CN102746989A - Leishmania liquid nutrient medium and preparation method thereof - Google Patents
Leishmania liquid nutrient medium and preparation method thereof Download PDFInfo
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- CN102746989A CN102746989A CN2012102509445A CN201210250944A CN102746989A CN 102746989 A CN102746989 A CN 102746989A CN 2012102509445 A CN2012102509445 A CN 2012102509445A CN 201210250944 A CN201210250944 A CN 201210250944A CN 102746989 A CN102746989 A CN 102746989A
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- leishmania
- liquid
- nutrient medium
- liquid nutrient
- hemolysate
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Abstract
The invention relates to the technical field of liquid culture media for visceral leishmaniasis epidemic situation monitoring and visceral leishmaniasis diagnosis and discloses a leishmania liquid culture medium and a preparation method thereof. Compared with a traditional 3N culture medium, the leishmania liquid culture medium used for culturing suspected marrows and enlarged animal spleens of patients suffering from visceral leishmaniasis and culturing and separating leishmania has the advantages that separation ratio in culture and separation of the desert type visceral leishmaniasis is increased from less than 1% to higher than 10%; results are obtained rapidly, and result obtaining time after culture and observation is shortened from 15 days to 5 days; and convenience in preservation is achieved, and period of validity is prolonged from 30 days to 2 years. The leishmania liquid culture medium and the preparation method thereof provide bases for visceral leishmaniasis clinical diagnosis, epidemiological investigation and leishmania drug resistance study.
Description
Technical field
The present invention relates to the liquid nutrient medium technical field of kala-azar epidemic monitoring and kala-azar diagnosis, is a kind of leishmania liquid nutrient medium and preparation method thereof.
Background technology
Kala-azar is one of Category B notifiable disease of China's " laws on contagious disease " regulation, is a kind of natural epidemic disease borne parasitic disease that is caused by leishmania, and sand fly is a communication media, and ill humans and animals is main contagium; Contagium and communication media that some is local are not also found out.WHO report in 2012, leishmaniasis comprises kala-azar, is distributed widely in 88 countries and regions, annual newly-increased case 200 ten thousand, total case has reached 1.2 hundred million, has 3.5 hundred million people to be in the threat of this disease.Global in recent years kala-azar patient's number is in rising trend, and the kala-azar patient of China and India accounts for 30% of world's leishmaniasis.Along with oil, agricultural and tourism development, the personnel in turnover epidemic-stricken area increase day by day, have increased the chance that kala-azar is propagated to external diffusion.Because treatment is not thorough or regimen is unreasonable, the leishmanial distribution range of resistance has expansion trend.
The culture of isolated leishmania is not only one of major criterion of judging kala-azar, and is the basis of carrying out study on prevention work in a deep going way.It is substratum and the culture of isolated technology that The World Health Organization (WHO) and China Ministry of Health kala-azar Case definition are recommended that the 3N culture medium culturing is separated leishmania.The 3N substratum is a biphasic culture, and lower floor is the solid agar gel, and the thawing back that freezes just can not be used, and can only preserve more than 0 ℃, and validity period is 30 days.Use the 3N culture medium culturing to separate leishmania, the kala-azar epidemic-stricken area in the Asia, kala-azar patient's leishmanial recall rate is less than 20%; In desert type kala-azar epidemic-stricken area, kala-azar patient's leishmania recall rate goes out the result for up to 15 days less than 1%.
Summary of the invention
The invention provides a kind of leishmania liquid nutrient medium and preparation method thereof; Overcome the deficiency of above-mentioned prior art, its can effectively solve use the 3N culture medium culturing separate leishmanial recall rate low, detect the problem that the time is long and validity period is short as a result.
One of technical scheme of the present invention realizes through following measure: a kind of leishmania liquid nutrient medium, this leishmania liquid nutrient medium contains NaCl 8.5g, KCl 0.4g, NaHCO
31.6g, KH
2PO
30.2g, CaCl
20.2g, glucose 5.0g, L-glutaminate 0.1g, Hepes 4.5g, RPMI-1640 10.4g, calf serum 200 ml, deionized water 1700ml, penicillium mould 1,000,000 units, Streptomycin sulphate 1,000,000 units, rabbit hemolysate 100 ml; This leishmania liquid nutrient medium obtains by following step: the first step, and successively with NaCl, KCl, the NaHCO of above-mentioned requirement
3, KH
2PO
3, CaCl
2, glucose and L-glutaminate dissolving mixing in the deionized water of above-mentioned requirement, with the NaOH solution adjusting pH to 7.2 of 0.1Mol/L, add Hepes, RPMI-1640 and the calf serum mixing of above-mentioned requirement; Second step was inserted into the liquid bottom with gas-filled valve, filled CO with gas-filled valve
2Gas becomes glassy yellow until liquid color by incarnadine in liquid, at this moment CO
2Gas reaches capacity in liquid; The 3rd goes on foot, and adds the abundant mixing of rabbit hemolysate, penicillium mould and Streptomycin sulphate of above-mentioned requirement; In the 4th step, the liquid behind the abundant mixing is placed in the sterilizing filter to use pressure be 0.4 kilogram/cm
2CO
2Obtain the leishmania liquid nutrient medium after the gas press filtration degerming, then leishmania liquid nutrient medium branch is installed in sterilization Plastic Bottle or the vial, airtight ,-20
OC preserves.
Be to the further optimization of one of foregoing invention technical scheme below or/and improve:
Above-mentioned rabbit hemolysate obtains by following step: the first step, select the doe of 1.3Kg to 1.5Kg, sterile blood sampling; The defiber of bead method; By 2 deionized waters extraordinarily of blood volume volume after the defiber, between temperature is for-5 ℃ to 5 ℃, freeze repeatedly to dissolve to erythrocyte and break, then centrifugal 20 minutes of 4000g in whizzer; Abandon deposition, supernatant is the rabbit hemolysate.
Two of technical scheme of the present invention realizes through following measure: a kind of preparation method of leishmania liquid nutrient medium is undertaken by following step: this leishmania liquid nutrient medium contains NaCl 8.5g, KCl 0.4g, NaHCO
31.6g, KH
2PO
30.2g, CaCl
20.2g, glucose 5.0g, L-glutaminate 0.1g, Hepes 4.5g, RPMI-1640 10.4g, calf serum 200 ml, deionized water 1700ml, penicillium mould 1,000,000 units, Streptomycin sulphate 1,000,000 units, rabbit hemolysate 100 ml; This leishmania liquid nutrient medium is undertaken by following step: the first step, and successively with NaCl, KCl, the NaHCO of above-mentioned requirement
3, KH
2PO
3, CaCl
2, glucose and L-glutaminate dissolving mixing in the deionized water of above-mentioned requirement, with the NaOH solution adjusting pH to 7.2 of 0.1Mol/L, add Hepes, RPMI-1640 and the calf serum mixing of above-mentioned requirement; Second step was inserted into the liquid bottom with gas-filled valve, filled CO with gas-filled valve
2Gas becomes glassy yellow until liquid color by incarnadine in liquid, at this moment CO
2Gas reaches capacity in liquid; The 3rd goes on foot, and adds the abundant mixing of rabbit hemolysate, penicillium mould and Streptomycin sulphate of above-mentioned requirement; In the 4th step, the liquid behind the abundant mixing is placed in the sterilizing filter to use pressure be 0.4 kilogram/cm
2CO
2Obtain the leishmania liquid nutrient medium after the gas press filtration degerming, then leishmania liquid nutrient medium branch is installed to airtight preservation in sterilization Plastic Bottle or the vial.
Be below two further optimization to the foregoing invention technical scheme or/and improve:
Above-mentioned rabbit hemolysate obtains by following step: the first step, select the doe of 1.3Kg to 1.5Kg, sterile blood sampling; The defiber of bead method; By 2 deionized waters extraordinarily of blood volume volume after the defiber, between temperature is for-5 ℃ to 5 ℃, freeze repeatedly to dissolve to erythrocyte and break, then centrifugal 20 minutes of 4000g in whizzer; Abandon deposition, supernatant is the rabbit hemolysate.
The present invention cultivates doubtful kala-azar patient's the marrow and the animal spleen of enlargement through the leishmania liquid nutrient medium; Cultivate and the separation leishmania; Turn out the leishmanial kala-azar of confirming as; Have the characteristics that the result is fast, preservation is convenient and validity period is long, improved leishmanial separation rate, for kala-azar clinical diagnosis, epidemiological survey and the research of leishmania resistance provide foundation.
Embodiment
The present invention does not receive the restriction of following embodiment, can confirm concrete embodiment according to technical scheme of the present invention and practical situation.
Embodiment 1: this leishmania liquid nutrient medium contains NaCl 8.5g, KCl 0.4g, NaHCO
31.6g, KH
2PO
30.2g, CaCl
20.2g, glucose 5.0g, L-glutaminate 0.1g, Hepes 4.5g, RPMI-1640 10.4g, calf serum 200 ml, deionized water 1700ml, penicillium mould 1,000,000 units, Streptomycin sulphate 1,000,000 units, rabbit hemolysate 100 ml; This leishmania liquid nutrient medium obtains by following step: the first step, and successively with NaCl, KCl, the NaHCO of above-mentioned requirement
3, KH
2PO
3, CaCl
2, glucose and L-glutaminate dissolving mixing in the deionized water of above-mentioned requirement, with the NaOH solution adjusting pH to 7.2 of 0.1Mol/L, add Hepes, RPMI-1640 and the calf serum mixing of above-mentioned requirement; Second step was inserted into the liquid bottom with gas-filled valve, filled CO with gas-filled valve
2Gas becomes glassy yellow until liquid color by incarnadine in liquid, at this moment CO
2Gas reaches capacity in liquid; The 3rd goes on foot, and adds the abundant mixing of rabbit hemolysate, penicillium mould and Streptomycin sulphate of above-mentioned requirement; In the 4th step, the liquid behind the abundant mixing is placed in the sterilizing filter to use pressure be 0.4 kilogram/cm
2CO
2Obtain the leishmania liquid nutrient medium after the gas press filtration degerming, then leishmania liquid nutrient medium branch is installed to airtight preservation in sterilization Plastic Bottle or the vial.The leishmania liquid nutrient medium is that can to preserve 3 months, temperature under 4 ℃ be can preserve under 25 ℃ 30 days in temperature for preserving 1.5 years, temperature under-20 ℃.
Embodiment 2: obtain by following step with the rabbit hemolysate of the difference of embodiment 1: embodiment 2: the first step, select the doe of 1.3Kg to 1.5Kg, sterile blood sampling; The defiber of bead method; By 2 deionized waters extraordinarily of blood volume volume after the defiber, between temperature is for-5 ℃ to 5 ℃, freeze repeatedly to dissolve to erythrocyte and break, then centrifugal 20 minutes of 4000g in whizzer; Abandon deposition, supernatant is the rabbit hemolysate.
The foregoing description gained leishmania liquid nutrient medium is used by following step:
In following practical application, strict by the enforcement of Biosafety operational provisions.
Leishmanial equipment of one culture of isolated and material are prepared
1. equipment and equipment: Biohazard Safety Equipment, inverted microscope, Tissue Culture Flask, capillary glass-tube, rk39ELISA test kit, rk39dipstick, disposable syringe, 0.9% saline water, eye scissors, 5ml Tissue Culture Flask.
2. leishmania liquid nutrient medium: the foregoing description gained leishmania liquid nutrient medium is adjusted to pH 7.2 leishmania liquid nutrient mediums is used to cultivate the leishmania promastigote; The foregoing description gained leishmania liquid nutrient medium is adjusted to pH 6.4 leishmania liquid nutrient mediums is used to cultivate the leishmania Donovan.
Face with before, the leishmania liquid nutrient medium that cryopreservation above-mentioned regulated melts and shakes up, aseptic technique is sub-packed in the 5ml Tissue Culture Flask, every bottle of 4ml.Can preserve 30 days for 4 ℃.
Two culture of isolated leishmania collections of specimens
1. gather doubtful kala-azar patient's marrow
Take the patients serum of doubtful kala-azar clinical symptom, press rk39dipstick or rk39ELISA working instructions and detect serum rk39 antibody, the patient of kala-azar antibody positive takes marrow before medicine for treatment.
2. gather marrow, spleen sample
Detect the animal of animal serum rk39 antibody positive with rk39dipstick or rk39ELISA, build bigger like domesticated dog etc., take marrow; Small-sized rodent also can be taked femur bone marrow or spleen sample, gets marrow or spleen 0.1g, on the shrinkage pool slide glass, grinds, and adds 0.2ml the foregoing description gained leishmania liquid nutrient medium, grinds to form suspension.
3. gather the sand fly sample
Tengsten lamp trapping sand fly after the evaluation classification, is dissected and gets gastric content, adds 0.1ml the foregoing description gained leishmania liquid nutrient medium, grinds to form suspension.
Three cultivations and observational technique
With doubtful kala-azar patient's marrow, animal spleen, sand fly gastric content suspension 0.01ml, be added to respectively in the capillary glass-tube or culturing bottle that the above-mentioned pH7.2 leishmania liquid nutrient medium that regulates is housed, seal back 25 ℃ of cultivations.From inoculating back beginning in the 2nd day, observe down with 200 times of mirrors of inverted microscope every day, finds that the amphitrichous body moves about, and just can define the promastigote growth; Observe the 15th day continuously, do not observe promastigote yet, handle by the leishmania negative culture results.
The above-mentioned pH6.4 leishmania liquid nutrient medium that regulates is used to cultivate Donovan; Be inoculated into the nutrient solution that the promastigote growth is arranged in the above-mentioned pH6.4 leishmania liquid nutrient medium that regulates; Get the nutrient solution smear after 3 days; Wright's staining or Ji's nurse Sa Albert'stain Albert, oily sem observation growing state.
Four leishmania enlarged culturing are planted with protecting
Observed the 8th day that promastigote is grown, and got the nutrient solution that part has promastigote, with 2 times of above-mentioned leishmania liquid nutrient mediums dilutions that regulate of amount, enlarged culturing.The leishmania that successful purifying is cultivated is diluted to every milliliter of 100,000 leishmanias with the above-mentioned leishmania liquid nutrient medium that regulates, and in vitro can preserve 40 days for 18 ℃-22 ℃, and aforesaid method can go down to posterity and preserve leishmania.
Claims (4)
1. a leishmania liquid nutrient medium is characterized in that this leishmania liquid nutrient medium contains NaCl 8.5g, KCl 0.4g, NaHCO
31.6g, KH
2PO
30.2g, CaCl
20.2g, glucose 5.0g, L-glutaminate 0.1g, Hepes 4.5g, RPMI-1640 10.4g, calf serum 200 ml, deionized water 1700ml, penicillium mould 1,000,000 units, Streptomycin sulphate 1,000,000 units, rabbit hemolysate 100 ml; This leishmania liquid nutrient medium obtains by following step: the first step, and successively with NaCl, KCl, the NaHCO of above-mentioned requirement
3, KH
2PO
3, CaCl
2, glucose and L-glutaminate dissolving mixing in the deionized water of above-mentioned requirement, with the NaOH solution adjusting pH to 7.2 of 0.1Mol/L, add Hepes, RPMI-1640 and the calf serum mixing of above-mentioned requirement; Second step was inserted into the liquid bottom with gas-filled valve, filled CO with gas-filled valve
2Gas becomes glassy yellow until liquid color by incarnadine in liquid, at this moment CO
2Gas reaches capacity in liquid; The 3rd goes on foot, and adds the abundant mixing of rabbit hemolysate, penicillium mould and Streptomycin sulphate of above-mentioned requirement; In the 4th step, the liquid behind the abundant mixing is placed in the sterilizing filter to use pressure be 0.4 kilogram/cm
2CO
2Obtain the leishmania liquid nutrient medium after the gas press filtration degerming, then leishmania liquid nutrient medium branch is installed in sterilization Plastic Bottle or the vial, airtight ,-20
OC preserves.
2. leishmania liquid nutrient medium according to claim 1 is characterized in that said rabbit hemolysate obtains by following step: the first step, select the doe of 1.3Kg to 1.5Kg; Sterile blood sampling, the defiber of bead method is by 2 deionized waters extraordinarily of blood volume volume after the defiber; Break for freezing repeatedly between-5 ℃ to 5 ℃ to dissolve to erythrocyte in temperature; Centrifugal 20 minutes of 4000g in whizzer abandons deposition then, and supernatant is the rabbit hemolysate.
3. the preparation method of a leishmania liquid nutrient medium, it is characterized in that being undertaken by following step: this leishmania liquid nutrient medium contains NaCl 8.5g, KCl 0.4g, NaHCO
31.6g, KH
2PO
30.2g, CaCl
20.2g, glucose 5.0g, L-glutaminate 0.1g, Hepes 4.5g, RPMI-1640 10.4g, calf serum 200 ml, deionized water 1700ml, penicillium mould 1,000,000 units, Streptomycin sulphate 1,000,000 units, rabbit hemolysate 100 ml; This leishmania liquid nutrient medium is undertaken by following step: the first step, and successively with NaCl, KCl, the NaHCO of above-mentioned requirement
3, KH
2PO
3, CaCl
2, glucose and L-glutaminate dissolving mixing in the deionized water of above-mentioned requirement, with the NaOH solution adjusting pH to 7.2 of 0.1Mol/L, add Hepes, RPMI-1640 and the calf serum mixing of above-mentioned requirement; Second step was inserted into the liquid bottom with gas-filled valve, filled CO with gas-filled valve
2Gas becomes glassy yellow until liquid color by incarnadine in liquid, at this moment CO
2Gas reaches capacity in liquid; The 3rd goes on foot, and adds the abundant mixing of rabbit hemolysate, penicillium mould and Streptomycin sulphate of above-mentioned requirement; In the 4th step, the liquid behind the abundant mixing is placed in the sterilizing filter to use pressure be 0.4 kilogram/cm
2CO
2Obtain the leishmania liquid nutrient medium after the gas press filtration degerming, then leishmania liquid nutrient medium branch is installed in sterilization Plastic Bottle or the vial, airtight ,-20
OC preserves.
4. the preparation method of leishmania liquid nutrient medium according to claim 3 is characterized in that said rabbit hemolysate obtains by following step: the first step, select the doe of 1.3Kg to 1.5Kg; Sterile blood sampling, the defiber of bead method is by 2 deionized waters extraordinarily of blood volume volume after the defiber; Break for freezing repeatedly between-5 ℃ to 5 ℃ to dissolve to erythrocyte in temperature; Centrifugal 20 minutes of 4000g in whizzer abandons deposition then, and supernatant is the rabbit hemolysate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3743579A (en) * | 1970-10-26 | 1973-07-03 | G Zilberblat | Method of cultivating leishmania |
| RU2086644C1 (en) * | 1992-09-01 | 1997-08-10 | Междидов Магомед Меджидович | Nutrient medium for leishmania culturing |
| DE102006041388A1 (en) * | 2006-08-29 | 2008-03-20 | Fachhochschule Jena | Nutrient medium for culturing protozoa comprises yeast extract, phosphate, hemin and glucose |
| CN102590508A (en) * | 2012-03-12 | 2012-07-18 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatographic strip for detecting viscerotropic leishmania infection and diagnosing kala-azar |
-
2012
- 2012-07-19 CN CN2012102509445A patent/CN102746989B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3743579A (en) * | 1970-10-26 | 1973-07-03 | G Zilberblat | Method of cultivating leishmania |
| RU2086644C1 (en) * | 1992-09-01 | 1997-08-10 | Междидов Магомед Меджидович | Nutrient medium for leishmania culturing |
| DE102006041388A1 (en) * | 2006-08-29 | 2008-03-20 | Fachhochschule Jena | Nutrient medium for culturing protozoa comprises yeast extract, phosphate, hemin and glucose |
| CN102590508A (en) * | 2012-03-12 | 2012-07-18 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatographic strip for detecting viscerotropic leishmania infection and diagnosing kala-azar |
Non-Patent Citations (2)
| Title |
|---|
| 廖力夫等: "从塔里木兔体内首次分离出婴儿利什曼原虫", 《中国媒介生物学及控制杂志》, vol. 20, no. 1, 28 February 2009 (2009-02-28), pages 45 - 47 * |
| 王克信等: "杜氏利什曼原虫前鞭毛体的培养和染色方法的改进", 《寄生虫与医学昆虫学报》, vol. 7, no. 2, 30 June 2000 (2000-06-30), pages 127 - 128 * |
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