Microalgae Isolation Techniques

• Isolation of microalgae from mixed cultures involves separating and purifying specific microalgal species from a complex mixture of microalgae, bacteria, and other contaminants.

Initial Culture Composition

• A mixed culture contains various identities of microalgae, bacteria, and other contaminants.
• The goal is to isolate a specific microalgal species, such as a unicellular cyanobacterium.

Isolation Process Overview

• The initial step involves obtaining the target algal species and eliminating any other contaminants.
• This process progresses from a mixed culture to a unialgal culture and finally to an axenic culture.
• A unialgal culture contains the target algae along with other contaminants, such as bacteria.
• An axenic culture is a highly purified culture containing only the targeted algae without any other contaminants.

Sample Handling and Dilution

• Collected samples are not directly cultured, they undergo serial dilution to avoid overly crowded cultures.
• Serial dilution involves creating different dilutions with varying concentrations on different plates.

Sample Examination and Characterization

• Samples are examined to determine the presence of cyanobacteria.
• Sample collection is the initial step, resulting in a mixed culture.
• Sample characterization involves assessing physical and chemical characteristics.
• The aim is to optimize the required conditions for culturing the targeted microalgae.

Sample Preparation

• Microscopic examination is carried out to identify the organisms.
• Serial dilution aids in isolation.
• Media preparation is based on previously characterized conditions.

Importance of Isolation

• Isolation of microalgae from mixed cultures is crucial to avoid contamination.
• It ensures pure cultures by excluding any organism that may cause confusion in the results.
• Isolation ensures efficiency and consistency in experiments.
• Optimizing growth conditions ensures that isolated organisms from the same source have the same equal requirements

Species Identification and Characterization

• Facilitates species identification and characterization. It requires molecular identification.
• Sequencing, primer design, and DNA analysis are essential steps.
• Primers are designed, sequencing is performed, and then alignment is done.
• The results are then analyzed.
• It involves biochemical characterization and analysis with specific dyes.

Research Support and Sterilization

• Supports application-specific research.
• The preparation step includes sterilization for tools and surfaces.

Isolation Challenges

• Contamination risk.
• Species identification complexities.
• Optimizing growth conditions.

Isolation Methods

• Serial dilution helps control the microbial load by creating single, separate colonies in the culture.
• A single colony is taken and purified on a new plate.
• Saline or sterilized water is prepared.
• 1 ml of the stock culture is transferred to the first test tube and mixed, then plated.
• The process continues to the second and third tubes.
• Screening is performed for the previously plated concentrations, purifying and isolating the target species.

Micromanipulation

• It involves the use of a fine needle and a high-resolution microscope.
• Two instruments are used: a micro manipulator and a high visualization resolution microscope.

Micromanipulation procedure

• A micropipette or fine capillary needle is used to pick up the desired organism.
• The injector is attached to a micromanipulator.
• A mixed culture is placed on a slide and a drop of sterilized is added.
• The slide is placed under the microscope.
• Micro manipulator axis is adjusted to target cell suction pressure.
• The cell is sterilized for culturing .
• Micromanipulation effectiveness relies on precise instruments.
• This includes a micro manipulator and a high-resolution microscope.

Centrifuge and Washing

• Centrifuge washing, which is a helpful process for isolation of target species.
• It depends on differences in densities.
• When the mixed culture is centrifuged, bacteria and cell debris will be in the supernatant.
• Microalgae are in the pellet due to their higher density.
• Centrifugation is performed.
• The supernatant is discarded.
• Cells and debris are washed with saline to maintain cell integrity, addressing osmotic pressure.
• Cells are then re-suspended in saline and re-centrifuged.
• Washing and discarding the supernatant is performed to obtain a pure.
• This method alone is not always effective.

Enrichment Culture

• Enrichment culture is used when the microalgae of choice is known.
• In Dunaliella culture salinity in the media is adjusted.
• High salinity allows for the selection of a specific organism to be attained.
• Conditions are adjusted, and the process enriches one species by compromising on another.

Sodium Alginate Procedure

• Sodium alginate is a polysaccharide mostly isolated from seaweed.
• It's found to do encapsulation, which is gel matrix.
• Sodium alginate is sterilized, not the mixed culture.
• Sodium alginate is mixed at a specific concentration that isn't as high as the cells
• Add the mixture to a glass pipette.
• A beaker and add Calcium solution.
• Add droplet wise of the alginate and calcium solution.
• The principle is that alginate finds divalent Calcium to form into a single beads.
• One sodium alginate finds the divalent calcium, which promotes the capsule formation.
• The capsule either has microalgae.

Post Alginate Procedure

• After the alginate procedure is done, the material beads are rinsed.
• Aspiration of the capsules is done next to confirm successful mixing.
• Capsule lysis is done with washing buffers.
• The final capsules are cultured under the best examination conditions and plated.

Agar Streak Plating

• Agar streak isolation is a method for creating isolated specimens on a plate.
• Samples are dispersed and placed on to the plates in streaks across media filled plates.
• By the end of the streak line it is expected that specimens are isolated onto new lines, helping to separate out a single colony.