A kind of preparation method of sterile Nannochloropsis
A technology of Nannochloropsis and algae liquid, which is applied in the field of aseptic preparation of microalgae, can solve the problems of tediousness, narrow scope of application, and heavy workload, and achieve the effects of simple process, strong repeatability, and easy operation
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Embodiment 1
[0061] The cultivation of embodiment 1 Nannochloropsis
[0062] The isolated Nannochloropsis gaditana was retransferred into fresh f / 2 medium, and the ratio of algae liquid to culture liquid was 1:10 for transplantation and activation. The culture temperature is 24±1 (° C.), the light intensity is 3000 lux, the light-dark ratio is 12L:12D, and the pH is 6.5-7.0.
Embodiment 2
[0063] Embodiment 2 preparation of sterile Nannochloropsis
[0064] 1. Screening of antibiotics
[0065] Take 1ml of activated algae fluid (cultured in fresh medium for 3 days), add it into 30ml of sterilized 2216E medium, and shake it in a biochemical incubator at 28°C for 18h to enrich the bacteria in the algae fluid. Take 0.2 mL of bacterial liquid and spread it on the 2216E solid medium, attach the drug-sensitive paper sheets containing different types of antibiotics to the surface of the plate, incubate in a biochemical incubator at 28°C for 3 days, and measure the diameter of the inhibition zone. Antibiotics with a diameter of inhibition zone ≥ 20 mm were selected as more sensitive types and selected as antibacterial drugs. As shown in Table 1, the present invention screens out 4 kinds of antibacterial drugs, chloramphenicol, tetracycline, ceftriaxone sodium and cefuroxime.
[0066] Table 1 The inhibitory effect of different antibiotics on bacteria in algae liquid
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Embodiment 3
[0077] Embodiment 3 sterilization effect test
[0078] Detection method 1: Take 0.2mL of the algae liquid grown for one week in the antibiotic-free medium after antibiotic treatment, spread it on 2216E solid medium, cultivate it in a biochemical incubator at 28°C for 3 days, and detect whether there are bacteria in the algae liquid;
[0079] Detection method 2: Take 1mL of the algae liquid grown for one week in the antibiotic-free medium after antibiotic treatment, add SYBR fluorescent dye in the dark and stain for 15min, and observe the sterilization effect with a fluorescence microscope after preparation;
[0080] Detection method 3: Take 10mL of the algae solution grown for one week in the antibiotic-free medium after antibiotic treatment, centrifuge at 12,000rpm for 10min, remove the supernatant, add 200μL TE buffer, mix well, and put it in a boiling water bath for 10min, and place it at -20°C for 30min Afterwards, thaw at room temperature, and centrifuge at 12,000 rpm for...
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