Académique Documents
Professionnel Documents
Culture Documents
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).
(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.
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
C.
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
Note
these guidelines are more strict than those for preparing media. Other
countries may have other regulations.
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.
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).
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
make concentrated f2 media 5mL of the 1L working stocks are used to make
100mL of concentrated media.
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)
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.
according
to
the
Redfield
Ratio
of
106:16:1
of
inorganic
Carbon
According to this ratio microalgae will need roughly 6 times more carbon
than nitrogen.
In some
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.
it
will
slowly
disassociate
and
be
available
for
uptake.
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
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
metal
complex
which
may
be
utilized
by
microalgae.
However
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
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
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
Phycological
Methods,
Culture
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
each
addition.
Then
make up to 1L.
2. CaCl2.2H2O
3. MgSO4.7H2O
4. K2HPO4
5. KH2PO4
6. NaCl
7. Na2EDTA
9. H3BO3
10. Micronutrients
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.
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)
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
of
dH2O
dissolve
additions.
second
and
between
solution
to
the
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
Na2MoO4 . 2H2O
CoCl2 . 6H2O
H2SeO3
Na3VO4 .nH2O
10 mg
4 mg
2 mg
1 mg
dissolving
between
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
stock
solutions.
After
all
Source
200 mg / L
24.7 g / 500 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
1.24 g / 500 mL
2.18 g / 500 mL
NaSiO3.5H2O
FeCl3.6H2O
0.79 g / 500 mL
CaCl2.2H2O
14.7 g / 500 mL
Vitamin B12
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
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
(1997).
Simple
procedures
for
In:
Mantoura
and
S.W.
Jeffrey,
S.W.
R.F.C.
Wright
(Eds)
Phytoplankton
pigments
in
oceanography;
Monographs
on
oceanographic
methodology
10,
Stock Solutions
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
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
Stocks
Vitamin
B12
Biotin
6. NaH2PO4.2H2O
7. Na2EDTA.2H2O
Store all stock solutions in the
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).
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.
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.
In our culturing
experience this is a good 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
(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
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
heat
to
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
5 mL
Soil Extract
5 mL
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
50 mL
Soil Extract
50 mL
Phosphate stock
10 mL
In our culturing
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
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
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
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
1000 mL
100 mL
25 mL
media
developed
for
range
of
microalgae
from
specific
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
150 g / L H2O
Volume
2.5
mL
2. Na2glyceroPO4 3.24 g / L H2O
3. Na2SiO3.9H2O 22.7 g / L H2O
5 mL
2.5
mL
4. Vitamins
5 mL
5. Trace Metals
FeNaEDTA
4.3 g / L H2O
5 mL
5 mL
6.2
100 g / L H2O
6.05
2.68 g / L H2O
5.0
mL
6. TRIS
mL
7. NH4Cl
mL
8. H2SeO3
1.29 mg / L H2O
9. Distilled water
5.0 mL
52.75 mL
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
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
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.
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
2. MgSO4.7H2O
3. NaHCO3
4. K2HPO4
5. NaNO3
f stock solution
0.5 mL
0.5 mL
6. Na2SiO3.5H2O
f stock solution
7. Na2EDTA
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
f stock solution
1.0 mL
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
Stock Solution
Add V (mL.L-1)
Source
2. K2HPO3
MJ stock solution
2.0 mL
20.0
50
0.1 mL
1.0
1.0 mL
10.0
5. Vitamin B12
6. Na2SiO3.5H2O
5.0
4.5 mL
45
7. EDTA
1.0 mL
10
Autoclave each stock solution except the Vitamin B12 which should be
0.2um filter sterilised.
(a)
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
fully
combine
dissolve.
each
solution
Then
and
make up to 1 L.
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.
up separately)
Primary Stocks
CuSO4.5H2O
ZnSO4.7H2O
CoCl2.6H2O
Na2MoO4.2H2O
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
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
973 mL
MLA
x40
concentrated
nutrients
(2)
25 mL
sterile NaHCO3 (3)
1 mL
1 mL
- preferred media for Nodularia, but always use highly purified agar e.g.
Difco Bacto purified agar.
- 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.
Autoclave
gelling
agent,
and
double-strength
nutrients.
Cool to 45 0C in a water bath, and in the same bath, warm the CaCl 2 and
containing the gel. Stir gently while aseptically adding nutrients (and for
saline media, adding seawater also).
***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
all
stock
solutions
in
Na2EDTA
and
the
refrigerator.
Source
1. NaNO3
2. K2HPO4
3. MgSO4.7H2O
4. CaCl2.2H2O
5. Na2CO3
Add V
1.0 mL
1.0 mL
6. Na2SiO3.5H2O
f stock solution
12.0 mL
7. Fe citrate:
f stock solution
0.66 mL
8. Na2EDTA
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
0.5 g
0.5 g
autoclace to sterilise
add aseptically
Alternatively Jump
Biomass
(cf
).
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.
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.
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
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.
Specific
Specific
Growth
Divisions
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
Schoen
Cell
Counting
in
C.S.
Lobban
et
al
(1988)
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:
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
volume).
2.
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.
Refill the pipette for each chamber. The time taken to fill the chamber
Allow cells to settle (~1min) and check grid under the microscope (x
haemacytometer and coverslip are rinsed with distilled water. Usually the
procedure is repeated twice more to give a total of 6 counts.
6.
, 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%