Abstract : The unicellular algal species such as Isochrysis galbana 3010, 3011, Zhanjiang Dichophyllum, Coccolithus alga, and Chaetoceros mutans cannot grow normally on solid medium with sodium nitrate as the nitrogen source. The source of solid culture medium grows good algae species and can be easily preserved.
Key words unicellular algae, solid medium, preservation, nitrogen source, single-cell (Unicelluler) preservation of algae species has always been an important factor that restricts the normal supply and long-term storage of monocytic algae in the production of factory nursery. The liquid preservation method is usually adopted, and the cycle is short, easy to be polluted, and preservation articles are published: it is difficult to control, and it is easy to cause the failure of algae species preservation. The use of solid medium preservation can extend the preservation period by 2 to 3 years under suitable conditions through the sustained release of nutrients. However, some key issues need to be solved. One is the purification technology of algae species, and the second is the selection of suitable nutrients for solid media, otherwise it is difficult to achieve good results with vaccination. There are not many detailed reports on practical technologies for the preservation of algae species in solid medium in China, most of which are only rough descriptions (1) and (2). Some have poor results in practical applications, such as the ball whip of Chrysophyta. Algae 3010, 3011, Zhanjiang dinoflagellates, Coccolithus algae, and dinoflagellates such as Chaetoceros mutans, etc. According to the data, the culture medium prepared with sodium nitrate as a nitrogen source and 2% agar is often used to culture and preserve algae species. . The change of Sichuan urea as a nitrogen source, 5 ~ 6 agar made of the culture medium, the algae growth is normal and easy to purify. Other marine unicellular algae species such as Chlorophyta, Bacillariophyta, and Xanthophyta can also be used for this method. The introduction is as follows:
l, materials and methods
1.1 Instruments and equipment: Clean bench, Nikon photomicroscope, biochemical incubator, electric steam pressure sterilizer, electronic balance, electric furnace, sterile sealing membrane, triangular conical flask, pipette, alcohol lamp, inoculation needle and so on.
1.2 Reagents Drugs: Purified agar powder (Chinese Academy of Sciences Shanghai Insect Science and Technology Development Co., Ltd.), urea, sodium nitrate, potassium dihydrogen phosphate, Lucog's solution, iron citrate, sodium silicate, sodium bicarbonate Potassium sorbate, EDTA-Na, VB1, VBl2, etc.
1.3 Algae materials: Isochrys galbana OA-3011, OA-3010, sorgysiszha sand iangensis Hu&Liu, Coccolithus algae, Pavlovaviridis Tseng, Chenet Zhang, Nguyen's Chaetocerosmiielleri Lemmermann, Heterogioeasp., Chloreiiaspp. Skeletonema costatum Greville.
1.4 Method:
1.4.1 Preparation of Algae Seed Solution: The above-mentioned unicellular algal species were inoculated aseptically one week prior to the inoculation of the solid medium in order to ensure that the algae were in an exponential growth phase when the solid medium was inoculated.
1.4.2 Preparation of solid medium: Preparation of solid medium In accordance with the following (1.4.3), the nutrient composition of the medium was used to prepare the culture solution. The order of addition of nutrients was N, P, Fe, Si, VB, NaHC03, potassium sorbate, EDTA-Na, etc. Every time you add a nutrient, stir it well and add the second one. Finally, agar is added, and the nutrient solution is placed in an adjustable temperature-heating furnace to heat and melt. The hot liquid is packed in a 100ml triangular conical flask, and the dispensing volume is about 30-40ml per bottle. The triangular conical bottle mouth is sealed with a sterile culture container sealing membrane.
1.4.3 Formulation of solid medium: The formulas of Chrysophyta, Chlorophyta, Diatomophyta and Cyanophyta are as follows (3):
1.4.4 Sterilization with solid medium Place the packed solid medium in an autoclave at a pressure of 15 pounds and a temperature of 121°C for 30 minutes.
1.4.5 Inoculation and culture On the clean bench or under the alcohol lamp, the above algae liquid is inoculated into the cooled solid medium, and the inoculated ring is generally used to pierce the surface of the culture medium to draw the word “of†or “wellâ€. Line method (4), the inoculated solid culture medium is placed in a temperature of 20-25 °C, natural light (3000-50001x or so, to avoid direct light) in the environmental conditions, about ten days, you can find the algae on the medium Falling growth.
1.4.6 Cryopreservation The solid culture medium with good growth of algae is placed in a low-temperature incubator at 10°C, and natural light can be used. Under this condition, algae species can be preserved for about 2-3 years.
2. results and analysis
2.1 Results: One week after the inoculation of the algae species, the algae cells in the solid medium of some algae species were gradually bred. After one month of pounding, the Chlorella of Chlorophyta Chrysosporium, Heterodera giganteum, Zymophyta was found. The species of algae, C. renifolia, and Chrysophyta, the species of algae in the phylum, can only grow. In experiment 2, except for Chrysophyta and Chaetoceros muelleri, other algae could grow normally.
2.2 Discussion
(1) The nutrient salts of the nitrogen source are different in the test No. 1 and No. 2 nutrients in the test No. 2 solid medium. The first experiment was urea, and the second experiment was sodium nitrate. It can be seen that different nitrogen sources play different roles in the process of seed protection by the unicellular algae. The reason for the analysis is that the single-celled algae such as stalks 3010 and 3011, Zhanjiang dinoflagellate, and Chaetoceros mutans have urease, which can decompose urea as a nitrogen source, and the use of sodium nitrate as a nitrogen source requires nitrate-nitrogen. Reducing to ammonium nitrogen (2), these algae contain less heterotrophic bacteria, and because they are in a sterile environment, algae species can normally grow in test one, but not normal growth in test two.
(2) Purification of the unicellular algae: Using the solid medium to inoculate the preservation method, it is often found that the algae and algae are present, and the desired algae can be re-inoculated into the preliminary solid medium for culture and preservation by using an inoculation loop. This is repeated several times. The algae were purified.
(3) Agar: The amount of 2% (1)(2) described in the data cannot be copied mechanically when agar is added. It should be used flexibly according to the quality of the agar. Several groups of media tests can be performed in advance to understand the characteristics of the agar. The amount of agar added is different. The medium is too hard to affect the sustained release of nutrients, the water is easily lost, the growth of algae is unfavorable, the medium is too soft and the solid is not stored at a low temperature, and the purpose of prolonging the preservation time is . In short, the specific analysis of specific issues can sometimes be based on the need to preserve the species to make semi-solid medium, on the one hand slow release of nutrients, on the other hand, high moisture content, the medium is not easy to dry, is conducive to algae species save.
(4) EDTA-Na EDTA-Na can bind heavy metal ions in seawater and seawater, but at the same time, it also removes the trace elements needed for the growth and reproduction of monocytic algae. The amount should be small, and generally cannot exceed 5 mg/l. At the time of seed maintenance, the solid medium can grow normally without using EDTA-Na algae.
references
1. Chen Mingyao, editor of biological bait cultivation, Beijing China Agricultural Press, 199533-71
2. Li Jinsong and Song Quanshan, Biofeeding Technology, Beijing China Agricultural Press 1999.40-50
3. Chen Zhufen, et al., Studies on the growth and major nutrients of Isochrysis galina 3011. Ocean and Limn. 1987 55-62
4. Jilin Agricultural University, editor of aquatic microbiology 1995.11
Key words unicellular algae, solid medium, preservation, nitrogen source, single-cell (Unicelluler) preservation of algae species has always been an important factor that restricts the normal supply and long-term storage of monocytic algae in the production of factory nursery. The liquid preservation method is usually adopted, and the cycle is short, easy to be polluted, and preservation articles are published: it is difficult to control, and it is easy to cause the failure of algae species preservation. The use of solid medium preservation can extend the preservation period by 2 to 3 years under suitable conditions through the sustained release of nutrients. However, some key issues need to be solved. One is the purification technology of algae species, and the second is the selection of suitable nutrients for solid media, otherwise it is difficult to achieve good results with vaccination. There are not many detailed reports on practical technologies for the preservation of algae species in solid medium in China, most of which are only rough descriptions (1) and (2). Some have poor results in practical applications, such as the ball whip of Chrysophyta. Algae 3010, 3011, Zhanjiang dinoflagellates, Coccolithus algae, and dinoflagellates such as Chaetoceros mutans, etc. According to the data, the culture medium prepared with sodium nitrate as a nitrogen source and 2% agar is often used to culture and preserve algae species. . The change of Sichuan urea as a nitrogen source, 5 ~ 6 agar made of the culture medium, the algae growth is normal and easy to purify. Other marine unicellular algae species such as Chlorophyta, Bacillariophyta, and Xanthophyta can also be used for this method. The introduction is as follows:
l, materials and methods
1.1 Instruments and equipment: Clean bench, Nikon photomicroscope, biochemical incubator, electric steam pressure sterilizer, electronic balance, electric furnace, sterile sealing membrane, triangular conical flask, pipette, alcohol lamp, inoculation needle and so on.
1.2 Reagents Drugs: Purified agar powder (Chinese Academy of Sciences Shanghai Insect Science and Technology Development Co., Ltd.), urea, sodium nitrate, potassium dihydrogen phosphate, Lucog's solution, iron citrate, sodium silicate, sodium bicarbonate Potassium sorbate, EDTA-Na, VB1, VBl2, etc.
1.3 Algae materials: Isochrys galbana OA-3011, OA-3010, sorgysiszha sand iangensis Hu&Liu, Coccolithus algae, Pavlovaviridis Tseng, Chenet Zhang, Nguyen's Chaetocerosmiielleri Lemmermann, Heterogioeasp., Chloreiiaspp. Skeletonema costatum Greville.
1.4 Method:
1.4.1 Preparation of Algae Seed Solution: The above-mentioned unicellular algal species were inoculated aseptically one week prior to the inoculation of the solid medium in order to ensure that the algae were in an exponential growth phase when the solid medium was inoculated.
1.4.2 Preparation of solid medium: Preparation of solid medium In accordance with the following (1.4.3), the nutrient composition of the medium was used to prepare the culture solution. The order of addition of nutrients was N, P, Fe, Si, VB, NaHC03, potassium sorbate, EDTA-Na, etc. Every time you add a nutrient, stir it well and add the second one. Finally, agar is added, and the nutrient solution is placed in an adjustable temperature-heating furnace to heat and melt. The hot liquid is packed in a 100ml triangular conical flask, and the dispensing volume is about 30-40ml per bottle. The triangular conical bottle mouth is sealed with a sterile culture container sealing membrane.
1.4.3 Formulation of solid medium: The formulas of Chrysophyta, Chlorophyta, Diatomophyta and Cyanophyta are as follows (3):
1.4.4 Sterilization with solid medium Place the packed solid medium in an autoclave at a pressure of 15 pounds and a temperature of 121°C for 30 minutes.
1.4.5 Inoculation and culture On the clean bench or under the alcohol lamp, the above algae liquid is inoculated into the cooled solid medium, and the inoculated ring is generally used to pierce the surface of the culture medium to draw the word “of†or “wellâ€. Line method (4), the inoculated solid culture medium is placed in a temperature of 20-25 °C, natural light (3000-50001x or so, to avoid direct light) in the environmental conditions, about ten days, you can find the algae on the medium Falling growth.
1.4.6 Cryopreservation The solid culture medium with good growth of algae is placed in a low-temperature incubator at 10°C, and natural light can be used. Under this condition, algae species can be preserved for about 2-3 years.
2. results and analysis
2.1 Results: One week after the inoculation of the algae species, the algae cells in the solid medium of some algae species were gradually bred. After one month of pounding, the Chlorella of Chlorophyta Chrysosporium, Heterodera giganteum, Zymophyta was found. The species of algae, C. renifolia, and Chrysophyta, the species of algae in the phylum, can only grow. In experiment 2, except for Chrysophyta and Chaetoceros muelleri, other algae could grow normally.
2.2 Discussion
(1) The nutrient salts of the nitrogen source are different in the test No. 1 and No. 2 nutrients in the test No. 2 solid medium. The first experiment was urea, and the second experiment was sodium nitrate. It can be seen that different nitrogen sources play different roles in the process of seed protection by the unicellular algae. The reason for the analysis is that the single-celled algae such as stalks 3010 and 3011, Zhanjiang dinoflagellate, and Chaetoceros mutans have urease, which can decompose urea as a nitrogen source, and the use of sodium nitrate as a nitrogen source requires nitrate-nitrogen. Reducing to ammonium nitrogen (2), these algae contain less heterotrophic bacteria, and because they are in a sterile environment, algae species can normally grow in test one, but not normal growth in test two.
(2) Purification of the unicellular algae: Using the solid medium to inoculate the preservation method, it is often found that the algae and algae are present, and the desired algae can be re-inoculated into the preliminary solid medium for culture and preservation by using an inoculation loop. This is repeated several times. The algae were purified.
(3) Agar: The amount of 2% (1)(2) described in the data cannot be copied mechanically when agar is added. It should be used flexibly according to the quality of the agar. Several groups of media tests can be performed in advance to understand the characteristics of the agar. The amount of agar added is different. The medium is too hard to affect the sustained release of nutrients, the water is easily lost, the growth of algae is unfavorable, the medium is too soft and the solid is not stored at a low temperature, and the purpose of prolonging the preservation time is . In short, the specific analysis of specific issues can sometimes be based on the need to preserve the species to make semi-solid medium, on the one hand slow release of nutrients, on the other hand, high moisture content, the medium is not easy to dry, is conducive to algae species save.
(4) EDTA-Na EDTA-Na can bind heavy metal ions in seawater and seawater, but at the same time, it also removes the trace elements needed for the growth and reproduction of monocytic algae. The amount should be small, and generally cannot exceed 5 mg/l. At the time of seed maintenance, the solid medium can grow normally without using EDTA-Na algae.
references
1. Chen Mingyao, editor of biological bait cultivation, Beijing China Agricultural Press, 199533-71
2. Li Jinsong and Song Quanshan, Biofeeding Technology, Beijing China Agricultural Press 1999.40-50
3. Chen Zhufen, et al., Studies on the growth and major nutrients of Isochrysis galina 3011. Ocean and Limn. 1987 55-62
4. Jilin Agricultural University, editor of aquatic microbiology 1995.11
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