Physiology and Cultivation of
Algae and Cyanobacteria
5.
Overview
• Collection of the samples
• Isolation & purification of algal culture
• Culturing
– Methods
– Equipments & material
– Conditions
– Culturing media
Collection of the samples
• purpose specific
• sample specifics (nature & environment)
• time
• concentration
• type of vessel/container
• removal of the unwanted organisms (filter)
• transfer conditions & storing
Isolation & purification of algal
culture
• Equipments & suplies
– Microscopes
– Filters & sieves
– Glassware, Plasticware, Utensils
• Methods
– Sterile manipulation
– Isolation techniques
Equipments & suplies
• Microscopes
– dissecting (80x,..)
– inverted
– lighting
• dark-field
• fiber optic light source
• fluorescent lamp
• epifluorescence
• Filters & sieves
– woven screens
– nylon netting
– membrane filters (material)
– differential filtration
• Flow-box
Glassware, Plasticware, Utensils
• borosicate glassware
• plasticware (ready-to-use, culture-grade)
• sterilization technique & sterility
• dust-proof cabinet / clean containers
• caps & splips
• sterile filtration apparatus
• sterile spatula
• pens, labels
• parafilm
• growth chambers
– test tubes, flask, culture flask, Erlenmeyer flask, Petri dish, plugs, two-steps screw
– cultivator, tank, bioreactor
Sterilization & sterile manipulation
Laminar flow-box
Example of tool arrangement and handling to avoid mistakes and contamination.
Sterilization types,
applicability and limits
Methods of isolation & purification
• Enrichment culture
• Single-cell isolation
• Size separation >> filtering
• Density separation >> centrifugation
• Dilution
• Isolation with use of agar
– streaking
– spray
• Isolation with use of phototaxis
• Automatization (flowcytometer)
purification steps of singlecell algae (after Guillard and Morton 2003)
purification steps for attached or colonial algae
Phototaxis apparatuses
(A) Schematic diagram of droplet cell
sorting, showing the joining of the
sample stream and sheath fluid
stream in the flow cell, forming the
jet-in-air flow stream with the cells
in single file. The laser intercepts the
cells at the interrogation point, where
measurements are made. If a cell
meets the criteria of the sort logic,
then the flow stream is charged just
before the break-off point of the
droplet containing the target cell. The
droplet retains the charge and is
deflected by the charge plates
toward the collection tube or plate.
(B) Image of flow stream
at the droplet break-off point with use
of a 70-mm tip.
Culturing techniques
• chemicals
• equipment – balances, ph-meter, autoclave, filtration,
ultrasonic washer, refrigerator, cultivator
• conditions – ph, temp, irradiation, photoperiode,..
• glasware – E. flask, reagent bottles, pipetes, flask, tubes, P.
dishes, spatulas, funels, filter holder, syringe, ..
• water
• agar
• soil
Media
• stock solutions
– macronutrients
– trace elements
– vitamins
– chelators
– soil extracts
• preparation of media
• synthetic media
• enriched media
• soil water media
• solidified media – agar
• Freshwater media
• Seawater media
• natural & artificial