By strictly following the above operating procedures, the bacterial density (typically up to OD600=8-12), product yield, and equipment lifespan can be significantly improved. In practical operation, it is necessary to establish a standardized process based on the physiological characteristics of specific strains, and keep a good record of the operation log for traceability and optimization.
The following are the usage details and key points of the liquid strain shaker:
1、 Preliminary preparation and container handling
1. Container selection and sterilization
Priority should be given to using triangular flasks (commonly 250-500mL) or baffle type fermentation tanks, and the liquid should be filled in a ratio not exceeding 70% of the container volume based on the culture volume. Glassware needs to be sterilized with high-pressure steam at 121 ℃ for 30 minutes, while plastic bottles can be fumigated with ethylene oxide to avoid residual inhibitors affecting bacterial growth.
2. Preparation of culture medium
Configure suitable culture medium according to the characteristics of the bacterial strain (such as LB medium for Escherichia coli), adjust the initial pH value (generally bacterial pH 6.8-7.2, fungal pH slightly acidic). If specific metabolites need to be enriched, inducers or precursor substances can be added.
2、 Vaccination and Loading Standards
1. Aseptic operation
Complete the inoculation in the biosafety cabinet, burn the bottle mouth with flame, remove the cotton stopper, add an appropriate amount of bacterial strain (inoculation amount is about 5% -10%), quickly seal and transfer it to the shaker through the transfer window. Pay attention to controlling the operation duration within the effective range of the alcohol lamp flame.
2. Symmetrical balanced loading
Place the container containing the bacterial solution symmetrically on the shaker to ensure load balance. Eccentric loading can cause abnormal motor vibration and shorten mechanical life. It is recommended to use counterweights to fill the gaps during high-capacity cultivation.
3、 Parameter setting and monitoring
1. Temperature control
According to the optimal growth temperature setting of the strain (such as 30 ℃ for mold and 55 ℃ for thermophilic bacteria), activate the preheating function to make the temperature difference inside the chamber ≤ ± 0.5 ℃. Verify the actual temperature every 4 hours during long-term operation to avoid temperature control failure and bacterial death.
2. Speed regulation
Balancing dissolved oxygen efficiency and shear damage: ordinary aerobic bacteria can operate at 200-300rpm, while filamentous fungi can operate at speeds below 150rpm; Anaerobic cultivation needs to be reduced to 80-120 rpm and replaced with nitrogen gas. Observe the production of foam, and add defoamer if necessary.
3. Time program management
Adopting a segmented timing strategy: slow adaptation during the delay period (0-6h), increasing to the target speed during the logarithmic period, and slowing down during the stable period to prolong the enzyme/spore formation stage. Set a deceleration program 15 minutes in advance to prevent sudden arrest from causing eddy current impact.
4、 Operation process management
1. Status inspection
Check the following items every hour: ① Check if the fixing device is loose; ② Smoothness of condensate discharge outlet; ③ Changes in liquid level inside the bottle (to determine evaporation rate); ④ Abnormal sound or odor appears.
2. Control the timing of material replenishment
Adopting an intermittent flow addition strategy, when the dissolved oxygen drops sharply on the DO electrode, a sterile silicone tube is used to supplement the carbon/nitrogen source. The replenishment volume should not exceed 10% of the working volume at a time to avoid sudden changes in osmotic pressure.
3. Pollution prevention and control
Immediately terminate the culture upon discovering an abnormal increase in turbidity, and confirm the contamination situation through microscopic examination of the sample. When there is severe pollution, the inner wall of the shaker should be wiped with disinfectant first, and then irradiated with ultraviolet light for 30 minutes before continuing to use.
5、 Harvest and aftermath management
1. Sampling techniques
Use siphon method or syringe lateral sampling to avoid disturbing the sediment at the bottom. Weigh and record the changes in wet weight before and after sampling, and convert the biomass accumulation curve.
2. Waste disposal
The pressurized container should be depressurized before opening, and the culture containing pathogenic factors should be inactivated at 121 ℃ for 30 minutes before being discharged. Metal fixtures are soaked in 75% ethanol for disinfection, and plastic parts are replaced with specialized garbage bags and sealed for disposal.
3. Equipment maintenance
Clean the dust accumulation on the fan filter every month and conduct quarterly maintenance on the tightness of the transmission belt. When not in use for a long time, drain the water tank and apply silicone grease to prevent rust. Cover the digital display screen with a protective cover to prevent dust.