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Australian

National

Algae

Culture

Collection - Methods
Making Media
Autoclaving, Cleaning / glassware preparation, Stock solutions and salts,
Seawater source and treatment, Notes for Aquaculturists Media Comparison
(constituents in moles)
Major nutrients, Carbon, Nitrogen, Phosphorus, Silica
Other nutrients, Boron,

Buffers,

Chelators,

Potassium,

Soil Extract,

Vitamins
The media used in CMARC are predominantly from published recipes, some
are well known and widely used in the phycological and aquaculture
communities such as f/2. All the media have several components in common
: sources of nitrogen, phosphorus, vitamins and trace metals. However the
specific types of these nutrients, their concentrations and ratios vary
between the media. Some media used in CMARC have been developed by us
over many years, for instance our medium of choice for freshwater
cyanobacteria is MLA (Bolch and Blackburn, 1996) which is a modification of
the ASM media developed by Gorham et al (1964).

Some media have

relatively unusual trace metals such as selenium in GSe (A modification of


GPM, Loeblich, 1975 and vanadium in MJ medium. Because of their unusual
nature it may be tempting to leave these out if stocks are difficult to come by
but this would defeat the very purpose of the media. A detailed description
of the common nutrients, buffers and chelators can be found by following
this link
Cleaning / glassware preparation
Proper cleaning of cultureware is as critical to successful maintenance
of microalgal cultures as it is for accurate results in analytical chemistry.
Some species, such as certain benthic diatoms, adhere to the glass and
autoclaving cultures before disposal can also lead to a crust on the glassware

at the meniscus which is not removed simply by a detergent bath but


requires scrubbing with a bristled brush. Our glassware preparation involves
a preparatory rinse in hot tap water, soaking overnight in a detergent bath
(e.g. Pyroneg), scrubbing,

a wash/rinse cycle in a Laboratory dishwasher

(wash cycle at 85 0C, 2 tapwater rinses, 2 Reverse Osmosis water rinses and
a drying cycle), followed by 3 hand rinses in Milli Q water and drying in a
laboratory oven.
Non-disposable plasticware such as ploycarbonate carboys and
polypropylene fittings are soaked in a compatible detergent such as Nalgene
L-900.
Autoclaving
We use autoclaving as the prime method for sterilizing media and
particularly for making media for axenic cultures. Many of our media recipes
refer to fully autoclaved media, where the last stage in media preparation is
for individual culture flasks to be autoclaved so that the flasks remain totally
unopened until culture transfer. However where maintaining axenic cultures
is not as critical then filter sterilizing particular components of the media and
adding them aseptically to presterilized seawater (or freshwater) can be
used. Concentrated nutrient solutions can be prepared in this way so that
phosphate stocks do not precipitate and vitamin activity is not impaired.
While autoclaving at 121 0C for 15 minutes appears to be a common
mantra, the required duration of autoclaving can cause some confusion. It
should be noted that the autoclave time refers to the duration the goods or
total volume of liquid is maintained at the set temperature, and not simply
the time in the autoclave or the time after 121 0C is reached. Autoclaves
come with different chamber sizes and with different capacities for
pressurization, air removal, vacuum generation, steam generation and air
exhaust, and all these factors influence the rate of temperature increase,
holding capacity and decrease.

Therefore it is difficult to give predefined

autoclave times for anything other than hard goods such as pipettes and
empty glassware or containers with up to 100mL of media where the typical

121 0C for 15 minutes is probably appropriate. Note, however that a carboy


with 10 L of media may require in excess of 60 minutes once the autoclave
reaches the set temperature of 121

C.

The only reliable means of

monitoring temperature in bulk media is to have a thermistor within a


reference container (e.g. PT100) and this would only have to be done
periodically to calibrate the system.

Relatively small benchtop pressure

cookers may be the sole autoclave in many aquaculture facilities or used as


backup autoclaves in laboratories.

In this case, where for example the

largest container that will fit may be a 1 L culture bottle, it is rare for more
than 800mL to be autoclaved and in this instance a time of 25-30 minutes
may be appropriate. Some confusion also exists around pressure and steam,
with a perception that these factors are important in lethality. This is not
true, temperature above 100 0C is the lethal factor and pressure and steam
are simply the means of delivering that temperature to the entire contents of
the autoclavable goods. Without steam, air pockets and voids can exist in
both glassware and within autoclave bags and these can persist at lower
temperatures than the chamber temperature but steam penetration into
these voids ensures the the required temperature is met.
Autoclaving

drives

off

carbon

dioxide,

needed

for

algal

photosynthesis, and raises the pH to undesirable levels. Leaving the media


for at least 1-2 days before use will allow sufficient time for CO2 equilibration.
Autoclaving is also a mandatory method for disposing of our microalgal
stock cultures under our Approved Premise for Quarantine Permit (provided
by the Australian Quarantine and Inspection Service, AQIS). Minimum draft
regulations set by AQIS June 2004 are 121 0C at 15psi for 30mins.

Note

these guidelines are more strict than those for preparing media. Other
countries may have other regulations.

The following two links are on

autoclaving culture media and on the myths of autoclaving commercial


source note
http://www.lab-m.com/TAutoclavingMedia.htm
http://www.800ezmicro.com/microBiology.asp?mb=88

Stock solutions and salts


In our recipes we refer to working stocks and primary stocks.
Working stocks are those whose aliquots contribute directly to making the
final media.

Primary stocks are normally made where several single

substance solutions are then combined to form the working stock, eg.
CuSO4.5H2O and ZnSO4.7H2O are two of the primary stocks used to make
up the Trace Metal working stock in F/2 medium.
Culture Collections and scientists undertaking experimental research
will use chemicals that are at least Analytical Reagent Grade, both to reduce
trace contaminants that may be harmful to some microalgae and also to
ensure experimental rigour. While we suggest all stock or starter cultures be
grown with AR grade chemicals it is understandable that in mass culture
applications (> 20 - 50 L) , particularly for aquaculture, these chemicals may
be too expensive when bought in bulk quantities.
Stock solutions are made up by accurately weighing the prescribed
amount of nutrient and dissolving in a specified volume of distilled water, if
possible in a volumetric flask. Some nutrients will readily dissolve, others
need heat and stirring to fully dissolve. In contrast vitamin stocks are heat
sensitive and should not be subjected to heat treatment and should also be
kept in the dark.

Failure to fully dissolve the primary stocks of some

nutrients such as EDTA can lead to gross precipitation when these stocks are
combined to make the media.
Nutrients come with different salts and hydration. For example, while
copper and zinc may be two desired active constituents they are readily
obtained from suppliers with either SO4 or Cl2 salts (ie CuSO4 or CuCl2 and
ZnSO4 or ZnCl2).

Some nutrients also come with different hydrations, ie

the .nH2O suffix. Substituting one form for another may have no effect on
the growth of some microalgae species, but it can lead to poor growth in
others and also lead to unwanted and time consuming precipitation problems
as the overall ratio of salts in the medium has changed. Therefore deviating

from the prescribed recipes is to be avoided and ordering the correct form is
recommended.
Seawater source and treatment
The marine microalgae species maintained in CMAR are grown
usingunpolluted oceanic seawater therefore, strictly speaking , we are using
an enrichment medium. Artificial seawater media in contrast is composed of
marine salts and nutrients added to pure freshwater. Artificial seawater is
only necessary where a clean natural seawater source is unavailable or in
particular research studies (e.g. trace metal studies) where the exact
composition needs to be controlled. Our collection source takes advantage of
CSIRO Hydrochemistry group's monitoring station on the seaward side of
Maria Island off the continental shelf along the east coast of Tasmania. The
salinity at this site is ~33-36 Practical Salinity Units. This off-shore site has
very low concentrations of metal and organic pollutants therefore making it
suitable as the base medium for a wide range of marine microalgae
species.
The seawater is collected in 20L black polyethylene carboys acid washed
prior to collection and then stored at 4 0C until needed. Then it is treated
using a filtration series incorporating three 12 Millipore cartridge filters (5
m prefilter, activated charcoal filter for organics removal and a Durapore
0.45 m filter) and finally a Millipak 40TM 0.22 m disc filter.
Notes for Aquaculturists
The

recipes

predominantly

and

preparation

designed

for

protocols

laboratory

scale

used

in

culturing.

CMARC

are

Even

the

concentrated nutrient solutions may not be of sufficient volume for regular


mass algal cultivation.

It is up to the user to modify the quantities of

material needed and determine the cost-effectiveness of using our proposed


media versus commercial alternatives. As an example in the procedure to

make concentrated f2 media 5mL of the 1L working stocks are used to make
100mL of concentrated media.

As the usage rate is 1mL per 100mL of

seawater, the 100mL stock is sufficient for a 10L culture. As there are 200
aliquots of working stock available (1L stock/5mL), the total culture volume
that can be supported is 2000L. The simplest modification to obtain useful
volumes for aquaculture is to increase the volumes of the concentrated stock
solution and its constituent working stocks e.g. for f2
Lab scale concentrated stock : 6 stocks x 5 ml each = 30 mL, made up to
100 ml with distilled H2O (d.H2O) This provides enough concentrated stock
for 10 L f2 medium.
Largescale : 6 stocks x 500 ml each = 3000 mL, made up to 10,000 ml (10 L)
with (d.H2O)

This provides enough concentrated stock for 1000 L f2

medium.
Note in our recipe only 100 ml of vitamin stock is made at any one
time. For large scale purposes that volume would need to be increased to
1000 ml in accordance with the other stocks.

Major Nutrients
Microalgae do not require equal amounts of all nutrients, therefore they are
added in different quantities. Natural seawater will supply enough nutrients
for limited growth of some marine species.

Most media recipes supply

nutrients vastly in excess of the concentrations normally found in order to


support high biomass cultures. Different species require different amounts of
some nutients but the requirement for the major nutrients is relatively
uniform

according

to

the

Redfield

carbon:nitrogen:phosphate (by moles).

Ratio

of

106:16:1

of

inorganic

Carbon
According to this ratio microalgae will need roughly 6 times more carbon
than nitrogen.

For marine species the carbon requirement in small batch

cultures is met by the 2 mM contained within seawater, and by allowing


atmospheric exchange then the carbon requirement can be supplied over
time. Carbon may be lost directly after autoclaving, therefore it is sensible to
allow freshly autoclaved media to stand for at least 1-2 days before
innoculating with microalgae. Large batch cultures, generally greater than
500 mL to 1 L may need to be aerated with either air or an air/CO 2 mix to
prevent carbon limitation. Typical CO2 concentrations are in the range 0.5 5.0 % v/v
Nitrogen (adapted from Thompson, 200?? course notes)
Nitrogen is most commonly added as nitrate. However some algal species
grow better on ammonium (Thompson et al, 1989). Unfortunately ammonium
is frequently toxic at >100 mM so that much lower concentrations of
ammonium must be used.

Guillard specifies 500 mM (Guillard, 1983) but

this concentration should be tested prior to widespread use.

In some

situations it is desirable to have ammonium available. This can be relatively


easily accomplished by adding it to the nitrate stock solution to provide both
nitrogen sources, for example 50 mM NH4 and standard NO3. Because algae
preferentially remove NH4 they are likely to deplete all NH 4 prior to using
nitrate for growth.

Phosphorus
Silica
Silica is only really required by diatoms (excepting the rarely cultured
silicoflagellates) and is therefore often left out of media which are selective
for other organisms. Diatoms only need trace quantities of silica because
they are extremely efficient at scouring silica from the environment. Some

diatoms may grow for many generations in seawater media which has no
added silica and some may continue to divide for several weeks, albeit with
poor frustule development. Media such as the G variety (GP, GSe) where
the only added silica, apart from the seawater, comes from soil extract may
readily support the growth of some diatom species. Enrichment cultures for
isolating microalgae devoid of added silicate may still become rich in
diatoms therefore if selection is for non-diatom species a diatom inhibitor is
advantageous.

Germanium dioxide (GeO 2) is toxic to diatoms because it

disrupts silica deposition and the addition of low concentrations (5 mg / L) of


GeO2 to a culture medium can inhibit diatom growth.
The silica stock may precipitate if made up in glass bottles so teflon bottles
can be used instead. Precipitation is a greater problem when the stock is
first added to seawater especially if autoclaved and in glass containers.
However

it

will

slowly

disassociate

and

be

available

for

uptake.

Polycarbonate carboys for larger volumes or teflon bottles (1 L) may be used


to limit the precipitation if it is problematic, noting that teflon is especially
expensive.
Other Nutrient components
Boron ;
Carrano etal, (2009) present a review on boron and marine life. Many studies
have shown boron is an essential trace element for both terrestrial higher
plants and freshwater and marine algae. Lewin (1965, 1966) showed that
Boron is essential for marine diatom growth based on results with 16 centric
and pennate species and essential for some but not all marine flagellate
species. Diatom cell division was much reduced at boron concentrations less
than 0.5 mgL1 or ~0.05mM (~ 10% of the typical 4.5 mg/L natural seawater
concentration). While boron is a component of many culture media it is not
present in some common media such as Guillards F or F/2 which have been
used widely to support marine microalgal growth including diatoms. The
presence of boron leaching into media from borosilicate glassware may

account for some of this positive diatom growth but scale-up of diatom
growth in plastic bags (e.g. 100 - 500 L) in mariculture operations argues
that boron may not be essential for all species.

Boron is required by

heterocystous cyanobacteria through its role in nitrogenase activity in


dinitrogen fixation and heterocyst morphology may alter without boron. Nonheterocystous cyanobacteria display no alteration in growth or nitrogen
fixation. (Bonilla et al, 1990).
Bonilla, I., M. Garcia-Gonzlez, et al. (1990). Boron requirement in
cyanobacteria. Its possible role in the early evolution of photosynthetic
organisms. Plant Physiol. 94: 1554-1560.
Carrano, C. J., S. Schellenberg, et al. (2009). Boron and Marine Life: A New
Look at an Enigmatic Bioelement. Marine Biotechnology 11(4): 431-440.
Lewin,

J.

C.

(1965).

Boron

Requirement

of

Marine

Diatom.

Naturwissenschaften 52(3): 70
Lewin, J. (1966). Boron as a Growth Requirement for Diatoms. Journal of
Phycology 2(4): 160-163
Buffers
Microalgal consumption of CO2 in batch culture systems (without added CO 2)
will increase the pH, and levels above pH 9 are toxic to many microalgal
species (cyanobacteria generally have higher tolerances for elevated pH
which may give them a competitive advantage under such conditions).
Buffers may give some control over the rise in pH and are suitable to low
volume stock cultures, however in large scale dense cultures pH control is
probably more readily met by the addition of CO 2 back into the culture by
aeration (air only or air/CO2 mix ..see Carbon).
TRIS buffer (tris (hydroxymethyl) aminomethane): Generally TRIS is used at
concentrations up to 5 mM but some microalage find it toxic. An example of
varying susceptibility is shown by a comparison between two frewshwater
cyanobacteria, Microcystis and Anabaena.
buffering between pH 7.4-7.7

TRIS at 10 mM provides good

in Microcystis; with increased tolerance

towards supraoptimal concentrations of monovalent but not divalent cations


(McLachlan and Gorham, 1961). However TRIS is toxic in Anabaena flosaquae at concentrations too low to provide suitable buffering but filament
coiling, indicative of good growth, is promoted by addition of 1 mM TRIS
(Gorham et al, 1964).
Glycylglycine: is used at concentrations up to 3.8 mM, however it is both
expensive and rapidly metabolised by bacteria and should only be used in
axenic cultures
Sodium bicarbonate (NaHCO3): buffers well in the pH range 7.5-8.5 and is
used in the freshwater medium MLA for cynaobacteria cultures.
Chelators
Metals are often insoluble in seawater, and if added may rapidly form
insoluble hydroxides and not be available for algal uptake. Various chelators
(organic chemicals) are available which bind with the free metal ions forming
a

metal

complex

which

may

be

utilized

by

microalgae.

Ethylenediaminetetraacetic acid (EDTA) is commonly used because it is not


readily metabolized by bacteria.

The di-sodium salt, Na 2EDTA.2H20 has

improved solubility characteristics and is the most widely used.

However

when making trace metal solutions where EDTA is required it is paramount to


completely dissolve the EDTA before adding further trace metals as
precipitation can still be a problem.

Citrate (or citric acid) has been

recommended because of its lower affinities for metals commonly found in


seawater (Ca, Mg). Thus citrate should produce a more constant degree of
chelation in situations where salinity fluctuates. A limitation of citrate is its
high biodegradability which also provides organic material for bacterial
growth. The theoretical chelator:metal ratio should be 1:1 but as some
chelator will be bound to Ca and Mg in the seawater, more chelator should
be added to ensure all metals stay in solution (ratios ranging up to 3:1;
McLaughlin 1973).
Potassium

Soil Extract (full preparation protocol detailed in media recipes)


Soil extract is an adaptation of E.G. Pringsheim's biphasic soil-water medium
and is a component of some of our media. As the chemical composition is
not well-defined and may vary from batch to batch its use in experimental
situations is not recommended, however in our experience certain species
will not grow well without it and soil extract is added for both culture
maintenance and experimental studies.

Soil must be collected from a

natural uncultivated environment or a rich garden loam may be suitable. No


fungicides, insecticides, garden fertilizers or fresh manure should be present.
At CSIRO, topsoil from a local sandy bushland environment has proved to
have particularly beneficial growth promoting properties. Soil from clay or
other soil types are less suitable in our experience. Soil should not be stored
or processed in the algal culture laboratory, since it is a potent source of
unwanted microorganisms. Soil should be aged under moist conditions
(preferably for 6 months or more) and then kept dry and away from light.An
extensive list of modified Soil Water media developed for a range of
microalgae from specific environments can be found on the UTEX website
(The

Culture

Collection

of

Algae

at

the

University

of

Texas)

http://www.utex.org/.
Vitamins
The three most widely used vitamins in order of significance to algae are
vitamin B12 (cobalamin or cyanocobalamin), thiamine and biotin. They are
most often prepared as a single stock solution.
Vitamin B12 (cobalamin or cyanocobalamin)
While vitamin B12 or cobalamin has been recognized as a necessary
component for algal growth, its metabolic role has been unclear, but recent
evidence suggests it is primarily a cofactor for vitamin B12-dependent
methionine synthase (Croft et al. 2005). A survey of 326 algal species
indicates that more than half of the algal kingdom are cobalamin auxotrophs
and that all algal phyla and even individual genera contain species that

require vitamin B12 and species that do not (Croft et al. 2005). Croft etal
argue that the source of cobalamin is through symbiosis with bacteria which
benefit from the extracellular carbon produced from algal photosynthesis.
This premise has been strongly criticized by Droop (2007), a pioneer of
marine vitamin ecology, on the basis that oceanic and coastal concentrations
of B12 should easily meet the low cell requirements of algal populations
without a direct symbiosis. Completing the genome sequence for the diatom
Thalassiosira pseudonana, has shown that this important species (ie
important

aquaculture

and

general

model

species

in

algal

physiology/ecology) is a cobalamin auxotroph. Other Thalassiosira species


however are not auxotrophic for vitamin B12.
Croft, M.T., Lawrence, A.D., Raux-Deery, E., Warren, M.J. and Alison G. Smith,
A.G. (2005) Algae acquire vitamin B12 through a symbiotic relationship with
bacteria. Nature 438 (3)90-93.
Droop, M. R. (2007). Vitamins, phytoplankton and bacteria: symbiosis or
scavenging? Journal of Plankton Research 29:107-13.
Thiamine........
Biotin..........
Preparing and using Vitamin stock solutions
Vitamins are known to be heat and light sensitive, primary stocks of Vitamin
B12 and biotin are dispensed in 10-20 mL aliquots in polycarbonate tubes and
frozen, where they may keep for long periods and may be refrozen. The
working stock solution, including the added thiamine, is normally only kept
for 3 months in alfoil wrapped refrigerated bottles before new solutions are
prepared. Despite the known degradation of vitamins when heat sterilized,
many media types call for total autoclaving.

Autoclaving is regularly

employed for making media for axenic cultures and rarely have we
encountered growth impedance that can be traced to vitamin deficiency.
Some algae may be able to use the decomposition products incurred after
autoclaving (Provasoli and Carlucci, 1974), however we recommend that for

important new isolates or poorly growing species 0.2 um filter-sterilized


vitamins should be added aseptically to presterilized media.
Provasoli, L. and Carlucci, A. F. (1974). Vitamins and growth regulators. In : W.
D. P. Stewart (ed) Algal Physiology and Biochemistry. Blackwell Scientific, UK,
741-87
Effect of nutrients on species
Filament coiling

in Anabaena flos-aquae : promoted by 4x > [Fe] or

deficiency in Manganese; filament straightening promoted by 4x > [Mn] or


iron deficiency. Too high a concentration of either causes filament
fragmentation into short pieces. Best growth occurs with slight coiling
(Gorham et al, 1964).

Culture maintenance procedures and transfer protocols.


The aim of maintaining a culture collection is to balance the needs of
sustaining healthy stock cultures whilst also reducing the growth rate to
provide for longer transfer intervals and less labour costs. In some situations
such as culture supply to external clients or for experimental studies we
optimize culture conditions to stimulate near maximum growth rates (one
measure of culture vigour).

This section, however, concentrates on the

maintenance of stock cultures.


At CMAR each strain is maintained for 3 generations in liquid media in
conical flasks with cotton sterristoppers (foil wrapped in axenic cultures) that
we denote as the daughter, parent and grandparent cultures (fig 1). Volumes
typically are 30-40 mL of culture in 50 mL flasks, 75ml culture in 125 mL
flasks or 125 mL culture in 250 mL flasks. Petriplates, test tubes, round flat
bottom flasks or larger conical flask volumes and agar medium may be used
for some strains. Transfer intervals vary from 2 weeks to 6 months depending
on species and strains, however most strains are transferred once every 4 to
8 weeks. When dealing with several hundred strains, it is not feasible to

grow every strain under optimal conditions for culture longevity or inter
transfer period. Therefore strains are grouped into functional transfer units. A
group of benthic diatom species at 20 0C and a group of prymnesiophytes at
10 0C that both will tolerate low light (5-10 mol. photons m-2 s

) can be

transferred once every 8 weeks compared with a group of oceanic diatoms


and estuarine dinoflagellates that need frequent transfers every 2-4 weeks.
Volumes transferred will also vary depending on the density of the culture
but generally 0.5 - 2.0 mL is transferred from the 50mL flask cultures into
new media.

Fig 1. Transfer generations


While many strains will grow from innoculum to harvest on low light we
generally give the fresh daughter innoculum 1-2 weeks of optimal light
intensity before decreasing it to maintenance levels. The lower maintenance
levels are achieved by placing sheets of glassine paper or paper towelling
beneath the cultures and surrounding the flasks with black cardboard
cylinders. Note that some types of paper towelling can cause spectral shifts
in the light quality that reaches the culture. Daughter and parent cultures
are kept together but the grandparents are removed to a separate location in
the event of a major room/cabinet malfunction (e.g. compressor failure and
dramatic increase in temperature). After the next transfer is completed the
grandparent cultures are autoclaved and removed for washing.

Media Recipes used in CMAR


Media Comparison Table (constituents in moles of some of the above
media)
Further help on making media including characteristics of individual media
components
See notes for Aquaculturists if large volumes of media are needed
Seawater source
Bolds
Basal
Medium
References:
Nichols, H. W. and Bold, H. C. (1965).
Trichosarcina polymorpha gen. Et sp.
Nov. J. Phycol. 1: 34-8.
Nichols, H. W. (1973) Growth media
freshwater. In Stein, J. (Ed.) Handbook
of

Phycological

Methods,

Culture

Methods and Growth Measurements,


Camb. Univ. Press pp. 7-24.

Adapted for freshwater algae


Stock Solutions
1. NaNO3
2. CaCl2.2H2O
3. MgSO4.7H2O
4. K2HPO4
5. KH2PO4
6. NaCl

per Litre distilled water (dH2O)


25.0 g
2.5 g
7.5 g
7.5 g
17.5 g
2.5 g

7. EDTA
KOH
8. FeSO4.7H2O
H2SO4
9. H3BO3
10. Micronutrients
ZnSO4.7H2O
MnCl2.4H2O
MoO3
CuSO4.5H2O
Co(NO3)2.6H2O

50.0 g
31.0g
4.98 g
1.0 mL
11.42 g
g.L-1
8.82 g
1.44 g
0.71 g
1.57 g
0.49 g

Add

each

constituent
separately

to

~800

of

mL

dH2O and fully


dissolve
between

each

addition.

Then

make up to 1L.

Store all stock solutions in the refrigerator.


To Prepare BB Medium standard method
Add 10 mL of each stock solution (1 6) to 940 mL distilled water.
Add 1 mL of stock solutions (7 10).
Autoclave at 121C (15PSI for 15 mins).
To Prepare BB Medium method combining stocks from
different media used in CMAR
1. NaNO3

2.5 mL D stock solution

2. CaCl2.2H2O

0.5 mL D stock solution

3. MgSO4.7H2O

0.94 mL MMII stock solution

4. K2HPO4

2.5 mL D stock solution

5. KH2PO4

10.0 mL BB stock solution

6. NaCl

10.0 mL BB stock solution

7. Na2EDTA

1.0 mL of 57.05gL-1H2O stock

8. FeSO4.7H2O with H2SO4

1.0 mL BB stock solution

9. H3BO3

1.0 mL BB stock solution

10. Micronutrients

1.0 mL BB stock solution

Add each stock solution (1 10) in the stated volume to 1 litre distilled
water.
Autoclave at 121C (15PSI for 15 mins).
BG (Blue - Green Medium)
Medium for Spirulina spp.
Source: Culture Collection of Algae and Protozoa Catalogue of Strains (1988).
Stock Solutions

per

litre

distilled

1. NaNO3
2. K2HPO4.3H2O
3. MgSO4.7H2O
4. CaCl2.2H2O
5. Citric acid
6. Ferric ammonium citrate
7. EDTA
8. Na2CO3
9. Trace metal mixture
H3BO3
MnCl2.4H2O
ZnSO4.7H2O
Na2MoO4.2H2O
CuSO4.5H2O
Co(NO3)2.6H2O

(dH2O)
15.0 g
4.0 g
7.5 g
3.6 g
0.6 g
0.6 g (Autoclave to dissolve)
0.1 g
2.0 g
g.L-1
Add
each
2.86 g
constituent
1.81 g
separately to ~800
0.222 g
0.39 g
mL of dH2O and
0.079 g
fully
dissolve
0.0494 g
between
each
addition.

water

Then

make up to 1 L.

To Prepare 1 litre of media


To 829 ml distilled water add:
Stock solution 1 100 ml
Stock solution 2-8

10 ml each

Stock solution 9 1 ml
pH should be adjusted to approximately 7.4 with 1M NaOH or HCl before
autoclaving.
BG Medium with Seawater - CSIRO modification
To prepare Medium
Step 1
Seawater mix
To 971 mL of Teflon sterilised seawater add the following:
4.0 ml per litre of G vitamins - filtered sterilised.
25 ml per litre sterile soil extract.

Step 2
Final BG/SW medium
Mix aseptically equal parts of Step 1 Seawater mix with BG medium recipe.
D MEDIUM
Distilled H2O
NaCl
MgSO4, 7H2O
KCl
NaNO3
CaCl2.2H2O
K2HPO4
FeCl3.6H20
TRIS
B12
Thiamine
P II Metal Mix
Adjust pH to 7.6-7.8

1.0 liter
18.0 g
5.0 g
6.0 mL of 10% solution
5.0 mL of 10% solution
2.77 mL of 10% solution
0.3 mL of 10% solution
0.05 mL of 10% solution
10.0 mL of 10% solution
3.0 mL of 1g/mL solution
1.0 mL of 1mg/mL solution
5.0 mL of stock solution (see below)
with 1 N HCl (approx.5-6 mls/liter)

P II Metal Mix Stock g / L

Add

Solution
Na2EDTA
FeCl3.6H2O
H3BO3
MnCl2.4H2O
ZnCl2
CoCl2.6H2O

~750mL of
6.0 g
0.29 g
6.85 g
0.86 g
0.06 g
0.026 g

the

Na2EDTA
dH2O

to

in a

volumetric flask and stir


over low heat to dissolve.
Add each of the other
constituents separately to
~200mL
fully

of

dH2O

dissolve

additions.
second

and

between

Then add this

solution

to

the

Na2EDTA and make up to


1 L.
(adjust pH to 7.8 - 8.0 with NaOH)
DYIY Medium (freshwater)
References

(Keller and Anderson unpubl.; modified


from Lehman 1976)
Source: In: Journal of Phycology, 1997,
supplement to Vol. 33, No. 6, CCMP Provasoli-Guillard National Center for
Culture of Marine Phytoplankton
Stock Solutions

Per

500mL

1. MgSO4 .7H2O
2. KCl
3. NH4NO3
4. NaNO3
5. Na2 glyceroPO4
6. H3BO3
7. Na2EDTA
8. NaSiO3. 9H2O
9. FeCl3 .6H2O
10. CaCl2 .2H2O
11. f/2 vitamins

water (dH2O)
25.0 g
1.5 g
1.33 g
2.5 g
5.0 g
0.4 g
4.0 g
7.5 g
1.35 g
37.5 g
(see 1 mL

below)
12. trace

(see 1 mL

metals

distilled

below)
f/2 vitamin Stock Solution
Working Stock
to one liter of distilled water, add the following:
Biotin
10.0 mL primary stock
Vitamin B12
1.0 mL primary stock
Thiamine HCL
200.0 mg
Primary Stocks
Biotin
0.1 mg. mLVitamin B12
1.0 mg. mL-1
DY trace metals mg per 500 mL distilled water
MnCl2. 4H2O
100
Add each constituent
ZnSO4.7H2O

mg
20 mg

to ~400ml dH2O, fully

Na2MoO4 . 2H2O
CoCl2 . 6H2O
H2SeO3
Na3VO4 .nH2O

10 mg
4 mg
2 mg
1 mg

dissolving

between

additions. Then make

To prepare 1L of medium
1. To 900 mL of dH2O add 200 mg of
MES buffer
2. Add 1 mL of the stock solutions (112).
3. After all additions, bring to 1 L and
adjust the pH to 6.8 (it will probably be
very acidic).
DYIY (freshwater) Medium - CSIRO
Modification
References

(Keller

and

Anderson

unpubl.; modified from Lehman 1976)


Source: In: Journal of Phycology, 1997,
supplement to Vol. 33, No. 6, CCMP Provasoli-Guillard National Center for
Culture of Marine Phytoplankton
To 900 ml of ddH2O add 200 mg of
MES buffer and dissolve. Add the
following

stock

solutions.

After

all

additions, make up to 1 L. Adjust the


pH to 6.8. Autoclave.
Stock Solution
MES
MgSO4.7H2O

Source
200 mg / L
24.7 g / 500 mL

MLA stock use 1.0 mL

KCl

1.5 g / 500 mL

NH4NO3

1.33 g / 500 mL

NaNO3

2.5 g / 500 mL

Na2 glyceroPO4

1.62 g / 500 mL

H3BO3
Na2EDTA

DYIY stock use 1.0 mL


DYIY stock use 1.0 mL
DYIY stock use 1.0 mL
K stock use 3.0 mL

1.24 g / 500 mL
2.18 g / 500 mL

MLA stock use 0.3 mL

MLA stock use 1.8 mL

NaSiO3.5H2O

11.35 g / 500 mL f stock use 0.66 mL

FeCl3.6H2O

0.79 g / 500 mL

MLA stock use 1.7 mL

CaCl2.2H2O

14.7 g / 500 mL

MLA stock use 2.6 mL

f/2 vitamins (see below) use 1.0 mL


trace metals (see below) use 1.0 mL
f/2 vitamin Stock Solution
Working Stock
to 100ml of distilled water, add the following:
Biotin

1.0 mL primary stock

Vitamin B12

1.0 mL primary stock

Thiamine HCL

200.0 mg

Primary Stocks
Vitamin B12

0.1 mg / mL

Biotin

0.1 mg / mL

DY trace metals
Add to 500 mL distilled water, first dissolving seperately:
MnCl2.4H2O

100 mg

ZnSO4.7H2O

20 mg

Na2MoO4.2H2O

10 mg

CoCl2.6H2O
Na3VO4 .nH2O

4 mg
1 mg

Add as the following stock solution:


H2SeO3

0.645 mg GSe stock use 3mL

Medium

(and

fE)

CSIRO

Modification
This medium is a slight modification of
the original f medium of Guillard and
Ryther (1962).

It is most commonly

prepared at half strength where it is


designated as f2 or f/2.
Reference: Jeffrey, S. W. and LeRoi, J.M.

(1997).

Simple

procedures

for

growing SCOR reference microalgal


cultures.

In:

Mantoura

and

S.W.

Jeffrey,

S.W.

R.F.C.

Wright

(Eds)

Phytoplankton

pigments

in

oceanography;

Monographs

on

oceanographic

methodology

10,

UNESCO, France, pp 181-205.


Guillard, R. R. L. and Ryther, J. H.
(1962) Canad. J. Microbiol., 8: 229239.

Stock Solutions

Per L distilled water

1. NaNO3
2. Trace metals
CuSO4.5H2O
ZnSO4.7H2O
CoCl2.6H2O
MnCl2.4H2O

(dH2O)
150.0 g
mg / L
19.6 mg
44.0 mg
22.0 mg
360.0
mg

Add each of the


constituents to
~750ml dH2O,
mixing

Na2MoO4.2H2O

12.6 mg

thoroughly
between
additions
3. Na2SiO3.5H2O
4. Fe citrate:
Ferric citrate
Citric acid

22.7 g
g/L
9.0 g
9.0 g

to

dissolve.
add

both

constituents to
1L of dH2O and
autoclave

to

dissolve
5. Vitamins
(i)Working

Stock to 100

mL

of distilled

Solution (make fresh solution water, add the following


every 3 months)
Biotin
Vitamin B12
Thiamine HCL
(ii)

1.0 mL primary stock


1.0 mL primary stock
20.0 mg
Primary

Stocks
Vitamin

10.0 mg /100 mL dH2O

B12
Biotin
6. NaH2PO4.2H2O
7. Na2EDTA.2H2O
Store all stock solutions in the

10.0 mg /100 mL dH2O


11.3 g
30.0 g
refrigerator.
Preparation Methods

1. To Prepare Medium f
Add 1 mL of each stock solution (1
5) to 1 litre seawater. Dispense to
flasks and autoclave at 121C (15PSI,
15 mins).

Phosphate (see Stock 6.- NaH2PO4.2H2O ). This must be sterilised


separately from seawater to prevent precipitation.

Dilute original

phosphate stock with distilled water such that 1 mL added to each flask of
sterile medium will give the required concentration of phosphate in the
medium. Autoclave dilute phosphate stock at 121C (15PSI, 15 mins). After
cooling, dispense aseptically with sterilised automatic dispenser.
For example:
For 100 x 125 mL Erlenmeyer flasks, each containing 75 mL medium,
prepare dilute phosphate stock as follows:
f and fE media:
Take 7.5 mL of original phosphate stock and make up to 100 mL with
distilled water.
Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL
per flask aseptically.

f2 and fE2 media:


Take 3.75 mL of original phosphate stock and make up to 100 mL with
distilled water.
Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL
per flask asepically.
Scale up in the same proportion for larger volumes.
To Prepare Medium fE
Prepare as medium f, but also add 1 mL of Na2EDTA.2H2O stock solution (7).
To Prepare Medium f2

Prepare as medium f, but using 0.5 mL of each stock solution instead of 1.0
mL of each.
To Prepare Medium fE2
Prepare as medium f2, but also add 0.5mL of Na2EDTA.2H2O stock solution.
2. To Prepare Medium f2 concentrated nutrients
Take 5mL of each stock solution (1 6) and make up to 100 mL with distilled
water.
Pour into a 250 mL Schott bottle.
Autoclave at 121C (15PSI, 15 mins).
Alternatively, filter sterilise using a 0.22 m filter into a sterile 250 mL
Schott bottle.
Use 1 mL per 100 mL sterile seawater adding correct amount of nutrient
aseptically.
See notes for Aquaculturists if a greater volume of concentrated nutrients is
needed
G. P. medium (CMAR uses the abbreviation G for GP medium)
Reference: Loeblich, A. R. and Smith, V. E. (1968) Lipids, 3: 5-13.
Note about Salinity: In medium G and derivatives the final preparation steps
require mixing of nutrients with seawater and distilled water in a 3:1 or 4:1
ratio.

The fully marine seawater (~33-36practical salinity units) used in

CMAR means the resulting media salinity is ~28psu.

In our culturing

experience this is a good salinity for estuarine and coastal flagellate species,
particularly dinoflagellates.
Stock Solutions

Per Litre distilled water

1. KNO3
2. K2HPO4
3. Vitamins
Working Stock Solution
Biotin
Vitamin B12
Thiamine HCL

(dH2O)
100.0 g
34.8 g
to 100 mL of distilled water,
add the following
2.0 mL primary stock
1.0 mL primary stock
100.0 mg (fresh solution
every 3 months)

Primary Stocks
Vitamin B12
Biotin
4. PII Metal Mix

10.0 mg /100 mL dH2O


10.0 mg /100 mL dH2O
g/L
Add the Na2EDTA
to ~750mL of dH2O

Na2EDTA
FeCl3.6H2O
H3BO3
MnCl2.4H2O
ZnCl2
CoCl2.6H2O

6.0 g
0.29 g
6.85 g
0.86 g
0.06 g
0.026 g

in

volumetric

flask and stir over


low

heat

to

dissolve. Add each


of

the

other

constituents
separately

to

~200mL of dH2O
and fully dissolve
between additions.
Then

add

this

second solution to
the Na2EDTA and
make up to 1L.
(adjust pH to 7.8 - 8.0 with NaOH)
5. Soil Extract see recipe at end
Store all stock solutions in the refrigerator.
G. P. medium
Preparation Methods

1. To Prepare G medium
To 750 mL seawater add:
Distilled water

250 mL

Nitrate stock

2 mL

Vitamin stock

1 mL

PII Metal Mix

5 mL

Soil Extract

5 mL

Dispense to flasks and autoclave at


121C (15PSI, 15 mins).
Phosphate must be sterilised separately from seawater to prevent
precipitation.
Dilute original phosphate stock with distilled water such that 1 mL added to
each flask of sterile medium will give the required concentration of
phosphate in the medium. Autoclave dilute phosphate stock at 121C
(15PSI, 15 mins). After cooling, dispense aseptically with sterilised automatic
dispenser.
For example:
For 100 x 125 mL Erlenmeyer flasks, each containing 75 mL medium,
prepare dilute phosphate stock as follows:
G medium:
Take 7.5 mL of original phosphate stock and make up to 100 mL with
distilled water.
Pour into a 250 mL Schott bottle and autoclave to sterilse. Dispense 1 mL
per flask aseptically.
G2 medium:
Take 3.75 mL of original phosphate stock and make up to 100 mL with
distilled water.
Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL
per flask aseptically.
Scale up in the same proportion for larger volumes.

To Prepare Medium G2
Make up dilutant, consisting of seawater and distilled water (3:1 ratio)
Dilute G medium by adding an appropriate volume of dilutant such that:
- medium is half of its original concentration (G2 medium).
2. To Prepare Medium G concentrated nutrients
Add Nitrate stock

20 mL

Vitamin stock

10 mL

PII Metal Mix

50 mL

Soil Extract

50 mL

Phosphate stock

10 mL

Make up to 200 mL with distilled water.


Pour into a 250 mL Schott bottle.
Autoclave at 121C (15PSI, 15 mins).
Alternatively, filter sterilise using a 0.22 m filter into a sterile 250 mL
Schott bottle.
Use 2mL /100mL sterile seawater and distilled water (3:1 ratio).
Add correct amount of nutrients aseptically.
To Prepare Medium G2 concentrated nutrients
Use G concentrated nutrients such that:
G2: use 1 mL /100mL sterile seawater and distilled water (3:1 ratio).
G5: use 0.4 mL /100mL sterile seawater and distilled water (3:1 ratio).
GSe medium Modification of G. P. medium
Reference: Blackburn, S. I.; Bolch, C. J. S.; Haskard, K. A., and Hallegraeff, G.
M. Reproductive Compatibility Among Four Global Populations of the Toxic

Dinoflagellate Gymnodinium Catenatum (Dinophyceae). Phycologia. 2001;


40(1):78-87.
Medium GSe selenium is added as an important trace metal (chelator).
Note about Salinity: In medium G and derivatives the final preparation steps
require mixing of nutrients with seawater and distilled water in a 3:1 or 4:1
ratio.

The fully marine seawater (~33-36 practical salinity units) used in

CMAR means the resulting media salinity is ~28 psu.

In our culturing

experience this is an optimal salinity for estuarine and coastal flagellate


species, particularly dinoflagellates.
Stock Solutions
1. KNO3
2. K2HPO4
3. Vitamins
Working Stock Solution
Biotin
Vitamin B12
Thiamine HCL

Per

Litre

distilled

water

(dH2O)
100.0 g
34.8 g
to 100 mL of distilled water,
add the following
2.0 mL primary stock
1.0 mL primary stock
100.0 mg (fresh solution every
3 months)

Primary Stocks
Vitamin B12
Biotin
4. PII Metal Mix

10.0 mg /100 mL dH2O


10.0 mg / 100 mL dH2O
g/L
Add the Na2EDTA to
~750 mL of dH2O in

Na2EDTA
FeCl3.6H2O
H3BO3
MnCl2.4H2O
ZnCl2
CoCl2.6H2O

6.0 g
0.29 g
6.85 g
0.86 g
0.06 g
0.026 g

volumetric

flask

and stir over low


heat

to

Add

each

other

dissolve.
of

the

constituents

separately to ~200
mL

of

dH2O

and

fully
between

dissolve
additions.

Then
add
this
(adjust pH to 7.8 - 8.0 with NaOH)
second solution to
5. Soil Extract
See soil extract protocol
6.
Selenium
(as
selenite) 1.29 mg
H2SeO3
Store all stock solutions in the refrigerator.
GSe medium
Preparation Method
1. Seawater
Autoclave filtered seawater in 1000 mL Teflon bottles to sterilise.
2. Distilled Water
Autoclave distilled water to sterilise.
3. To Prepare GSe medium concentrated nutrients (excluding soil
extract)
Add Nitrate stock

20 mL

Phosphate stock

10 mL

Vitamin stock

10 mL

PII Metal Mix

50 mL

Selenium stock
Make up to 200 mL with distilled water.
Pour into a 250 mL Schott bottle.
Autoclave at 121C (15PSI, 15 mins).

10 mL

Alternatively, filter sterilise using a 0.22 m filter into a sterile 250 mL


Schott bottle.
Use 2 mL /100mL sterile seawater and distilled water (3:1 ratio).
Add correct amount of nutrients aseptically.
4. Soil Extract Solution
See soil extract protocol for details.
Use soil extract at a concentration of 0.5 mL per 100 mL medium.
To Prepare GSe medium
For example to make 5000 mL Medium GSe:
In a sterile 5000 mL Schott bottle add aseptically
sterile seawater (1)
4000 mL
sterile distilled water (2)

1000 mL

sterile GSe concentrated nutrients (3)

100 mL

sterile soil extract solution (4)

25 mL

Mix. This medium is now ready to be decanted aseptically into sterile


culture flasks.
Soil Extract
Soil Extract is an adaptation of E.G. Pringsheim's biphasic soil-water medium
and is a component of some of our media. As the chemical composition is
not well-defined and may vary from batch to batch its use in experimental
situations is not recommended, however in our experience certain species
will not grow well without it and soil extract is added for both culture
maintenance and experimental studies. An extensive list of modified Soil
Water

media

developed

for

range

of

microalgae

from

specific

environments can be found on the UTEX website (The Culture Collection of


Algae at the University of Texas).

http://www.utex.org/
CMAR protocol
Soil must be collected from a natural uncultivated environment or a rich
garden loam may be suitable. No fungicides, insecticides, garden fertilizers
or fresh manure should be present. At CSIRO, topsoil from a local sandy
bushland environment has proved to have particularly beneficial growth
promoting properties. Soil from clay or other soil types are less suitable in
our experience.
Soil should not be stored or processed in the algal culture laboratory, since
it is a potent source of unwanted microorganisms. Soil should be aged under
moist conditions (preferably for 6 months or more) and then kept dry and
away from light.
To Prepare Soil Extract
1. Sift dry soil (not recently treated with fertilizer or herbicide) once through
a coarse sieve and twice through a fine sieve (1mm mesh).
2. Mix 1 kg of soil into 2 litres of distilled water.
3. Autoclave for 60 minutes at 121C and cool overnight.
4. Filter through absorbant cotton wool packed into the stem of a glass filter
funnel.
5. Centrifuge at 5000 rpm for 20 minutes in 250 ml polyethylene centrifuge
tubes and collect the deep brown supernatant.
6. Filter again through absorbant cotton wool.
7. Dispense the supernatant (50 mL aliquots) into 100 mL Schott bottles or
100 mL media bottles.
8. Autoclave for 15 minutes at 121C.
9. After cooling, wrap caps with parafilm to prevent airborne contamination
from fungal spores or bacteria.
10. Store sterile soil extract at 4C (cold room or refrigerator).

JM (Jaworskis Medium)
Reference: Culture Collection of Algae and Protozoa (CCAP) Catalogue of
Strains 1988.
Adapted for freshwater Algae

Stock Solutions
1. Ca(NO3)2.4H2O
2. KH2PO4
3. MgSO4.7H2O
4. NaHCO3
5. EDTA FeNa
EDTA Na2
6. H3BO3
MnCl2.4H2O
(NH4)6MO7O24.4H2O
7. Vitamins
Cyanocobalamin
(Vitamin B12)
Thiamine

HCl

Per

Litre

distilled

water (dH2O)
20.0 g
12.4 g
50.0 g
15.9 g
2.25 g
2.25 g
2.48 g
1.39 g
1.00 g
0.04 g
(Vitamin 0.04 g

B1)
Biotin
0.04 g
8. NaNO3
80.0 g
9. Na4HPO4.12H2O
36.0 g
Store all stock solutions in the refrigerator.
To Prepare JM - fully autoclaved media for axenic cultures
Add 1 mL of each stock solution (1 9) to 1 litre distilled water.
Autoclave at 121C (15 PSI for 15 mins).
K Medium
Reference: Adapted from Table 2; Keller, M. D., Selvin, R. C., Claus, W. and
Guillard, R. R. L. (1987). Media for the culture of oceanic ultraplankton. J.
Phycol. 23, 633-638

Note: Specific K stock solutions and stock solutions from other media are
used in CMAR to prepare this media. A concentrated working stock solution
is made up and used at a concentration of 2mL per 100ml of seawater..
To prepare 100 ml working stock : (use at 2 mL per 100 mL
seawater)
Stock Solutions primary stock

source

1. NaNO3

f stock solution, use

150 g / L H2O

Volume
2.5

mL
2. Na2glyceroPO4 3.24 g / L H2O
3. Na2SiO3.9H2O 22.7 g / L H2O

K stock solution, use

5 mL

f stock solution, use

2.5

mL
4. Vitamins

f stock solution, use

5 mL

5. Trace Metals
FeNaEDTA

4.3 g / L H2O

K stock solution, use

K-Primary Trace mix (see below) K stock solution, use


Na2EDTA.2H2O 30 g / L H2O

5 mL
5 mL

fE stock solution, use

6.2

100 g / L H2O

K stock solution, use

6.05

2.68 g / L H2O

K stock solution, use

5.0

mL
6. TRIS
mL
7. NH4Cl
mL
8. H2SeO3

1.29 mg / L H2O

9. Distilled water

GSe stock solution, use

5.0 mL
52.75 mL

Filter-sterilise through 0.22 m sterile filter under aseptic conditions.


K-Primary Trace mix

CuSO4.5H2O

4.9 mg / L H2O

ZnSO4.7H2O

22.0 mg / L H2O

CoCl2.6H2O

11.0 mg / L H2O

MnCl2.4H2O

180.0 mg / L H2O

Na2MoO4.2H2O

6.3 mg / L H2O

Add each ingredient to ~750 mL of distilled water, mixing


thouroughly between additions and then make up to 1 L.
All stock solutions are made up in distilled water.
Store all stock solutions in the refrigerator.
MBL Medium - Woods Hole
Reference: Nichols, H. W. (1973) in
Handbook of Phycological Methods,
Ed. J. R. Stein, pp. 16-17. Camb. Univ.
Press. (R.

R. L. Guillard,

personal

communication).
Adapted for freshwater Algae
Stock solutions
1.
2.
3.
4.
5.
6.
7.
8.
9.

CaCl2.2H2O
MgSO4.7H2O
NaHCO3
K2HPO4
NaNO3
Na2SiO3.9H2O
Na2EDTA
FeCl3.6H2O
Metal Mix
CuSO4.5H2O
ZnSO4.7H2O
CoCl2.6H2O
MnCl2.4H2O

Per Litre distilled water


(dH2O)
36.76 g
36.97 g
12.60 g
8.71 g
85.01 g
28.42 g
4.36 g
3.15 g
Add
0.01
0.022
0.01
0.18

each

g
constituent
g
g separately
g

to

Na2MoO4.2H2O

0.006 g

~750mL
dH2O,

of
fully

dissolving
between
10. Vitamin stock
Cyanocobalamin
(Vitamin B12)
Thiamine

HCl

aditions.
0.0005 g / L dH2O
(Vitamin

0.10 g / L dH2O

B1)
Biotin
11. Tris stock
Store all stock

solutions

0.0005 g / L dH2O
250.0 g / L dH2O
in the

refrigerator.

To Prepare MBL Medium


Add 1mL of each stock solution (1 11) to 1 litre distilled water.
(For species which cannot use nitrate substitute 1mL of NH 4Cl made up to
5.4 g /L H2O)
Adjust pH to 7.2 with HCl.
Autoclave at 121C (15PSI for 15 mins).
To Prepare MBL/NB2 Medium
Make MBL medium as above and add 2.5 g Oxiod nutrient broth No. 2
before autoclaving.
MBL Medium - Woods Hole: CSIRO working modification

This recipe closely resembles the Woods Hole MBL Medium listed
previously. However it is made up using stocks from other media used in
CMAR.
Stock Solution

Source

Add V

1. CaCl2.2H2O

D stock solution 0.8 mL

2. MgSO4.7H2O

MMII stock solution

3. NaHCO3

12.60 g / L-1H2O 1.0 mL

4. K2HPO4

G stock solution 0.2 mL

5. NaNO3

f stock solution

0.5 mL

0.5 mL

6. Na2SiO3.5H2O

f stock solution

7. Na2EDTA

fE stock solution 0.15 mL

8.

4.5 mL

Fe citrate:
Ferric citrate
Citric acid

9. Trace Metals

f stock solution

1.0 mL

f stock solution

0.5 mL

10. Vitamin stock

f stock solution

1.0 mL

11. Tris stock

D stock solution 2.0 mL

Store all stock solutions in the refrigerator.


For species which cannot use nitrate substitute 1mL of NH 4Cl made up to
5.4 g / L H2O
Adjust pH to 7.2 with HCl.

To Prepare MBL Medium


Add each stock solution (1 11) in the volumes indicated to 1 litre distilled
water.
Autoclave at 121C (15PSI for 15 mins).
To Prepare MBL/NB2 Medium

Make MBL Medium as above and add 2.5g Oxiod nutrient broth No. 2 before
autoclaving.
MJ Medium (Modified Jorgensens media for diatoms)
Based on recipe obtained from University of Tasmania Harmful Algae
Culture Collection
This

media

has

been

used

predominantly for the culture of Pseudonitzschia species and also in trials


of some Chaetoceros species where biomass and maintenance of normal
morphology seems to be better than in other media. Preparation of the
media is based on the utilization of MJ and f-stock solutions.

Stock Solution

Add V (mL.L-1)

Source

(a) Normal (b) concentrated


1. NaNO3

f stock solution (150.0 g / L )

2. K2HPO3

MJ stock solution

2.0 mL

(make up at 2.0 g / L ) 5.0 mL

20.0
50

3. Trace Metals I MJ stock solution (see below)

0.1 mL

1.0

4. Trace Metals II MJ stock solution (see below)

1.0 mL

10.0

5. Vitamin B12

f stock solution (1.0 mg / L ) 0.5 mL

6. Na2SiO3.5H2O

f stock solution (22.7 g / L )

5.0

4.5 mL

45
7. EDTA

MJ stock solution (10 g / L )

1.0 mL

10

Autoclave each stock solution except the Vitamin B12 which should be
0.2um filter sterilised.
(a)

lists the normal stock volumes needed to prepare 1L of media. (b)


lists the volumes to prepare a concentrated nutrient solution as
described under Preparation below.

3. Trace Metals I
Na2EDTA
CoCl2.6H2O
CuSO4.5H2O
(NH4)6Mo7O24
NH4VO3
ZnSO4.7H2O

2.5 g / L H2O
Add
the
Na2EDTA
to
150 mg / L
~750mL of dH2O in a
H2O
volumetric flask and stir
800 mg / L
over low heat to dissolve.
H2O
150 mg / L Add each of the other
constituents separately to
H2O
250 mg / L ~200 mL of dH2O and fully
H2O
dissolve
between
250 mg / L
additions. Then add this
H2O
second solution to the
Na2EDTA and make up to 1
L.

4. Trace Metals II
FeSO4.7H2O
Citric acid
H3Bo3
MnCl2.4H2O

3.0
3.0
1.5
1.0

g
g
g
g

/
/
/
/

L
L
L
L

Make

up

each

constituent

separately in ~200 mL of dH2O


and

fully

combine

dissolve.
each

solution

Then
and

make up to 1 L.

To Prepare concentrated nutrient stock (enough for 10L media)


Add aseptically each of the prepared sterile stock solutions to 59 mL of
sterile distilled water in a 200 mL Schott bottle (making total volume 200
mL).

To make media, add 20 ml of this final nutrient solution per 1itre of


seawater.
MLA Medium
Reference: Bolch, C. J. S. and Blackburn S. I. (1996). Isolation and
purification of Australian isolates of the toxic cyanobacterium Microcystis
aeruginosa Ktz. Journal of Applied Phycology 8, 5-13
MLA is derived from ASM-1 medium reported in Gorham etal, (1964).
Isolation and culture of toxic strains of Anabaena flos-aquae (Lyngb.) de
Brb. Verh. int. Ver. Limnol 15, 796-804
For Cyanobacterial Cultures
Stock Solutions

Per

Litre

distilled

water (dH2O)
MgSO4.7H2O
49.4 g
NaNO3
85.0 g
K2HPO4
6.96 g
H3BO3
2.47 g
H2SeO3
1.29 mg
Vitamins
Working Stock Solution
to 100mL of distilled water, add the following:
Biotin
0.05 mL primary stock
Vitamin B12
0.05 mL primary stock
Thiamine HCl
10.0 mg
Primary Stocks
Biotin
10.0 mg / 100 mLH2O
Vitamin B12
10.0 mg / 100 mLH2O
7. Micronutrients
Stock Solution
.
to 800mL of distilled water add each of the following
1.
2.
3.
4.
5.
6.

constituents separately, mixing to dissolve each addition


Na2EDTA
4.36 g (add first & stir on
low heat to fully dissolve)
FeCl3.6H2O
1.58 g
NaHCO3
0.60 g
MnCl2.4H2O
0.36 g
then add 10mL of the following primary stocks (each made

up separately)
Primary Stocks
CuSO4.5H2O
ZnSO4.7H2O
CoCl2.6H2O
Na2MoO4.2H2O

(per Litre dH20)


1.0 g.
2.2 g.
1.0 g.
0.6 g.

Finally, make up the micronutrient stock to 1 litre with


distilled water
If precipitate forms increase pH up to 7.
(If precipitation becomes an issue then replacing the two
sulphate stocks with equimolar amounts of the trace metal in
the chloride form has proven useful; Ben Long, pers comm)
8. NaHCO3
16.9 g
9. CaCl2.2H2O
29.4 g
Store all stock solutions in the refrigerator.
MLA Medium Preparation Methods
There

are

components

as

follows:
1. Distilled Water
Autoclave to sterilise
2. To Prepare MLA Medium x40
concentrated

nutrients

(250mL

volume)
To 130mL distilled water add
MgSO4.7H2O

10 mL

NaNO3

20 mL

K2HPO4

50 mL

H3BO3
H2SeO3
Vitamin stock

10 mL
10 mL
10 mL

Micronutrient stock

10 mL

Filter sterilise using a 0.22 m filter


into a sterile 250 mL Schott bottle.
3. NaHCO3 16.9 g / L H2O
Autoclave to sterilise.
4. CaCl2.2H2O 29.4 g / L H2O
Autoclave to sterilse.

To Prepare MLA Medium


For example to make 1000 mL MLA Medium:
In a sterile 1000 mL Schott bottle add aseptically
sterile distilled water (1)
964 mL
sterile

MLA

x40

concentrated

nutrients

(2)

25 mL
sterile NaHCO3 (3)
10 mL
sterile CaCl2.2H2O (4)
1 mL
Mix well after each addition.
This medium is now ready to be decanted aseptically into sterile culture
flasks.
To Prepare MLA Medium (Fully Autoclaved)
For axenic cultures. The media is essentially the same but due to the
autoclaving process the NaHCO3 concentration is adjusted.
For example to make 1000 mL autoclaved MLA Medium add

distilled water (1)


sterile

973 mL

MLA

x40

concentrated

nutrients

(2)

25 mL
sterile NaHCO3 (3)

1 mL

sterile CaCl2.2H2O (4)

1 mL

Adjust pH to 7.5 to 8.0 with HCl (often no adjustment is necessary).


Dispense to flasks and autoclave at 121C (15PSI, 15 mins).
Allow to cool in autoclave overnight. Helps to minimise the amount of
precipitate.

Protocols for solid MLA medium using a X40 concentrate


MLA for solid media is modified by the addition of Na 2SO3, as this can
improve survival of cyanobacteria on solid media (Parker, 1982). A stock
solution of Na2SO3 (12.6 g/L) is sterilised by autoclaving. This is then added
aseptically just prior to pouring to give a final concentration 0.1 mM Na 2SO3
(i.e. 1 ml / L of Na2SO3stock).

Agar - final concentration of 10 g / L (1%)

- preferred media for Nodularia, but always use highly purified agar e.g.
Difco Bacto purified agar.

Agarose - Final concentration of 5 g / L (0.5%)

- preferred media for Microcystis and Anabaena. (although the latter grows
only very slowly, or not at all on solid media).

To

prepare

500

ml

of

non-saline

solid

MLA

medium:

two components are mixed, each 250 mls and double-strength Add the gelling agent (5 g agar or 2.5 g agarose) to 250 ml MQ
water

in

500

ml

Schott

bottle

with

magnetic

stirrer.

Add 12.5 mls of filter-sterile x40 concentrate to 250 ml sterile MQ


water in a 250 ml Schott bottle. (full strength = 25ml/L in liquid
media)

Autoclave

gelling

agent,

and

double-strength

nutrients.

Final steps detailed below


To prepare 500 ml of saline solid MLA medium:
three components are mixed, each 167 mls and triple-strength Add the gelling agent (5 g agar or 2.5 g agarose) to 167 ml
MQ water in a 500 ml Schott bottle with a magnetic stirrer.
Add 12.5 mls of filter-sterile x40 concentrate to 167 mls MQ
water.
Autoclave the gelling agent, the triple-strength nutrients and
167 mls of triple strength saline
-------------------------------------------------------------------------------------------------Final steps in preparation of solid media:

Cool to 45 0C in a water bath, and in the same bath, warm the CaCl 2 and

NaHCO3 and if appropriate Na2SO3.

On a magnetic stirrer plate in the laminar flow, place the bottle

containing the gel. Stir gently while aseptically adding nutrients (and for
saline media, adding seawater also).

Aseptically add 1 ml each of vitamins and CaCl 2 and if appropriate

Na2SO3, and 10 ml of NaHCO3.

Pour media into sterile petri plates or McCartney bottles***.

Dry agar/agarose plates in the laminar flow for 20 minutes prior to

storing; and/or place McCartneys in a basket on a sharp angle until slopes


are solid.
---------------------------

***If using a dispenser, and in case problems arise during dispensing the
solid media, it is advised to keep some hot sterile water for rinsing the
dispenser aseptically.
Mineral Medium II
Reference: Hughes, E. O., Gorham, P.
R. and Zehnder, A. (1958) Canad. J.
Microbiol. 4: 225-236.
Freshwater Algae
Stock Solutions
1.
2.
3.
4.
5.
6.
7.

NaNO3
K2HPO4
MgSO4.7H2O
CaCl2.2H2O
Na2CO3
Na2SiO3.9H2O
Fe citrate:
Ferric citrate
Citric acid
8. EDTA
9. PII Metal Mix
Na2EDTA
FeCl3.6H2O
H3BO3
MnCl2.4H2O
ZnCl2
CoCl2.6H2O

Per

Liter

distilled

water

(dH2O)
1500.0 g
370.0 g
80.0 g
40.0 g
20.0 g
60.0 g
6.0 g
6.0 g (autoclave to dissolve)
1.0 g
g / L dH2O
Add the Na2EDTA to
6.0 g
~750mL of dH2O in
0.29 g
a volumetric flask
6.85 g
0.86 g
and stir over low
0.06 g
heat to dissolve.
0.026 g
Add each of the
other

constituents

separately
~200mL
and

to

of

fully

dH2O

dissolve

between additions.
Then

add

this

second solution to
the

Adjust pH to 7.8 - 8.0 with NaOH


Store

all

stock

solutions

in

Na2EDTA

and

the

refrigerator.

To Prepare Mineral Medium II


To 1 litre distilled water
Add 1mL of each stock solution (1 8).
Add 0.5mL PII Metal Mix stock solution (9)
Autoclave at 121C (15PSI, 15 mins).
Mineral Medium II CSIRO working
modification
This recipe closely resembles the Mineral Medium II listed previously.
However it is made up using stocks from other media used in CMAR.
Stock Solution

Source

1. NaNO3

D stock solution 15.0 mL

2. K2HPO4

G stock solution 10.0 mL

3. MgSO4.7H2O

MMII stock solution

4. CaCl2.2H2O
5. Na2CO3

Add V

1.0 mL

D stock solution 0.8 mL


MMII stock solution

1.0 mL

6. Na2SiO3.5H2O

f stock solution

12.0 mL

7. Fe citrate:

f stock solution

0.66 mL

8. Na2EDTA

fE stock solution 0.14 mL

9. PII Metal Mix

G stock solution 0.5 mL

Store all stock solutions in the refrigerator.

To Prepare Mineral Medium II


Add each stock solution (1 9) to 950
mL distilled water.
Autoclave at 121C (15PSI, 15 mins).

Porphyridium Medium
Medium

for

dinoflagellate

the

heterotrophic

Crypthecodinium

cohnii.
Reference: Starr, R. C. and Zeikus, J. A. (1993). UTEX - The Culture
Collection of Algae at the University of Texas at Austin. J. Phycol. 29 (2)
p94. (E.G. Pringsheim, pers. comm.)
To prepare medium:
For each 500ml of medium required combine:
distilled water

200 ml

flitered seawater 250 ml


yeast extract
tryptone

0.5 g
0.5 g

autoclace to sterilise
add aseptically

50 ml sterile soil extract.

Optional ingredients: agar at 7.5 g per litre to solidify.


Australian National Algae Culture Collection - Methods
Algal growth phases including determination of the growth rate and
population doubling time
There are 5 reasonably well defined phases of algal growth in batch cultures
(Fogg and Thake, 1987)

1 lag; 2 exponential; 3 declining growth rate; 4 stationary; 5 death. Each of


the phases is described below and in Fig 1 (goto fig).

Alternatively Jump

straight to growth rate equation


Lag phase
The condition of the innoculum has a strong bearing on the duration of the
lag phase (Spencer, 1954). An innoculum taken from a healthy exponentially
growing culture is unlikely to have any lag phase when transferred to fresh
medium under similar growth conditions of light, temperature and salinity. In
general the length of the lag phase will be proportional to the length of time
the innoculum has been in phases 3-5. A lag phase may also occur if the
innoculum is transferred from one set of growth conditions to another.
Exponential phase and calculating growth rates
The growth rate of a microalgal population is a measure of the increase in
biomass over time and it is determined from the exponential phase. Growth
rate is one important way of expressing the relative ecological success of a
species or strain in adapting to its natural environment or the experimental
environment imposed upon it. The duration of exponential phase in cultures
depends upon the size of the innoculum, the growth rate and the capacity of
the medium and culturing conditions to support algal growth.

Biomass

estimates need to be plotted over time, and logistical constraints determine


their frequency but once every one to two days is generally acceptable. Cell
count and dry weight are common units of biomass determination. In-vivo
fluorescence and turbidity can be used as surrogate measures which enable
higher temporal resolution due to the logistical ease of measurement
(correlations between fluorescence or turbidity and cell count can be
established but they will become less accurate as experimental conditions
are varied. For example cell fluorescence may vary with temperature so an
experiment with several test temperatures may need correlations to be
determined for each temperature. Correlations also become innacurate as

cultures move into stationary phase so fluorescence can not be used as a


substitute for cell counts where an estimate of final cell yield is needed).
Once the growth phase has been plotted (time on x-axis and biomass on
logarthmic y-axis) careful determination of the exponential (straightline)
phase of growth is needed. Two points, N1 and N2, at the extremes of this
linear phase (see fig below) are taken and substituted into the equation
Growth rate ; K' = Ln (N2 / N1) / (t2 - t1)
Where N1 and N2 = biomass at time1 (t1) and time2 (t2) respectively;
Levasseur et al (1993).
Divisions per day and the generation or doubling time can also be calculated
once the specific growth rate is known.
Divisions per day ; Div.day-1 = K' / Ln2
Generation time ; Gen' t = 1 / Div.day-1
For healthy cells of a robust species, small innoculums equal to 0.5 % of the
volume of the new culture will normally generate new healthy cultures. If
the species is delicate or the culture less healthy then a larger innoculum of
~ 10% may be needed to support a new culture. (Many of the stock cultures
in CMARC are transferred with a 0.5 to 1 mL innoculum into 40 mL fresh
medium representing a 1.25 % to 2.5% innoculum).
Declining growth
Declining growth normally occurs in cultures when either a specific
requirement for cell division is limiting or something else is inhibiting
reproduction.

In this phase of growth biomass is often very high and

exhaustion of a nutrient salt, limiting carbon dioxide or light limitation


become the primary causes of declining growth. When biomass is increasing
exponentially a constant supply of air (or air plus CO 2) will only be in balance
with growth at one point during exponential phase. At low cell densities too
much CO2 may lower the pH and depress growth. CO 2 limitation at high cell

densities causes any further biomass increase to be linear rather than


exponential (with respect to time) and proportional to the input of CO2.
Light limitation at high biomass occurs when the cells absorb most of
the incoming irradiation and individual cells shade each other (hence the
often quoted term self-shading).

Growth in most phytoplankton is

saturated at relatively low irradiances of 50-200 mol. photons m -2 s

(cf

noontime irradiance at the water surface in the tropics of 2000 mol.


photons m-2 s

).

Microalgae are therefore generally well adapted to

surviving conditions of low incident light and may survive for extended
periods under these conditions.
Stationary phase
Cultures enter stationary phase when net growth is zero, and within a matter
of hours cells may undergo dramatic biochemical changes. The nature of the
changes depends upon the growth limiting factor. Nitrogen limitation may
result in the reduction in protein content and relative or absolute changes in
lipid and carbohydrate content.

Light limitation will result in increasing

pigment content of most species and shifts in fatty acid composition. Light
intensities that were adequate or optimal for growth in the first 3 phases can
now become stressful and lead to a conditon known as photoinhibition. It is
important that while the measured light intensity within the culture will
decrease with increasing biomass if the incident illumination is maintained
relatively high then a large proportion of cells may become stressed,
photoinhibit and the culture can be pushed into the death phase.

This is

especially the case if the culture is also nutrient stressed. It is preferable for
many species to halve or further reduce the incident light intensity when
cultures enter stationary phase to avoid photoinhibition. Some green algae
and cyanobacteria may survive in the vegetative state (ie not as cysts) for
over 6 12 months under very low illumination.

For many species lower

temperature combined with lower irradiance can further reduce stress.


Survival is inversely proportional to temperature but only in darkness. Some

algal species may form long lived cysts or temporary resting cysts with
greatly reduced metabolism under different conditions of stress. The shut
down of many biochemical pathways as stationary phase proceeds means
that the longer the cells are held in this condition the longer the lag phase
will be when cells are returned to good growth conditions.
Death phase
When vegetative cell metabolism can no longer be maintained the death
phase of a culture is generally very rapid, hence the term culture crash is
often used. The steepness of the decline is often more marked than that
represented in the accompanying growth figure. Cultures of some species
will lose their pigmentation and appear washed out or cloudy, whereas cells
of other species may lyse (no recognizable cells) but the culture colour will
be maintained. The latter is an important consideration and one reason why
colour should not be relied upon to guage culture health.

Bacteria which

may have been kept in check during exponential and early stationary phase
may

explode

as

cell

membrane

integrity

become

progressivley

compromised or leaky and a rich carbon source for bacterial growth is


released.

Free pigment and bacterial growth are further reasons why

measures of turbidity or fluorescence should not be used beyond early


stationary phase as surrogate biomass indicators, or especially as indicators
of culture health.

Occassionally cell growth of some species can reoccur

after a culture has apparently died. In this instance most vegetative cells will
have died, and possibly most of the bacteria, releasing nutrients back into
the media.

Then either the very few remaining vegetative cells or more

likely germination of cysts or temporary cysts will be able to fund this


secondary growth.

Fig 1 General pattern of microalgal growth in batch cultures

Specific

Specific

Growth

Divisions

Generation time =Growth

Divisions Generation

rate
K'
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.69
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00

per day
Div.day-1
0.144
0.216
0.289
0.361
0.433
0.505
0.577
0.649
0.721
0.793
0.866
1.000
0.938
1.010
1.082
1.154
1.226
1.298
1.371
1.443

Doubling
days
6.931
4.621
3.466
2.773
2.310
1.980
1.733
1.540
1.386
1.260
1.155
1.000
1.066
0.990
0.924
0.866
0.815
0.770
0.730
0.693

per day
Div.day-1
1.515
1.587
1.659
1.731
1.803
1.876
1.948
2.020
2.092
2.164
2.236
2.308
2.380
2.453
2.525
2.597
2.669
2.741
2.813
2.885

time
hours
166.36
110.90
83.18
66.54
55.45
47.53
41.59
36.97
33.27
30.25
27.73
24.00
25.59
23.77
22.18
20.79
19.57
18.48
17.51
16.64

rate
K'
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
1.55
1.60
1.65
1.70
1.75
1.80
1.85
1.90
1.95
2.00

time

= Doubling time
days
hours
0.660
15.84
0.630
15.12
0.603
14.47
0.578
13.86
0.555
13.31
0.533
12.80
0.513
12.32
0.495
11.88
0.478
11.47
0.462
11.09
0.447
10.73
0.433
10.40
0.420
10.08
0.408
9.79
0.396
9.51
0.385
9.24
0.375
8.99
0.365
8.76
0.355
8.53
0.347
8.32

Cell counting using a Haemacytometer


Reference S.

Schoen

Cell

Counting

in

C.S.

Lobban

et

al

(1988)

Experimental Phycology a Laboratory Manual


R.R.L. Guillard Counting Slides in A. Sournia (1978) Phytoplankton Manual.
UNESCO. p. 182
Haemacytometer
As the name suggests these counting chambers have been developed for
counting blood cells but they can be used to calculate the cell density of an
algal culture providing the cells are relatively small (~ 5-50m and either
single cells or short chains.

Larger cells or long chains of cells are more

appropriately counted using a Sedgewick Rafter cell or settling chamber. A


haemacytometer is used for cell densities >10 4 cells/ml. The size of these
chambers can vary with manufacturer but we use a Neubauer brand which

consists of two chambers, each with a volume of 0.1mm 3, containing a


marked counting grid 1mm2 in area .

Be extremely careful when handling haemacytometers as they are


fragile if dropped and very expensive items of equipment costing up
to $Aus 200.00 each
Method
1.

Algal Sample Non-motile cells which do not need fixing can be

counted as soon as the sample is collected. However, if there will be a delay


between sample collection and counting, or if the cells are motile then the
sample will need to be preserved.

The most common fixative used

for

marine microalgae is Lugols Solution. The recipe for the acidic form is given
here but note in the case of microalgae with calcium carbonate scales, such
as the coccolithophorids, the acid will destroy the organisms and a basic
solution should be prepared instead.
For cultures add 1 drop of 1-2% Lugols solution to 1 ml sample, for field
samples 10 drops per 200 mL of sample or until the colour of weak tea.
Overuse of Lugols will cause some delicate flagellate species to overstain,
lose flagella or blow up entirely.
Lugols is made by dissolving 100 g Potassium Iodide (KI) in 1L of
distilled water, then 50 g crystalline iodine (I 2) is dissolved in this solution
and then100 ml glacial acetic acid is added. Lugols should be stored in the
dark as the iodine is light sensitive and will degrade. It should also be stored
with a tight fitting lid and kept away from the general culture environment.
Note: Sample dilution or concentration:

The haemacytometer can be

used where cell densities are in the range 5 x10 4 - 107 cells / mL. It is more
likely that cultures will be less rather than more dense than this range but
occasionally very dense cultures, such as nanoplanktonic flagellates and

some cyanobacteria, may need to be counted. It is both inefficient and very


difficult to accurately count these cultures without first diluting the sample
(contrast with the Sedgwick-Rafter Cell where cell density range is 30 104
cells / mL). Therefore dilute with a known volume of culture media and then
fix. Alternatively a Lugols Dilution Solution (LDS) may be used where 100 mL
of 0.2m filtered seawater or distilled water (depending on whether the
culture is marine or freshwater) is prestained with concentrated Lugols until
it is a weak tea colour. Then a known volume of culture can be added to a
known volume of the LDS. Using LDS also means that all cells are exposed
to the optimum concentration of Lugols whereas adding concentrated
Lugols could destroy some cell types when it mixes into the sample. For
example a culture with a density above 1 x10 7 cells / mL will have >1000
cells in the counting region of a heamocytometer (see detailed explanation
below) and a 1 in 5 dilution (1 mL of culture + 4 mL of LDS) will allow the
sample to be counted more readily.
If the culture is dilute, concentrate by centrifuging or settling in a flat
bottom measuring cylinder (allow 1 hour of sinking for each 10mm of
cylinder height; therefore overnight is a practical solution). For either method
once concentrated, remove and discard up to 90% of the clear supernatant
(upper portion of the liquid) without disturbing the settled biomass.
Homogenize the remaining sample and count, bearing in mind the need to
integrate the concentration factor

= final count x (settled volume / initial

volume).
2.

To fill haemacytometer chambers, place the thick coverglass over both

grids and take a Pasteur pipette and fill its tip by capillary action with
sample. Hold the pipette at an angle of ~450 (higher or lower to control flow
rate) and place the tip at the leading edge of the coverslip. With very gentle
pressure, allow the sample to flow quickly and evenly into the chamber,
exactly filling it. The chamber surface in the Neubauer brand is a flat mirrorlike rectangle and the sample must cover this rectangle but not flow over its

edges. It is useful to rest your hand on a bench and steady the pipette tip
with a finger.

If flooding occurs, rinse haemacytometer and coverslip with distilled

water, and repeat procedure.

Refill the pipette for each chamber. The time taken to fill the chamber

should be short, to minimize setting of cells in the pipette.


3.

Allow cells to settle (~1min) and check grid under the microscope (x

20 objective) for satisfactory distribution of cells, i.e. evenly spread.


4. The Haemacytometer grid in detail
The grid is divided into 9 large squares, each 1mm x 1mm, by triple
lines.
Each large square is divided into 25 medium squares, each 0.23mm on a
side, and each medium square is further divided into 16 small squares, each
0.05mm on a side.

For all haemacytometers, the fundamental measurement is the average


number of cells per 1mm square, so the centre large square is usually
counted. To obtain the total number of cells in this large square, the number
of cells in each of the 25 medium squares are counted, recorded then added
(see sample cell count)
Note: When counting cells bordering on triple rulings, the convention is to
count only those cells touching the top and left-hand side rulings of each
square.
5.

After counting each of the two haemacytometer chambers, the

haemacytometer and coverslip are rinsed with distilled water. Usually the
procedure is repeated twice more to give a total of 6 counts.
6.

To obtain the cell density, calculate the average cell


count and multiply by the conversion factor (for Neubauer = x104)

Sample Cell Count


Isochrysis sp. (Tahitian) CS-177
Age; 8 days
Growth conditions; 250C, light intensity 50mol. photons m-2 s

, 12:12 light:

dark cycle
1

3
6
6
14
4
Total

6
3
5
9

5
5
8
6
10
no. =
9
6
9
7

12
6
4
8
7
175

8
8
8
5
8

6
5
5
9
9

4
12
8
10
7

7
6
7
9
11
Total

4
8
6
4

5
10
9
3

12
10
8
3

7
3
3
7

3
3
9
9

8
4
4
6
13
no. =
4
5
6
10

10
8
4
13
6
189

6
4
5
4
13

7
6
10
9

4
6
6
15

5
12
12
Total no. = 181

8
8
9
4
9
Total

5
4
9
10
6

13
6
11
5
9

1
5
7
6
10
no. =

8
5
4
7
5
174

10

2
8
3
Total no. = 162

5
5
8
8
5

5
13
8
6
6
Total

5
9
3
2
15

6
5
8
6
10
no. =

6
2
6
7
6
165

Each block 1-6 represents the total number of cells in the large centre
square.

Mean =
Where = total no.
= sum of all totals
& n= no. of counts
Cell Count = x ccf (chamber conversion factor for Neubauer = x104)
Standard deviation S= where variance =
=
% Error =
from above example
cell count ; = 174.3 x 104 x ccf
= 1.74 x 106 cells/mL
standard deviation =
=
=9.99
%error = x 100 = 5.7%
Note: %error should be below 10%