Phytoplankton Measuring and Culture Techniques

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Phytoplankton Measuring and Culture Techniques

Phytoplankton Ecology; 24 April 2007; KLS

  1. Field Sampling

    1. How do we collect phytoplankton?

      1. Water sampling bottles at discrete depths

        1. Examples of gear: Nansen, Nisken, Van Dorn, Kimmerer…

        2. Advantages

          1. Collects all phytoplankton (including nanoplankton)

          2. Can determine detailed vertical profiles if take samples at different depths

          3. Quantitative (can sample known volume)

        3. Disadvantages

          1. Need many samples to get a vertical profile

          2. May miss phytoplankton in between sampling points (and thin layers of phytoplankton are found in many lakes)

          3. Samples often need to be concentrated for counting (settling)

        4. Caveats/variations – can use vertical profilers (e.g., measures of in vivo fluorescence on samplers) to determine where to collect discrete samples so don’t miss phytoplankton layers.

      2. Integrated water column sampling

        1. Examples of gear: Coliwasa (column integrated water sample), tube with plug (‘snake’), pump and tube

        2. Advantages

          1. Collects all phytoplankton (including nanoplankton)

          2. Collects phytoplankton from all depths

          3. Quantitative (sample known volume)

          4. Need fewer samples to represent phytoplankton at many depths

        3. Disadvantages

          1. Lose vertical resolution

          2. Often only can sample top 10 m at most with this method (good for shallow lakes or epilimnion samples in many lakes)

          3. Samples often need to be concentrated for counting

      3. Phytoplankton nets

        1. Gear: fine meshed (often 10 m) phytoplankton nets

        2. Advantages

          1. Concentrates large algae, so often settling isn’t needed to identify samples

          2. Easily deployed from shore (tossing) or boats

        3. Disadvantages

          1. A large % of phytoplankton is small enough to pass through even the finest mesh nets (more than 50% by biomass and often more by productivity) – so this is not a good qualitative measure of phytoplankton

          2. The volume of water passing through the net is hard to measure – it’s hard to get good flow meter reading and net clogs, changing efficiency – so is not possible to get a quantitative measure

          3. Force of towing can break up and destroy some larger algae

          4. Once the standard for phytoplankton collection (before 1950) phytoplankton nets are rarely used now except to collect ‘net’ plankton for demonstration purposes or to isolate for culturing (not the best method for that either due to cell damage)

    2. How do we preserve phytoplankton for counting?

      1. Don’t preserve; count fresh

        1. Advantages

          1. Don’t lose color, a good diagnostic for division level identification

          2. Don’t lose motility – can also help with identification

        2. Disadvantages

          1. Need to do right away or algae will die or grow, so counts will be inaccurate

      2. Lugol’s solution (10 g I2 and 20 g KI in 200 mL distilled water and 20 mL glacial acetic acid; use ~ 1 drop – 0.05 mL per 5 mL sample)

        1. Advantages

          1. Adds weight to cells and helps phytoplankton cells sink to bottom of settling chamber (see below)

          2. Is not very toxic

          3. Is a relatively good preservative for both ‘soft’ and loricate algae

        2. Disadvantages

          1. Breaks down in sunlight – need to keep samples and stock solution in the dark; if storing long term need to replenish lugols in samples periodically

          2. Turns the cells brown, making it harder to identify them to division (no color cues). This is not a problem with algal cultures, where you know what algae are there and are just enumerating, so is good for that use. Can also enhance contrast under light microscopy, which can aid in Image Analysis.

      3. Buffered glutaraldehyde

        1. Advantages

          1. Preserves much of the color

        2. Disadvantages

          1. Is quite toxic

    1. How do we concentrate algae for counting?

      1. Phytoplankton nets (see above I.A.3)

      2. Sedimentation chambers

        1. Examples: Utermöhl chambers; modified sedimenting chambers

        2. How they work

          1. Settle all algae in a known volume of water (settle by gravity – need to settle 3 hours for every 1 cm height to allow sinking of all cells)

          2. Either count on the cover slip (on bottom of Utermöhl chamber through an inverted scope – water can limit magnification to ~500X; or with special sedimenting chamber can count on either inverted or compound scope)

        3. Advantages

          1. Relatively easy to use

          2. Standard method since 1950s. Lots of comparable data with the same or similar protocols

        4. Disadvantages

          1. Can be hard to find large volume cells (e.g., 50 ml) necessary to concentrate the lower number of cells per volume from oligotrophic lakes

          2. Often chambers are expensive and you can only settle a few samples at a time

          3. Some setups don’t allow you to make permanent slides for record keeping (this is less important with the advent of digital cameras that can be used to keep a permanent record of fields of view)

      3. Settling chambers with removal of water – can keep preserved samples

        1. Advantages

          1. Can concentrate higher volumes of water

          2. Can preserve the concentrated cells for future use

          3. Can use alternate counting trays (not just Utermöhl chambers) for counting cells (not restricted to inverted microscopes)

          4. Often is cheap to concentrate many cells at once

        2. Disadvantages

          1. Need to remove surface water carefully (gentle aspiration) to avoid resuspending cells.

          2. A few cells may be lost in transfer from the settling chamber if not careful

        3. Tip: Add a drop of surfactant (soap) to surface of material to be settled, so as to release any phytoplankton stuck in the surface film

      4. Concentration by water displacement – remove water from sample and leave algae by gluing a fine mesh over a plastic tube, and submerging the tube gently into the sample. Can remove water in tube by aspiration (as above). Determine volume of concentrate after removal of water to calculate the concentration factor.

        1. Advantages

          1. Can concentrate higher volumes of water than Utermöhl

          2. Can preserve the concentrated cells for future use

          3. Can use alternate counting trays (not just Utermöhl chambers) for counting cells

        2. Disadvantages

          1. Need to remove surface water carefully (gentle aspiration) to avoid resuspending cells.

          2. Some small phytoplankton may pass through mesh, biasing sample

          3. More time consuming than method 3

          4. A few cells may be lost in transfer from the settling chamber if not careful

      5. Centrifugation

        1. Advantages

          1. Quick

          2. Cheap if you have a centrifuge

        2. Disadvantages

          1. Often doesn’t centrifuge out all algae – not a good quantitative technique

          2. Some fragile algae may lyse – not a good qualitative technique

          3. Sometimes hard to get all algae out of centrifuge tube so that the sample remains quantitative

          4. Rarely used now as a technique for processing field samples

    1. How do we estimate phytoplankton biomass from field samples?

      1. Chlorophyll a

        1. What it tells you

          1. a rough estimate of algal biomass

        2. Advantages

          1. relatively quick

          2. easy

          3. relatively inexpensive

          4. long records of chlorophyll a values for comparison

          5. unlike seston carbon, doesn’t include non-phytoplankton components of seston (e.g., zooplankton or soil-derived detritus, inorganic carbon)

        3. Disadvantages

          1. can’t tell type of phytoplankton (all have chl. a)

          2. chlorophyll per cell and per carbon varies with many other factors including light climate, type of phytoplankton…(Reynolds discusses this)

        4. Methods for measuring chlorophyll a

          1. Fluorometry

            1. in vivo fluorescence –

              1. Advantages – can be done quickly and can get continuous profiles both vertically with lowered instruments and horizontally with pumped samples while cruising

              2. Disadvantages -- often not a very good estimator of extracted chlorophyll a because the relationship between in vivo chlorophyll a and biomass varies with algal taxon, cell condition (how much energy actually gets captured by cell vs. fluorescing), light conditions, etc.

              3. Caveats: Need to calibrate each sampling date/site with some extracted chlorophyll estimates

            2. Extracted chlorophyll – filter water sample onto membrane (often 0.2 or 0.45 m) or glass fiber (<0.7 m) filters; extract into solvent (varies depending on method used – 90% buffered acetone is most common now); some methods require grinding of filters

              1. Advantages – very accurate means of determining chlorophyll a in seston; relatively easy; can detect low levels of chlorophyll when sample is dilute; before extraction filtered samples can be stored for a few weeks in ultra-freezer (-20 to -80 oC) or a few days in freezer

              2. Disadvantage – is some time involved in extraction and cost in buying filters – can’t get as much spatial/temporal detail as with in vivo fluorescence

          2. Spectrophotometry

            1. Advantages

              1. Accurate

              2. Can estimate phaeophytins (degraded chlorophyll) and other pigments (see below)

              3. before extraction filtered samples can be stored for a few weeks in ultra-freezer (-20 to -80 oC) or a few days in freezer

            2. Disadvantage

              1. Requires more concentrated sample (may need to collect and filter lots of water in an oligotrophic lake)

              2. Is some time involved in extraction and cost in buying filters – can’t get as much spatial/temporal detail as with in vivo fluorescence

          3. Satellite and other remote sensing

            1. Advantages –

              1. Can get huge spatial scales (horizontal)

            2. Disadvantages

              1. Still need ground truthing with season and lake

              2. Expense and availability of images

              3. Lose vertical resolution

              4. Need large water body

      2. Seston Carbon

        1. What it tells you – amount of carbon in seston

        2. Advantages

          1. Can be relatively quick and easy

          2. Carbon is often a basic currency for food web and biogeochemical studies

        3. Disadvantages

          1. Need to collect and filter water samples for method (1)

          2. Can include detrital carbon or inorganic carbon depending on method (sometimes not a problem, sometimes substantial – e.g., in small eutrophic lakes)

        4. Methods

          1. Elemental analyzer (EA) – take filter and put in tin boat. EA combusts and reduces to carbon and nitrogen gas – get C and N content of seston

            1. Advantages

              1. Simple

              2. Get carbon and nitrogen content

              3. Direct measure of seston

            2. Disadvantages

              1. Can include detrital carbon or inorganic carbon (if treat with acid fumes you can eliminate inorganic C contamination)

              2. Need an elemental analyzer

              3. Cost of filters and running samples

              4. Processing time of filtering, preparing and running samples

          2. Estimate from: (cell counts for each taxon) X (cell volume per taxon) X (cell density for each taxon)

            1. Advantages

              1. Estimates carbon in algae only (not detritus)

              2. Get species-specific carbon estimates

            2. Disadvantages

              1. Incredibly time consuming

              2. Errors involved in cell counts, biovolume estimates and cell density compound in this calculation – you are making lots of assumptions here (see methods below for cell counts and for biovolume)

              3. Generally cell densities are taken from literature, not measured for that study

    1. How do we estimate phytoplankton composition from field samples?

      1. Microscope counts, identification, and biomass

        1. Advantages

          1. Tells you exactly who’s there

          2. You can decide on level of taxonomic resolution – anything from division to species (hard!)

          3. Can make permanent slides or digital images to have a long-term record of counts

        2. Disadvantages

          1. Requires a lot of training

          2. Very time consuming

          3. Depending on desired taxonomic resolution may require high quality high powered compound scopes with specialized features (DIC, phase, inverted scopes, epifluorescence)

        3. Caveats – need to make sure you count enough random fields or strips so that you get a good sample (>100 cells of each major taxon) – often cells are not distributed evenly on slides or other counting devices.

        4. Methods

          1. Utermöhl chamber or modified settling chambers

            1. Concentration of sample occurs on chamber; sample is counted with an inverted scope; can use a whipple grid (grid in ocular) or count random fields or strips to determine number of cells per area of cover slip and convert to cells per volume

            2. Advantages

              1. No need to transfer sample from another concentrating chamber

              2. Long a standard method

            3. Disadvantages

              1. Some diminishment of resolution if cell has large volume of water overlying the sample

              2. Limit to how much can concentrate samples

          2. Counting cells that can be used with concentrated samples (or lab cultures – often more concentrated than in the field)

            1. Sedgwick-Rafter (S-R) cell – only can use on low power (too deep); good for large algae and processing lots of sample

            2. Hemocytometer – used for counting blood cells; has a grid; contains a known volume of water (generally small); allows you to choose random squares of different sizes for counting; need to have concentrated sample; can use with relatively high power

            3. Palmer-Maloney (P-M) cell – circular chamber with 2 narrow channels; holds 0.1 ml; can be used on higher power than S-R (is thinner); need concentrated sample

          3. Filtration onto membrane filter (often 0.45 m) and clearing of membrane (with immersion oil)

            1. Advantages

              1. can use v. high magnification

              2. can make permanent slides

            2. but even low vacuum (never use >0.5 atm) can rupture and deform cells (e.g. flagellates)

            3. distribution of cells on filter is non-random (often are concentrated at edge) – again, need to count strips across the membrane or many fields for an accurate count

          4. Staining with a fluorescent dye (e.g., AO or DAPI), filtration and examination under epi-fluorescent scope (need 2 filter sets – one for stain and one for autofluorescence)

            1. Advantages

              1. really only way to count tiny phytoplankton (nano and picoplankton)

              2. can count heterotrophic bacteria at same time

              3. process can be sped up with image analysis (take digital images of fields – also get permanent records)

            2. Disadvantages

              1. Time consuming – need to stain and count cells

              2. Stain and epifluorescence scope and bulbs can be expensive – need at least 2 filter sets

      2. Flow cytometry/particle counting

        1. Advantages

          1. Automates the counting process

          2. Faster than optical counting

          3. Sometimes can give lots of useful information – cell volumes, DNA content

        2. Disadvantages

          1. Can be very expensive

          2. Can get signals from detritus and other particles

          3. Don’t have the same taxonomic resolution as counts (but that’s ok for some things)

        3. Examples

          1. Coulter counter – particles pass through opening between 2 electrodes, breaking the electric field; calculates number and assumes objects are spheres to calculate estimated volumes. Can’t count very small cells (<2 m); interference problems with non-algal particles. Don’t get taxonomic information (only sorts by size); good for cultures.

          2. Optical beam counters (similar problems and advantages); some new ones allow imaging

          3. Flow cytometers – cells pass in fluid in a thin laminar stream past lasers or mercury lamps – light is scattered based on physical properties of cell (e.g., size, shape, refractive index); cells may also emit fluorescence. These signals are detected and analyzed.

            1. Advantages

              1. Can tell number, size, pigments of major divisions

              2. Can count picoplankton

              3. Some really expensive ones can sort out particular cells to culture or analyze!

              4. Can combine with fluorescent probes to look at bacteria and viruses or at specific cytochemical compounds – e.g., proteins, lipids, nucleic acids

              5. Can also use fluorescently labeled antibodies to tag certain taxa – many of these are being developed

            2. Disadvantages

              1. Generally need fresh samples (<12 hours) or deep frozen or specially preserved samples

              2. Very expensive

              3. Can require lots of tweaking and calibration

              4. Often still not high taxonomic resolution unless using other probes or techniques

      3. Pigment analysis

        1. Advantages

          1. Because different algal divisions have different accessory pigments, analyzing seston for these pigments can help determine broad taxonomic composition of a phytoplankton community

          2. Easier and faster than counting – requires less time and skill/training

          3. Samples can be stored at -80oC for months

        2. Disadvantages

          1. Gives you data at a broad taxonomic level

          2. Pigment expression varies with environmental conditions, so this really is more often like a presence/absence of a taxon rather than quantitative

          3. Can require lots of sample

          4. Can be expensive and time-consuming (HPLC)

          5. People still debate the correct/standard methods for both spectrophotometric equations and appropriate HPLC techniques

        3. Methods

          1. Spectrophotometric (as in chlorophyll a) – extract pigments from filtered algae; use a scanning spectrophotometer to get absorbance at different wavelengths. Empirically derived equations can be used to separate chlorophylls a, b and c, as well as to estimate phaeophytin (with an acidification step).

            1. Advantages – faster and cheaper than HPLC

            2. Disadvantages – requires lots of sample; fewer pigments determined than HPLC; can have complications from sample turbidity

          2. HPLC (high performance liquid chromatography) – extract pigments from filtered algae; pigment separation is obtained by using different columns; pigments are identified by comparing retention time with standards, by UV/VIS spectroscopy, or by liquid chromatography/mass spectrometry (LC/MS) and comparing the mass spectra with those of pigment standards or known libraries.

            1. Advantages – can identify many accessory pigments; can provide optical and structural data about unknown pigments

            2. Disadvantages – methods need to be calibrated and perfected for each set of pigments with internal standards and extensive calibration and controls; requires expensive equipment; quantification can require lots of QA/QC.

          3. Various types of field ‘fluoroprobes’

            1. Advantages -- Allows use in field and may sample continuously over broad spatial scales

            2. Disadvantages -- Is in vivo measurement and may be altered by water conditions, algal community composition and condition; need lots of calibrations with extracted pigments and/or cell counts

    1. How do we estimate cell biovolume?

      1. Microscopically

        1. Using ocular micrometer measure cell dimensions and approximate cell shapes for each type of cell (usually estimate for ~30 cells per taxon per sample)

          1. Advantages

            1. Cell size varies greatly season to season and lake to lake – think about diatom reproduction, for example, so using tables of standard volumes for each species/genus will introduce a lot of error into biovolume calculations; direct measurement is superior

          2. Disadvantages

            1. Very time consuming

            2. Are still estimating volume by approximating cell shape (not really measuring it in 3D)

        2. Automated image analysis of digital images from microscope fields (e.g., ImagePro, Image J)

          1. Advantages

            1. Permanent digital image

            2. Saves time (automated measuring functions or macros)

            3. As above, is more accurate than assuming all cells of species X are always the same size

          2. Disadvantages

            1. Is still time consuming

            2. Still not 3D – can get area but not volume of cells directly

      2. Particle counters/flow cytometry – provide equivalent spherical volumes

        1. Advantages

          1. Get data along with counts – quick

        2. Disadvantage

          1. Often not taxon-specific volumes

          2. Is still an estimation of volume (not all cells are spheres)

    1. How do we estimate phytoplankton productivity in the field?

      1. Cell number changes – problems with losses of cells due to grazers and other mortality – generally not a good indicator of growth rate

      2. Chlorophyll changes – as above

      3. Whole lake/stream oxygen changes – diurnal pattern or hypolimnetic oxygen debt give reasonable estimates of community metabolism (community primary productivity and respiration); need to make assumptions about exchanges of gas with the atmosphere (not a closed system)

      4. Light-dark bottle incubation – closed system so avoids atmospheric exchange; bottle effects may be a problem with long incubations

        1. oxygen change – can do anywhere; simple; microtechniques allow for some low-level measurements, but lower limit of detection still relatively high

        2. 14C uptake – permit increasingly hard to obtain for field work; allows measurements of low levels of primary production; can’t easily separate gross and net production

      5. Cell condition – methods being used more; e.g., fluorometry

  1. Phytoplankton culturing

    1. How do I acquire phytoplankton cultures?

      1. Can order known strains from culture collections (or trade with friends!)

        1. Advantages

          1. Known source (avoid cheap sources like wards unless for class only)

          2. Often is other work done on that strain (genetic, biochemical, ecological)

          3. Can get same clone if you happen to kill your culture

          4. Is selected to grow well in lab

          5. Often comes with good ideas about which culture media it will grow on best

        2. Disadvantages

          1. May be a ‘lab rat’ not representative of ‘wild’ algae – many, many generations for further selection for traits that are beneficial in lab

          2. Many kinds of algae lose ‘wild’ traits in culture collections – e.g., growth pattern becomes unicellular instead of colonial

      2. Isolate your own algae

        1. Some algae are sensitive to collection techniques and do not do well if captured by net

        2. Some algae die quickly in a sample (even in 1 hour). Collect water and isolate immediately for best results

        3. Always record site and date of sample

        4. Need to use sterile technique and have clean, non-toxic glassware (see below)

        5. Use a dissecting or inverted microscope – easier to move hand/pipette in correct direction to isolate cells

        6. Micropipette isolation of algae –

          1. Use either sterilized glass (borosilicate) or pre-sterilized plastic micropipettes or capillary tubes. You can ‘pull’ glass pipettes after heating on a Bunsen burner to make your own micropipettes

          2. Pick up a cell you want to culture by sucking it up into the pipette (while looking through the microscope

          3. Place cell into sterile lake water or culture medium droplet

          4. Repeat transfer into new separate droplets at least 2X – need to be sure you have only that cell and have rinsed any contaminants

          5. Can either suck up cells with capillary action or by gentle mouth suctioning on tube attached to pipette; blow out to move to next droplet. If using capillary action you want to fill the pipette with sterile water so that it doesn’t suck up too much of the sample from which you are trying to isolate a single cell – there will still be enough capillary action when you place it into the sample to suck up a cell

          6. When have a single cell isolated, add it to the culture tube in which you will grow it

        7. Plating on agar

          1. Streak a sample across the agar and let grow

          2. Remove cells from individual colonies with micropipette or transfer loop

          3. Identify and verify you have uni-algal cultures

        8. Miscellaneous techniques

          1. Multiple serial dilutions until you have on average 1 cell per tube – grow and see what you have; make uni-algal if not already

          2. Can use phototaxis or centrifugation as an initial concentrating step before isolation

          3. Flow cytometry (see above)

        9. Often get greater success by first isolating into ‘enrichment’ media (see below) and then transferring to defined media once culture is successful

    1. How do I culture my algae once I have them?

      1. Culture media

        1. Agar – may need to wash to remove impurities (ordinary agar can lyse some cyanobacteria) or buy molecular biology grade agarose

          1. Advantages – can often maintain slow growing cells on agar – good for stocks/backups

          2. Disadvantages – often algae don’t grow like ‘wild’ algae on agar; may need to add nutrients to promote growth

        2. Soil media -- Needs to be ground and dried. Distilled water is then added to the soil (2 parts water to 1 part soil) and then the mixture is autoclaved (often 2 hours) and filtered. The filtrate is autoclaved again.

          1. Advantages – useful when requirements of algae are not fully known (also may add DOC for mixotrophs); algae often grow more like ‘wild’ algae on these media

          2. Disadvantages – not defined; need to avoid soils with fertilizer and pesticides; generally loamy and not clay soil is best. The medium may change as source of soil changes

        3. Enriched media – add nutrients to natural lake/stream water

          1. Advantages – algae often grow more like ‘wild’ algae on these media

          2. Disadvantages – all components not defined

        4. Defined/synthetic media – using a ultrapure distilled water, various macronutrients, trace elements and vitamins are added from stock solutions.

          1. Advantages – know all components of media; constant through time

          2. Disadvantages – time consuming to produce; many options – need to find right medium for your alga; can be toxic to zooplankton

      2. Sterile technique

        1. Vital for maintaining uni-algal cultures – otherwise cultures will be contaminated – can never expose containers or medium to air – will be contaminated with dust, spores and microorganisms (including algae). Never touch or expose open surfaces!

        2. Methods

          1. Wipe all surfaces and gloved hands with ethanol before handling cultures or sterilized equipment

          2. Acid wash, rinse and then sterilize all glassware in an autoclave

          3. Autoclave or heat in a flame all transfer pipettes or loops

          4. Do culture transfers under a laminar flow hood, preferably with UV light to pre-sterilize surfaces

          5. Sterilize culture medium

            1. Autoclave on ‘liquid’ setting – this will heat under pressure to temperatures over boiling

              1. Be sure to let exhaust slowly (don’t end cycle early) or you will boil the liquid and precipitate ingredients (often bound with iron or silicate)

              2. Don’t autoclave temperature sensitive materials – e.g., vitamins. Add these later and sterilize by filtering

            2. Sterile filter

              1. Pore size <0.2 m

              2. Can get autoclavable large volume filters

              3. Autoclave/sterilize filter apparatus

              4. Pump (usually peristaltic or vacuum pump) medium through filter into autoclaved container – do this under a laminar flow hood if possible. Otherwise make sure all components and table top are wiped off with ethanol and everything is covered and not exposed to air

              5. Won’t kill viruses

      1. Axenic culture – growing algae with no bacteria (or viruses); take unialgal culture and either screen filter gently (allowing bacteria to pass), centrifuge gently (collecting heavy fraction), or use repeated dilutions into sterile medium and antibiotics to kill bacteria

        1. Advantages

          1. You have pure algae

        2. Disadvantages

          1. Very difficult and time consuming

          2. Need to check repeatedly that you don’t have bacterial contaminants

          3. Not all algae have been successfully grown axenically

      2. Batch vs. continuous culture

        1. Continuous (chemostat) - medium pumped into algal culture at slow, continuous rate

          1. Advantages

            1. Cultures can remain in exponential growth for long periods

            2. Can control growth rate (must match the dilution rate once the cells are at equilibrium)

          2. Disadvantage

            1. Requires more expensive equipment (e.g., pumps)

            2. Requires daily checking

        2. Batch

          1. (semi-continuous) – remove and add a percentage of the medium at known intervals. The faster the interval, the more this approximates continuous culture.

            1. Advantages

              1. Cheaper than chemostats

            2. Disadvantages

              1. Balanced growth occurs only during exponential growth (in chemostats is the standard condition) so cell condition will change between fresh medium additions

              2. Need to keep good track of fluorescence and other parameters to make sure you are in exponential growth phase

          2. Batch – transfer exponentially growing cells to new medium to keep growing exponentially; requires transfer often enough that growth is unlimited

      3. General advice

        1. Need to be careful to avoid light limitation – common in dense cultures; most common problem. Use full spectrum fluorescent bulbs to match natural light (old standard was ‘cool-white, but not as natural a spectrum). Make sure you measure light (should have >10 moles quanta m-1 sec-1, but probably <200 moles quanta m-1 sec-1 to avoid photo-inhibition)

        2. May need to bubble dense cultures to avoid C limitation, but be sure to have sterile filter on air line to avoid contamination

        3. Can grow in Erlenmeyer (or similar) flasks, glass tubes, etc. Tops must allow gas exchange, but prevent contamination

        4. Use a light: dark cycle

        5. Need to ‘feed’ algae new medium at constant intervals to maintain cultures in same phase of growth and same general biochemical composition (pigments, nutrient content…)

        6. Must pre-wash all new glassware in dilute acid and rinse copiously

        7. Avoid using any glassware that has been used for poisons (e.g., phenol, formaldehyde, mercury)

        8. Collect and feed algae at same time of day (diurnal rhythms of cell division)

Useful References
American Public Health Association. 2005. Standard Methods for the Examination of Water and Wastewater. 21st edition. American Public Health Association, Washington, D.C. 1386 pages.

Anderson, R.A., editor. 2005. Algal Culturing Techniques. Elsevier.

* This is the current methods book sponsored by the Phycological Society of America

Fogg, G.E. and B. Thake. 1987. Algal Cultures and Phytoplankton Ecology. 3rd edition. The University of Wisconsin Press, Madison.

Kemp, P.E., B.F. Sheer, E.B. Sheer and J.J. Cole, editors. 1993. Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers.

Stein, J.R., editor. 1973. Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press, Cambridge.

Strickland, J.D.H. and T.R. Parsons. 1978. A Practical Handbook of Seawater Analysis. 2nd edition. Fisheries Research Board of Canada.

Wetzel, R.G. and G.E. Likens. 2000. Limnological Analyses. 3rd edition. Springer-Verlag, New York.

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