratum (sigma company product), counts by dark-field microscope, determine that its concentration reaches 10
7-10
8Cells/ml.With BSK-H conventional liq substratum gradient dilution, obtaining respectively concentration is 10,10
2, 10
3, 10
4, 10
5The bacteria suspension of cells/ml.
(4) regulate water-bath temperature to 55 ℃, by 5% sepharose as for wherein melting gradually.Then in sterilizing centrifuge tube, mix 7.5 milliliters of BSK stock solution substratum and 7.5 milliliter of 5% sepharose, the burgdorferi bacteria suspension to be separated that adds 1 milliliter of different concns after fully mixing, turn upside down after mixing for 10-20 time and be poured onto in the bottom solid medium prepared.Standing 12 hours of room temperature, solidify, put into after surperficial evaporated condensation water 37 ℃ of CO2gas incubator (5%) fully until substratum and cultivate.
(5) observe substratum and bacterial growth situation, determine the pollution without bacterium or mould, occur after macroscopic single bacterium colony with toothpick picking colony dark-field microscopy.
Described BSK stock solution substratum, its composition is as follows:
The application of a kind of burgdorferi list bacterium colony separation purification method in preparing the burgdorferi pure growth, its application process is:
(1) preparation of burgdorferi pure growth:
With toothpick picking burgdorferi list bacterium colony, be seeded to 12 milliliters of BSK-H conventional liq substratum (sigma company product), cultivate in 37 ℃ of CO2gas incubator (5%), observe the burgdorferi growing state, strain growth is to logarithmic phase, and bacterium colony concentration reaches 10
7More than cells/ml, can obtain single bacterium colony pure growth of burgdorferi.
(2) evaluation of burgdorferi endogenous plasmid:
Burgdorferi contains the wire that differs in size or the plasmid of ring-type forms, and according to the specific primer of the sequences Design of each plasmid, adopts the method for PCR to identify in this bacterium colony whether contain certain plasmid.
(3) endogenous plasmid collection of illustrative plates:
The PCR product of acquisition is carried out to agarose gel electrophoresis, if specific band is arranged, illustrate that this bacterium colony contains the purpose plasmid.The single bacterium colony pure growth filtered out, can be used for follow-up study and prepare the bacterial strain standard substance.
Microorganism strains involved in the present invention includes but are not limited to Borrelia burgdoyferi, allly can all can be applicable to the present invention with the Borrelia bacterium of BSK-H conventional liq culture medium culturing, comprise borrelia obermeyri (Borrelia recurrentis), the logical burgdorferi (Borrelia duttonii) of Du, Hermes burgdorferi (Borrelia hermsii), borrelia persica (Borrelia persica) and borrelia latyschewii (Borrelia latyschewii).
The present invention compares with routine techniques, has the following advantages and effect:
(1) single bacterium colony separation of burgdorferi is difficult to carry out according to conventional spread plate method and plate streak, because Borrelia burgdoyferi is obligatory parasitism at nature in life cycle, conventional spread plate method and plate streak carry out single bacterium colony while separating, planar surface exposes fully, is unfavorable for the growth of burgdorferi; And the microanaerobic environment that the double-layer plate method is created is more conducive to the propagation of burgdorferi.
(2) with the single bacterium colony of dilution process acquisition by unlimited, compare, separation purification method Simple fast of the present invention, result is distinct, can see single bacterium colony by naked eyes.
(3) method of the present invention is utilized 5% low melting-point agarose configuration solid BSK substratum, and low melting-point agarose has lower melt temperature, like this can be not because excess Temperature affects the existence of the burgdorferi cell in bacteria suspension when pouring into culture dish.In addition, low melting-point agarose has higher purity, can be because of the growth of impurity effect burgdorferi cell, and also the substratum of configuration is transparent, is convenient to observe.And the low melting-point agarose degree of crosslinking is moderate, be applicable to the growth of burgdorferi, can form macroscopic single bacterium colony.The present invention adopts double-deck flat board to carry out single bacterium colony separation and pure culture, in order that be the microanaerobic environment of growth creation of burgdorferi, is convenient to the single bacterium colony of Growth of Cells, field planting and formation.
The accompanying drawing explanation:
Fig. 1 is the schematic diagram of single bacterium colony of the Borrelia burgdoyferi B31 bacterial strain that obtains of a kind of double-layer plate tipping.
Left figure is solid plate, and the impermeable single bacterium colony of visible white produces, and colony diameter is about the 0.2-1.5 millimeter, and right figure is that amplify the part of partial left figure, and three single bacterium colonies are clearly arranged.
Embodiment
Substratum in all embodiment and molecular biology working method are familiar with by these those skilled in the art, can be with reference to (the laboratory manual such as Sambrook " molecular cloning ", the cold spring port, 1989) and the reagent in " Z ü ckert; W.R.2007.Laboratory Maintenance of Borrelia burgdorferi. Current Protocols in Microbiology.4:12C.1.1 – 12C.1.10. " embodiment of the present invention be more than analytical pure, purchased from U.S. Sigma company.
The invention will be further described below in conjunction with embodiment, but the present invention is not subjected to the restriction of embodiment.
Embodiment 1:
A kind of burgdorferi BSK stock solution substratum, it is made by the raw material of following weight part:
A kind of preparation method of burgdorferi BSK stock solution substratum, the steps include:
Said components is added in distilled water in order one by one, and room temperature (20-30 ℃) stirs to dissolving fully, adds next component after a upper component is dissolved fully again.Finally adjust pH constant volume.With the filter filtration sterilization of 0.22 micron pore size, as for-20 ℃ of Refrigerator stores.
Embodiment 2:
Borrelia burgdoyferi B31 solid plate pours into and single bacterium colony separation and purification:
(1) sepharose preparation: with low melting-point agarose configuration 5%(weightmeasurement ratio, below identical) sepharose, 121 ℃, 103.4 kPas sterilizing 20 minutes, be placed in 25 ℃ standby.
(2) preparation of bottom solid medium: regulate water-bath temperature to 55 ℃, by 5% sepharose as for wherein melting gradually.Then according to volume ratio, be that the BSK stock solution substratum made in ratio and the embodiment 1 of 1:1 mixes, adding kantlex (sigma company) to final concentration is every milliliter of 200 microgram.Be poured onto in culture dish, for the culture dish of 10 centimetres of diameters, each needs 10 milliliters of bottom solid mediums.Standing 12 hours of room temperature, until substratum solidify fully, stand-by in 4 ℃ of Refrigerator stores after surperficial evaporated condensation water.
(3) Borrelia burgdoyferi B31 bacterial strain is cultured to logarithmic phase with BSK-H conventional liq substratum (sigma company product), counts by dark-field microscope, determine that its concentration reaches 10
7-10
8Cells/ml.Then use BSK-H conventional liq substratum gradient dilution, obtaining respectively concentration is 10,10
2, 10
3, 10
4, 10
5The bacteria suspension of cells/ml.
(4) regulate water-bath temperature to 55 degree, by 5% sepharose as for wherein melting gradually.Then in sterilizing centrifuge tube, mix BSK stock solution substratum and 7.5 milliliter of 5% sepharose made in 7.5 milliliters of embodiment 1, the Borrelia burgdoyferi bacteria suspension that adds 1 milliliter of different concns after fully mixing, adding kantlex (sigma company) to final concentration is every milliliter of 200 microgram, turn upside down after mixing for 20 times and be poured onto in the bottom solid medium prepared, standing 12 hours of room temperature, solidify, put into after surperficial evaporated condensation water 37 ℃ of CO2gas incubator (5%) fully until substratum and cultivate.
(5) observe substratum and bacterial growth situation every day, determine the pollution without bacterium or mould, for Borrelia burgdoyferi B31 bacterial strain, need approximately macroscopic single bacterium colony to occur in 10 days later, rear single bacterium colony became more obvious in 14 days.Colony diameter is about the 0.5-1.5 millimeter, circle, and smooth surface, colony edge is smooth or coarse, as shown in Figure 1.Colony growth, in Double-Medium, with the careful picking colony dark-field microscopy of toothpick, can be observed typical Borrelia burgdoyferi bacterial strain.The unicellular number difference that the bacteria suspension of different concns obtains, and obvious Gradient Effect is arranged.For Borrelia burgdoyferi B31 bacterial strain, the bacteria suspension (10 of overrich
4, 10
5Cells/ml) plate poured into, single bacterium colony may be too intensive and become inseparable.
Embodiment 3:
The application of a kind of burgdorferi list bacterium colony separation purification method in preparing the Borrelia burgdoyferi pure growth, its application process is:
(1) preparation of Borrelia burgdoyferi pure growth:
With 5 Borrelia burgdoyferi B31 bacterial strains of the careful picking of toothpick, be seeded in 12 milliliters of BSK-H conventional liq substratum (sigma company product), in 37 degree CO2gas incubator (5%), cultivate 3-4 days, observe the Borrelia burgdoyferi growing state, strain growth is to logarithmic phase, and bacterium colony concentration reaches 10
7More than cells/ml, can obtain single bacterium colony pure growth of Borrelia burgdoyferi.
(2) evaluation of Borrelia burgdoyferi endogenous plasmid:
Borrelia burgdoyferi contains wire that 20 5-56kb differ in size or the plasmid of ring-type forms, some plasmids are unsettled in the subculture in vitro separately culturing process, yet be but the important phenotypic characteristic of having encoded, therefore, the endogenous plasmid of Borrelia burgdoyferi is identified strain identification somatotype, clinical diagnosis etc. is had great importance.Usually according to the specific primer of the sequences Design of each plasmid, adopt the method for PCR to identify in this bacterium colony whether contain certain plasmid.The sequence of primer is referring to bibliographical information, " Analysis of Mechanisms Associated with Loss of Infectivity of Clonal Populations of Borrelia burgdorferi B31M1 " .Infection and Immunity.June2001, p.3670-3677.
Get single bacterium colony pure growth of 1 milliliter of acquisition, 8000g is centrifugal 10, adds 100 microliters of water resuspended, in boiling water, boils 10 minutes, centrifugal collection supernatant is cell pyrolysis liquid, identifies for PCR whether the Borrelia burgdoyferi list bacterium colony obtained also contains various endogenous plasmids.
The PCR reactive component is as follows: 10 times of damping fluids, 5 microlitres; The 10 milli dNTP that rub, 4 microlitres; 10 mmole upstream primers, 1 microlitre; 10 mmole downstream primers, 1 microlitre; The cell pyrolysis liquid template, 1 microlitre; RTaq enzyme 0.2 microlitre; Moisturizing is to cumulative volume 50 microlitres.
The PCR reaction conditions: 94 ℃ of denaturations 5 minutes, 94 ℃ of sex change of 30 circulations 30 seconds, 51 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, and then 72 ℃ are continued to extend 10 minutes.
(3) endogenous plasmid collection of illustrative plates:
The PCR product of acquisition is carried out to agarose gel electrophoresis, if specific band is arranged, illustrate that this bacterium colony contains the purpose plasmid.As shown in the table to 5 monoclonal detected results that obtain ,+meaning to contain this plasmid, this plasmid of-expression lacks.
As can be known by result, only have mono-clonal 5# bacterial strain to contain the plasmid of all detections, the one or more plasmids of other several mono-clonal disappearance, Borrelia burgdoyferi mono-clonal 5# has all endogenous plasmids, be applicable to follow-up research and as the standard substance bacterial strain, this also illustrates that initial Borrelia burgdoyferi bacterial strain is a mixed bacterial before single bacterium colony separates, and a plurality of single bacterium colonies with different plasmid maps have been mixed in the inside.
Embodiment 4:
The application of a kind of burgdorferi list bacterium colony separation purification method in preparing the burgdorferi pure growth, its application process is:
(1) use respectively BSK-H conventional liq substratum (sigma company product) to be cultured to logarithmic phase borrelia obermeyri (Borrelia recurrentis) and Hermes burgdorferi (Borrelia hermsii), by dark-field microscope, count, determine that its concentration reaches 10
7-10
8Cells/ml.Then use BSK-H conventional liq substratum gradient dilution, obtaining concentration is 10
3The bacteria suspension of cells/ml.
(2) regulate water-bath temperature to 55 degree, by 5% sepharose as for wherein melting gradually.Then in sterilizing centrifuge tube, mix BSK stock solution substratum and 7.5 milliliter of 5% sepharose made in 7.5 milliliters of embodiment 1, be poured onto in the bottom solid medium prepared in embodiment 2 after after fully mixing, adding the burgdorferi bacteria suspension of 1 milliliter of different concns to turn upside down to mix for 10 times.Standing 12 hours of room temperature, solidify, put into after surperficial evaporated condensation water 37 ℃ of CO2gas incubator (5%) fully until substratum and cultivate.
(3) observe substratum and bacterial growth situation every day, determine the pollution without bacterium or mould, colony growth is in Double-Medium, with the careful picking colony dark-field microscopy of toothpick.For the borrelia obermeyri bacterial strain, need approximately macroscopic single bacterium colony to occur in 14 days later, rear single bacterium colony became more obvious in 20 days.Colony diameter is about the 0.2-1.0 millimeter, circle, and smooth surface, colony edge is smooth or coarse.Colony growth, in Double-Medium, with the careful picking colony dark-field microscopy of toothpick, can be observed typical borrelia obermeyri bacterial strain.For Hermes burgdorferi bacterial strain, need approximately macroscopic single bacterium colony to occur in 12 days later, rear single bacterium colony became more obvious in 15 days.Colony diameter is about the 0.5-2.0 millimeter, circle or subcircular, and smooth surface, colony edge is smooth or coarse.Colony growth, in Double-Medium, with the careful picking colony dark-field microscopy of toothpick, can be observed typical Hermes burgdorferi bacterial strain.
Claims (6)
1. BSK stock solution substratum, it is made by the raw material of following weight part:
Parts by weight of raw materials
CMRL-1066 8－11 g；
Bovine serum albumin V 45-55 g;
New peptone peptone 3-6 g;
Hydroxyethyl piperazine second thiosulfonic acid 5.5-7.5 g;
Trisodium Citrate 0.4-1.2 g;
Glucose 3.5-10 g;
Yeast powder 1-3 g;
Sodium bicarbonate 1.5-3.2 g;
Sodium.alpha.-ketopropionate 0.5-1.2 g;
Acetyl glucosamine ammonia 0.2-0.6 g;
Rabbit anteserum 30-70 milliliter;
Sodium hydroxide is adjusted Medium's PH Value 7-7.8;
Distilled water is settled to 500 milliliters.
2. BSK stock solution substratum according to claim 1 is characterized in that:
Parts by weight of raw materials
CMRL-1066 9－11g；
Bovine serum albumin V 48-52 g;
New peptone peptone 4-65g;
Hydroxyethyl piperazine second thiosulfonic acid 6-7 g;
Trisodium Citrate 0.7-1g;
Glucose 5.5-8 g;
Yeast powder 1.5-2.5 g;
Sodium bicarbonate 1.8-3g;
Sodium.alpha.-ketopropionate 0.8-1 g;
Acetyl glucosamine ammonia 0.3-0.5 g;
Rabbit anteserum 35-65 milliliter;
Sodium hydroxide is adjusted Medium's PH Value to 7.2-7.6;
Distilled water is settled to 500 milliliters.
3. BSK stock solution substratum according to claim 1 is characterized in that:
Parts by weight of raw materials
CMRL-1066 9.2 g；
Bovine serum albumin V 50.6 g;
New peptone peptone 4.0 g;
Hydroxyethyl piperazine second thiosulfonic acid 7.4g;
Trisodium Citrate 0.8 g;
Glucose 5.2g;
Yeast powder 2.2g;
Sodium bicarbonate 2 g;
Sodium.alpha.-ketopropionate 1g;
Acetyl glucosamine ammonia 0.5 g;
66 milliliters of rabbit anteserums;
Sodium hydroxide is adjusted Medium's PH Value to 7.6;
Distilled water is settled to 500 milliliters.
4. BSK stock solution substratum according to claim 1 is characterized in that:
Parts by weight of raw materials
CMRL-1066 9.7 g；
Bovine serum albumin V 50.0 g;
New peptone peptone 5.0 g;
Hydroxyethyl piperazine second thiosulfonic acid 6.6 g;
Trisodium Citrate 0.7 g;
Glucose 5.0 g;
Yeast powder 2.0 g;
Sodium bicarbonate 2.2 g;
Sodium.alpha.-ketopropionate 0.8 g;
Acetyl glucosamine ammonia 0.4 g;
64 milliliters of rabbit anteserums;
Sodium hydroxide is adjusted Medium's PH Value 7.4;
Distilled water is settled to 500 milliliters.
5. method of utilizing the single bacterium colony burgdorferi of BSK stock solution substratum separation and purification claimed in claim 1, its step is as follows:
(1) sepharose preparation: with low melting-point agarose configuration 5% W/V sepharose, 121 ℃, 103.4 kPas of sterilizing 20-40 minute, be placed in 20-30 ℃ standby;
(2) preparation of bottom solid medium: regulating water-bath temperature to 55 ℃, by the thawing that is placed in one of 5%W/V sepharose, is that the ratio of 1:1 mixes with BSK stock solution substratum according to volume ratio, is poured onto culture dish; 20-30 ℃ of standing 12-24 hour, until substratum solidify fully, stand-by in 4 ℃ of Refrigerator stores after surperficial evaporated condensation water;
(3) burgdorferi bacterial strain to be separated is used BSK-H conventional liq culture medium culturing to logarithmic phase, counted by dark-field microscope, determine that its concentration reaches 10
7-10
8Cells/ml, with BSK-H conventional liq substratum gradient dilution, obtaining respectively concentration is 10,10
2, 10
3, 10
4, 10
5The bacteria suspension of cells/ml;
(4) regulate water-bath temperature to 55 ℃, by 5% W/V sepharose as for wherein melting, then in sterilizing centrifuge tube, mix 7.5 milliliters of BSK stock solution substratum and 7.5 milliliter of 5% W/V sepharose, the burgdorferi bacteria suspension to be separated that adds 1 milliliter of different concns after mixing, turn upside down after mixing for 10-20 time and be poured onto in the bottom solid medium prepared; 20-30 ℃ standing 12 hours, until substratum, solidify fully, put into 37 ℃ after surperficial evaporated condensation water, in 5% V/V CO2gas incubator, cultivate;
(5) observe substratum and bacterial growth situation, determine the pollution without bacterium or mould, occur using toothpick picking colony dark-field microscopy after macroscopic single bacterium colony;
Described BSK stock solution substratum, its composition is as follows:
Parts by weight of raw materials
CMRL-1066 8－11 g；
Bovine serum albumin V 45-55 g;
New peptone peptone 3-6 g;
Hydroxyethyl piperazine second thiosulfonic acid 5.5-7.5 g;
Trisodium Citrate 0.4-1.2 g;
Glucose 3.5-10 g;
Yeast powder 1-3 g;
Sodium bicarbonate 1.5-3.2 g;
Sodium.alpha.-ketopropionate 0.5-1.2 g;
Acetyl glucosamine ammonia 0.2-0.6 g;
Rabbit anteserum 30-70 milliliter;
Sodium hydroxide is adjusted Medium's PH Value 7-7.8;
Distilled water is settled to 500 milliliters.
6. the application of burgdorferi list bacterium colony separation purification method claimed in claim 5 in preparing the burgdorferi pure growth.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101613757A (en) * | 2009-07-09 | 2009-12-30 | 中国农业科学院兰州兽医研究所 | Detect the test kit and the detection method of Lyme disease cause of disease in the tick body |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101613757A (en) * | 2009-07-09 | 2009-12-30 | 中国农业科学院兰州兽医研究所 | Detect the test kit and the detection method of Lyme disease cause of disease in the tick body |
Non-Patent Citations (6)
| Title |
|---|
| BILAL ASLAM 等: "Evaluation of BSK-H Complete Medium Supplemented with Rabbit Serum and Sodium Bicarbonate for the Growth of Borrelia anserina", 《PAKISTAN VETERINARY JOURNAL》, 31 May 2013 (2013-05-31) * |
| 尚德秋: "《防疫检验》", 31 May 1991, article "第三章 螺旋体检验" * |
| 张习坦、马静: "《莱姆病及其防治》", 31 March 1996, article "第二章 莱姆病的病原学" * |
| 杨正时，房海: "《人及动物病原细菌学》", 31 December 2002, article "第71章 布氏疏螺旋体" * |
| 王昭孝 等: "莱姆病伯氏疏螺旋体分离培养方法的改良", 《贵州医药》, vol. 26, no. 3, 31 December 2002 (2002-12-31) * |
| 罗海波等: "《现代医学细菌学》", 31 December 1995, article "第二十二章 伯氏疏螺旋体" * |
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