australian national algae culture collection-making media

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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)

Maor nutrients, Car!on, Nitrogen, "#osp#orus, Silica

$t#er nutrients, %oron, %u&ers, C#elators, "otassium, Soil 'tract,

itamins

*#e media used in CMA+C are predominantl from pu!lis#ed recipes, some

are well known and widel used in t#e p#cological and aquaculture

communities suc# as f/-. All t#e media #ave several components in common

sources of nitrogen, p#osp#orus, vitamins and trace metals. 0owever t#e

speci1c tpes of t#ese nutrients, t#eir concentrations and ratios var

!etween t#e media. Some media used in CMA+C #ave !een developed ! us

over man ears, for instance our medium of c#oice for fres#water

cano!acteria is M2A (%olc# and %lack!urn, 3445) w#ic# is a modi1cation of

t#e ASM media developed ! 6or#am et al (3457). Some media #averelativel unusual trace metals suc# as selenium in 6Se (A modi1cation of

6"M, 2oe!lic#, 3489 and vanadium in M: medium. %ecause of t#eir unusual

nature it ma !e tempting to leave t#ese out if stocks are di;cult to come !

!ut t#is would defeat t#e ver purpose of t#e media. A detailed description

of t#e common nutrients, !u&ers and c#elators can !e found ! following

t#is link

Cleaning / glassware preparation

"roper cleaning of cultureware is as critical to successful maintenance

of microalgal cultures as it is for accurate results in analtical c#emistr.

Some species, suc# as certain !ent#ic diatoms, ad#ere to t#e glass and

autoclaving cultures !efore disposal can also lead to a crust on t#e glassware
http://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Autoclavinghttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Cleaninghttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Stocksolutionshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#seawatersourcehttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#aquaculturistshttp://www.marine.csiro.au/microalgae/methods/Media%20comparison.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#carbonhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#nitrogenhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#phosphorushttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#silicahttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#boronhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#buffershttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#chelatorshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#potassiumhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#soilextracthttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#vitaminshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#majornutrientshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#majornutrientshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Cleaninghttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Stocksolutionshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#seawatersourcehttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#aquaculturistshttp://www.marine.csiro.au/microalgae/methods/Media%20comparison.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#carbonhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#nitrogenhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#phosphorushttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#silicahttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#boronhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#buffershttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#chelatorshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#potassiumhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#soilextracthttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#vitaminshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#majornutrientshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#majornutrientshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#Autoclaving


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at t#e meniscus w#ic# is not removed simpl ! a detergent !at# !ut

requires scru!!ing wit# a !ristled !rus#. $ur glassware preparation involves

a preparator rinse in #ot tap water, soaking overnig#t in a detergent !at#

(e.g. "roneg), scru!!ing, a was#/rinse ccle in a 2a!orator dis#was#er

(was# ccle at #and rinses in Milli ? water and dring in a

la!orator oven.

Non@disposa!le plasticware suc# as plocar!onate car!os and

polproplene 1ttings are soaked in a compati!le detergent suc# as Nalgene

2@4==.

Autoclaving

e use autoclaving as t#e prime met#od for steriliBing media and

particularl for making media for aenic cultures. Man of our media recipes

refer to full autoclaved media, w#ere t#e last stage in media preparation is

for individual culture asks to !e autoclaved so t#at t#e asks remain totall

unopened until culture transfer. 0owever w#ere maintaining aenic cultures

is not as critical t#en 1lter steriliBing particular components of t#e media and

adding t#em asepticall to presteriliBed seawater (or fres#water) can !e

used. Concentrated nutrient solutions can !e prepared in t#is wa so t#atp#osp#ate stocks do not precipitate and vitaminactivit is not impaired.

#ile autoclaving at 3-3 =C for 39 minutes appears to !e a common

mantra, t#e required duration of autoclaving can cause some confusion. Dt

s#ould !e noted t#at t#e autoclave time refers to t#e duration t#e goods or

total volume of liquid is maintained at t#e set temperature, and not simpl

t#e time in t#e autoclave or t#e time after 3-3 =C is reac#ed. Autoclaves

come wit# di&erent c#am!er siBes and wit# di&erent capacities for

pressuriBation, air removal, vacuum generation, steam generation and air

e#aust, and all t#ese factors inuence t#e rate of temperature increase,

#olding capacit and decrease. *#erefore it is di;cult to give prede1ned

autoclave times for ant#ing ot#er t#an #ard goods suc# as pipettes and

empt glassware or containers wit# up to 3==m2 of media w#ere t#e tpical
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3-3 =C for 39 minutes is pro!a!l appropriate. Note, #owever t#at a car!o

wit# 3= 2 of media ma require in ecess of 5= minutes once t#e autoclave

reac#es t#e set temperature of 3-3 =C. *#e onl relia!le means of

monitoring temperature in !ulk media is to #ave a t#ermistor wit#in a

reference container (e.g. "*3==) and t#is would onl #ave to !e done

periodicall to Ecali!rateF t#e sstem. +elativel small !enc#top pressure

cookers ma !e t#e sole autoclave in man aquaculture facilities or used as

!ackup autoclaves in la!oratories. Dn t#is case, w#ere for eample t#e

largest container t#at will 1t ma !e a 3 2 culture !ottle, it is rare for more

t#an = minutes

ma !e appropriate. Some confusion also eists around pressure and steam,

wit# a perception t#at t#ese factors are important in let#alit. *#is is not

true, temperature a!ove 3== =C is t#e let#al factor and pressure and steam

are simpl t#e means of delivering t#at temperature to t#e entire contents of

t#e autoclava!le goods. it#out steam, air pockets and voids can eist in

!ot# glassware and wit#in autoclave !ags and t#ese can persist at lower

temperatures t#an t#e c#am!er temperature !ut steam penetration into

t#ese voids ensures t#e t#e required temperature is met.

Autoclaving drives o& car!on dioide, needed for algalp#otosnt#esis, and raises t#e p0 to undesira!le levels. 2eaving t#e media

for at least 3@- das !efore use will allow su;cient time for C$-equili!ration.

Autoclaving is also a mandator met#od for disposing of our microalgal

stock cultures under our Approved "remise for ?uarantine "ermit (provided

! t#e Australian ?uarantine and Dnspection Service, A?DS). Minimum draft

regulations set ! A?DS :une -==7 are 3-3 =C at 39psi for >=mins. Note

t#ese guidelines are more strict t#an t#ose for preparing media. $t#er

countries ma #ave ot#er regulations. *#e following two links are on

autoclaving culture media and on t#e mt#s of autoclaving G commercial

source note

#ttp//[email protected]/*AutoclavingMedia.#tm

#ttp//www.


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Stock solutions and salts

Dn our recipes we refer to Eworking stocksF and Eprimar stocksF.

orking stocks are t#ose w#ose aliquots contri!ute directl to making t#e

1nal media. "rimar stocks are normall made w#ere several single

su!stance solutions are t#en com!ined to form t#e working stock, eg.

CuS$7.90-$ and JnS$7.80-$ are two of t#e primar stocks used to make

up t#e *race Metal working stock in K/- medium.

Culture Collections and scientists undertaking eperimental researc#

will use c#emicals t#at are at least Analtical +eagent 6rade, !ot# to reduce

trace contaminants t#at ma !e #armful to some microalgae and also to

ensure eperimental rigour. #ile we suggest all stock or starter cultures !e

grown wit# A+ grade c#emicals it is understanda!le t#at in mass culture

applications (L -= @ 9= 2) , particularl for aquaculture, t#ese c#emicals ma

!e too epensive w#en !oug#t in !ulk quantities.

Stock solutions are made up ! accuratel weig#ing t#e prescri!ed

amount of nutrient and dissolving in a speci1ed volume of distilled water, if

possi!le in a volumetric ask. Some nutrients will readil dissolve, ot#ers

need #eat and stirring to full dissolve. Dn contrast vitamin stocks are #eatsensitive and s#ould not !e su!ected to #eat treatment and s#ould also !e

kept in t#e dark. Kailure to full dissolve t#e primar stocks of some

nutrients suc# as '*A can lead to gross precipitation w#en t#ese stocks are

com!ined to make t#e media.

Nutrients come wit# di&erent salts and #dration. Kor eample, w#ile

copper and Binc ma !e two desired active constituents t#e are readil

o!tained from suppliers wit# eit#er S$7 or Cl- salts (ie CuS$7 or CuCl- and

JnS$7 or JnCl-). Some nutrients also come wit# di&erent #drations, ie

t#e .n0-$ su;. Su!stituting one form for anot#er ma #ave no e&ect on

t#e growt# of some microalgae species, !ut it can lead to poor growt# in

ot#ers and also lead to unwanted and time consuming precipitation pro!lems

as t#e overall ratio of salts in t#e medium #as c#anged. *#erefore deviating


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from t#e prescri!ed recipes is to !e avoided and ordering t#e correct form is

recommended.

Seawater source and treatment

*#e marine microalgae species maintained in CMA+ are grown

usingunpolluted oceanic seawater t#erefore, strictl speaking , we are using

an enric#ment medium. Arti1cial seawater media in contrast is composed of

marine salts and nutrients added to pure fres#water. Arti1cial seawater is

onl necessar w#ere a clean natural seawater source is unavaila!le or in

particular researc# studies (e.g. trace metal studies) w#ere t#e eact

composition needs to !e controlled. $ur collection source takes advantage of

CSD+$ 0droc#emistr groups monitoring station on t#e seaward side of

Maria Dsland o& t#e continental s#elf along t#e east coast of *asmania. *#e

salinit at t#is site is O>>@>5 "ractical Salinit Pnits. *#is o&@s#ore site #as

ver low concentrations of metal and organic pollutants t#erefore making it

suita!le as t#e !ase medium for a wide range of marine microalgae

species.

*#e seawater is collected in -=2 !lack polet#lene car!os acid was#ed

prior to collection and t#en stored at 7=

C until needed. *#en it is treatedusing a 1ltration series incorporating t#ree 3-F MilliporeQ cartridge 1lters (9

m pre1lter, activated c#arcoal 1lter for organics removal and a urapore

=.79m 1lter) and 1nall a Millipak 7=*M=.-- m disc 1lter.

Notes for Aquaculturists

*#e recipes and preparation protocols used in CMA+C are

predominantl designed for la!orator scale culturing. 'ven t#econcentrated nutrient solutions ma not !e of su;cient volume for regular

mass algal cultivation. Dt is up to t#e user to modif t#e quantities of

material needed and determine t#e cost@e&ectiveness of using our proposed

media versus commercial alternatives. As an eample in t#e procedure to


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make concentrated f-media 9m2 of t#e 32 working stocks are used to make

3==m2 of concentrated media. As t#e usage rate is 3m2 per 3==m2 of

seawater, t#e 3==m2 stock is su;cient for a 3=2 culture. As t#ere are -==

aliquots of working stock availa!le (32 stock/9m2), t#e total culture volume

t#at can !e supported is -===2. *#e simplest modi1cation to o!tain useful

volumes for aquaculture is to increase t#e volumes of t#e concentrated stock

solution and its constituent working stocks e.g. for f-

2a! scale concentrated stock 5 stocks 9 ml eac# I >= m2, made up to

3== ml wit# distilled 0-$ (d.0-$) *#is provides enoug# concentrated stock

for 3= 2 f- medium.

2argescale 5 stocks 9== ml eac# I >=== m2, made up to 3=,=== ml (3= 2)

wit# (d.0-$) . *#is provides enoug# concentrated stock for 3=== 2 f-

medium.

Note in our recipe onl 3== ml of vitamin stock is made at an one

time. Kor large scale purposes t#at volume would need to !e increased to

3=== ml in accordance wit# t#e ot#er stocks.

Major Nutrients

Microalgae do not require equal amounts of all nutrients, t#erefore t#e are

added in di&erent quantities. Natural seawater will suppl enoug# nutrients

for limited growt# of some marine species. Most media recipes suppl

nutrients vastl in ecess of t#e concentrations normall found in order to

support #ig# !iomass cultures. i&erent species require di&erent amounts of

some nutients !ut t#e requirement for t#e maor nutrients is relativel

uniform according to t#e +ed1eld +atio of 3=5353 of inorganic

car!onnitrogenp#osp#ate (! moles).
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Carbon

According to t#is ratio microalgae will need roug#l 5 times more car!on

t#an nitrogen. Kor marine species t#e car!on requirement in small !atc#

cultures is met ! t#e - mM contained wit#in seawater, and ! allowing

atmosp#eric ec#ange t#en t#e car!on requirement can !e supplied over

time. Car!on ma !e lost directl after autoclaving, t#erefore it is sensi!le to

allow fres#l autoclaved media to stand for at least 3@- das !efore

innoculating wit# microalgae. 2arge !atc# cultures, generall greater t#an

9== m2 to 3 2 ma need to !e aerated wit# eit#er air or an air/C$ -mi to

prevent car!on limitation. *pical C$-concentrations are in t#e range =.9 @

9.= R v/v

Nitrogen adapted from !hompson" #$$%% course notes&

Nitrogen is most commonl added as nitrate. 0owever some algal species

grow !etter on ammonium (*#ompson et al, 34. %ecause algae

preferentiall remove N07 t#e are likel to deplete all N07prior to using

nitrate for growt#.

'hosphorus

Silica

Silica is onl reall required ! diatoms (ecepting t#e rarel cultured

silicoagellates) and is t#erefore often left out of media w#ic# are selective

for ot#er organisms. iatoms onl need trace quantities of silica !ecause

t#e are etremel e;cient at scouring silica from t#e environment. Some


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diatoms ma grow for man generations in seawater media w#ic# #as no

added silica and some ma continue to divide for several weeks, al!eit wit#

poor frustule development. Media suc# as t#e 6 variet (6", 6Se) w#ere

t#e onl added silica, apart from t#e seawater, comes from soil etract ma

readil support t#e growt# of some diatom species. 'nric#ment cultures for

isolating microalgae devoid of added silicate ma still !ecome ric# in

diatoms t#erefore if selection is for non@diatom species a diatom in#i!itor is

advantageous. 6ermanium dioide (6e$-) is toic to diatoms !ecause it

disrupts silica deposition and t#e addition of low concentrations (9 mg / 2) of

6e$- to a culture medium can in#i!it diatom growt#.

*#e silica stock ma precipitate if made up in glass !ottles so teon !ottles

can !e used instead. "recipitation is a greater pro!lem w#en t#e stock is

1rst added to seawater especiall if autoclaved and in glass containers.

0owever it will slowl disassociate and !e availa!le for uptake.

"olcar!onate car!os for larger volumes or teon !ottles (3 2) ma !e used

to limit t#e precipitation if it is pro!lematic, noting t#at teon is especiall

epensive.

$t#er Nutrient components(oron )

Carrano etal, (-==4) present a review on !oron and marine life. Man studies

#ave s#own !oron is an essential trace element for !ot# terrestrial #ig#er

plants and fres#water and marine algae. 2ewin (3459, 3455) s#owed t#at

%oron is essential for marine diatom growt# !ased on results wit# 35 centric

and pennate species and essential for some !ut not all marine agellate

species. iatom cell division was muc# reduced at !oron concentrations less

t#an =.9 mg23or O=.=9mM (O 3=R of t#e tpical 7.9 mg/2 natural seawater

concentration). #ile !oron is a component of man culture media it is not

present in some common media suc# as 6uillards K or K/- w#ic# #ave !een

used widel to support marine microalgal growt# including diatoms. *#e

presence of !oron leac#ing into media from !orosilicate glassware ma
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account for some of t#is positive diatom growt# !ut scale@up of diatom

growt# in plastic !ags (e.g. 3== @ 9== 2) in mariculture operations argues

t#at !oron ma not !e essential for all species. %oron is required !

#eterocstous cano!acteria t#roug# its role in nitrogenase activit in

dinitrogen 1ation and #eterocst morp#olog ma alter wit#out !oron. Non@

#eterocstous cano!acteria displa no alteration in growt# or nitrogen

1ation. (%onilla et al, 344=).

%onilla, D., M. 6arcia@6onBTleB, et al. (344=). %oron requirement in

cano!acteria. Dts possi!le role in t#e earl evolution of p#otosnt#etic

organisms. "lant "#siol. *+ 3997@395=.

Carrano, C. :., S. Sc#ellen!erg, et al. (-==4). %oron and Marine 2ife A New

2ook at an 'nigmatic %ioelement. Marine %iotec#nolog ,,(7) 7>3@77=.

2ewin, :. C. (3459). %oron +equirement of a Marine iatom.

Naturwissensc#aften #(>) 8=

2ewin, :. (3455). %oron as a 6rowt# +equirement for iatoms. :ournal of

"#colog #(7) 35=@35>

(u.ers

Microalgal consumption of C$-in !atc# culture sstems (wit#out added C$-)will increase t#e p0, and levels a!ove p0 4 are toic to man microalgal

species (cano!acteria generall #ave #ig#er tolerances for elevated p0

w#ic# ma give t#em a competitive advantage under suc# conditions).

%u&ers ma give some control over t#e rise in p0 and are suita!le to low

volume stock cultures, #owever in large scale dense cultures p0 control is

pro!a!l more readil met ! t#e addition of C$-!ack into t#e culture !

aeration (air onl or air/C$-mi ..see Car!on).

!0S bu.er (tris (#dromet#l) aminomet#ane)1 6enerall *+DS is used at

concentrations up to 9 mM !ut some microalage 1nd it toic. An eample of

varing suscepti!ilit is s#own ! a comparison !etween two frews#water

cano!acteria, Microcystis and Anabaena. *+DS at 3= mM provides good

!u&ering !etween p0 [email protected] in MicrocystisU wit# increased tolerance
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towards supraoptimal concentrations of monovalent !ut not divalent cations

(Mc2ac#lan and 6or#am, 3453). 0owever *+DS is toic in Anabaena fos-

aquaeat concentrations too low to provide suita!le !u&ering !ut 1lament

coiling, indicative of good growt#, is promoted ! addition of 3 mM *+DS

(6or#am et al, 3457).

2l3c3lgl3cine is used at concentrations up to >.< mM, #owever it is !ot#

epensive and rapidl meta!olised ! !acteria and s#ould onl !e used in

aenic cultures

Sodium bicarbonate(Na0C$>) !u&ers well in t#e p0 range 8.9@


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Soil 45tract full preparation protocol detailed in media recipes&

Soil etract is an adaptation of '.6. "rings#eims !ip#asic soil@water medium

and is a component of some of our media. As t#e c#emical composition is

not well@de1ned and ma var from !atc# to !atc# its use in eperimental

situations is not recommended, #owever in our eperience certain species

will not grow well wit#out it and soil etract is added for !ot# culture

maintenance and eperimental studies. Soil must !e collected from a

natural uncultivated environment or a ric# garden loam ma !e suita!le. No

fungicides, insecticides, garden fertiliBers or fres# manure s#ould !e present.

At CSD+$, topsoil from a local sand !us#land environment #as proved to

#ave particularl !ene1cial growt# promoting properties. Soil from cla or

ot#er soil tpes are less suita!le in our eperience. Soil s#ould not !e stored

or processed in t#e algal culture la!orator, since it is a potent source of

unwanted microorganisms. Soil s#ould !e aged under moist conditions

(prefera!l for 5 mont#s or more) and t#en kept dr and awa from lig#t.An

etensive list of modi1ed Soil ater media developed for a range of

microalgae from speci1c environments can !e found on t#e P*'V we!site

(*#e Culture Collection of Algae at t#e Pniversit of *eas)

#ttp//www.ute.org/.

6itamins

*#e t#ree most widel used vitamins in order of signi1cance to algae are

vitamin %3- (co!alamin or canoco!alamin), t#iamine and !iotin. *#e are

most often prepared as a single stock solution.

itamin %3- (co!alamin or canoco!alamin)

#ile vitamin %3- or co!alamin #as !een recogniBed as a necessar

component for algal growt#, its meta!olic role #as !een unclear, !ut recent

evidence suggests it is primaril a cofactor for vitamin %3-@dependent

met#ionine snt#ase (Croft et al. -==9). A surve of >-5 algal species

indicates t#at more t#an #alf of t#e algal kingdom are co!alamin auotrop#s

and t#at all algal p#la and even individual genera contain species t#at
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require vitamin %3- and species t#at do not (Croft et al. -==9). Croft etal

argue t#at t#e source of co!alamin is t#roug# sm!iosis wit# !acteria w#ic#

!ene1t from t#e etracellular car!on produced from algal p#otosnt#esis.

*#is premise #as !een strongl criticiBed ! roop (-==8), a pioneer of

marine vitamin ecolog, on t#e !asis t#at oceanic and coastal concentrations

of %3- s#ould easil meet t#e low cell requirements of algal populations

wit#out a direct sm!iosis. Completing t#e genome sequence for t#e diatom

Thalassiosira pseudonana, #as s#own t#at t#is important species (ie

important aquaculture and general model species in algal

p#siolog/ecolog) is a co!alamin auotrop#. $t#er Thalassiosira species

#owever are not auotrop#ic for vitamin %3-.

Croft, M.*., 2awrence, A.., +au@eer, '., arren, M.:. and Alison 6. Smit#,

A.6. (-==9) Algae acquire vitamin %3- t#roug# a sm!iotic relations#ip wit#

!acteria. Nature+78(>)4=@4>.

roop, M. +. (-==8). itamins, p#toplankton and !acteria sm!iosis or

scavengingHJournal o Plankton Research-43=8@3>.

*#iamine........

%iotin..........

'reparing and using 6itamin stock solutions

itamins are known to !e #eat and lig#t sensitive, primar stocks of itamin

%3- and !iotin are dispensed in 3=@-= m2 aliquots in polcar!onate tu!es and

froBen, w#ere t#e ma keep for long periods and ma !e refroBen. *#e

working stock solution, including t#e added t#iamine, is normall onl kept

for > mont#s in alfoil wrapped refrigerated !ottles !efore new solutions are

prepared. espite t#e known degradation of vitamins w#en #eat steriliBed,

man media tpes call for total autoclaving. Autoclaving is regularl

emploed for making media for aenic cultures and rarel #ave we

encountered growt# impedance t#at can !e traced to vitamin de1cienc.

Some algae ma !e a!le to use t#e decomposition products incurred after

autoclaving ("rovasoli and Carlucci, 3487), #owever we recommend t#at for


13/62

important new isolates or poorl growing species =.- um 1lter@steriliBed

vitamins s#ould !e added asepticall to presteriliBed media.

"rovasoli, 2. and Carlucci, A. K. (3487). itamins and growt# regulators. Dn .

. ". Stewart (ed) Algal "#siolog and %ioc#emistr. %lackwell Scienti1c, PW,

873@


14/62

grow ever strain under optimal conditions for culture longevit or inter

transfer period. *#erefore strains are grouped into functional transfer units. A

group of !ent#ic diatom species at -= =C and a group of prmnesiop#tes at

3= =C t#at !ot# will tolerate low lig#t (9@3= Zmol. p#otons m@- s G3) can !e

transferred once ever < weeks compared wit# a group of oceanic diatoms

and estuarine dinoagellates t#at need frequent transfers ever -@7 weeks.

olumes transferred will also var depending on t#e densit of t#e culture

!ut generall =.9 @ -.= m2 is transferred from t#e 9=m2 ask cultures into

new media.

Kig 3. *ransfer generations

#ile man strains will grow from innoculum to #arvest on low lig#t we

generall give t#e fres# daug#ter innoculum 3@- weeks of optimal lig#t

intensit !efore decreasing it to maintenance levels. *#e lower maintenance

levels are ac#ieved ! placing s#eets of glassine paper or paper towelling

!eneat# t#e cultures and surrounding t#e asks wit# !lack card!oard

clinders. Note t#at some tpes of paper towelling can cause spectral s#ifts

in t#e lig#t qualit t#at reac#es t#e culture. aug#ter and parent cultures

are kept toget#er !ut t#e grandparents are removed to a separate location in

t#e event of a maor room/ca!inet malfunction (e.g. compressor failure and

dramatic increase in temperature). After t#e net transfer is completed t#e

grandparent cultures are autoclaved and removed for was#ing.


15/62

Media ecipes used in CMA

Media Comparison *a!le (constituents in Zmoles of some of t#e a!ove

media)

urther help on !akin" !edia includin" characteristics o indi#idual !edia

co!ponents

$ee notes or Aquaculturistsi lar"e #olu!es o !edia are needed

$ea%ater source

(olds

(asal

Medium

+eferences

Nic#ols, 0. . and %old, 0. C. (3459).

Trichosarcina poly!orpha gen. 't sp.

Nov.J. Phycol. , >7@) 6rowt# media G

fres#water. Dn Stein, :. ('d.) &andbook

o Phycolo"ical Methods, 'ulture

Methods and (ro%th Measure!ents,Cam!. Pniv. "ress pp. 8@-7.

Adapted for fres#water algae

Stock Solutions per :itre distilled water d;# -9.= g

#9 CaCl-.-0-$ -.9 g79 MgS$7.80-$ 8.9 g+9 W-0"$7 8.9 g9 W0-"$7 38.9 g=9 NaCl -.9 g
http://www.marine.csiro.au/microalgae/methods/Media%20comparison.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#aquaculturistshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#seawatersourcehttp://www.marine.csiro.au/microalgae/methods/Media%20comparison.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htmhttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#aquaculturistshttp://www.marine.csiro.au/microalgae/methods/Making%20Media.htm#seawatersource


16/62

>9 '*A

W$0

9=.= g

>3.=g

89 KeS$7.80-$

0-S$7

7.4< g

3.= m2

*9 0>%$> 33.7- g,$9 Micronutrients g.2@3 Add eac#

constituent

separatel to

O


17/62

=9 NaCl 3=.= m2 %% stock solution

>9 Na-'*A 3.= m2 of 98.=9g2@30-$ stock

89 KeS$7.80-$ wit# 0-S$7 3.= m2 %% stock solution

*9 0>%$> 3.= m2 %% stock solution

,$9 Micronutrients 3.= m2 %% stock solution

Add eac# stock solution (, ? ,$) in t#e stated volume to 3 litre distilled

water.

Autoclave at 3-3[C (39"SD for 39 mins).

(2 (lue - 2reen Medium&

Medium for Spirulinaspp9

Source Culture Collection of Algae and "rotoBoa Catalogue of Strains (34


18/62

!o 'repare , litre of media

*o


19/62

Step #

inal (2/SB medium

Mi asepticall equal parts of Step 3 Seawater mi wit# %6 medium recipe.

M'DPM

istilled 0-$ 3.= liter

NaCl 3 9.= m2 of 3=R solution

CaCl-.-0-$ -.88 m2 of 3=R solution

W-0"$7 =.> m2 of 3=R solution

KeCl>.50-= =.=9 m2 of 3=R solution

*+DS 3=.= m2 of 3=R solution

%3- >.= m2 of 3g/m2 solution*#iamine 3.= m2 of 3mg/m2 solution

" DD Metal Mi 9.= m2 of stock solution (see !elow)

Adust p0 to 8.5@8.< wit# 3 N 0Cl (appro.9@5 mls/liter)

' 00 Metal Mi5 Stock

Solution

g / 2

Add t#e Na-'*A to

O89=m2 of d0-$ in a

volumetric ask and stir

over low #eat to dissolve.

Add eac# of t#e ot#er

constituents separatel to

O-==m2 of d0-$ and

full dissolve !etween

additions. *#en add t#is

second solution to t#e

Na-'*A and make up to3 2.

Na-'*A 5.= gKeCl>.50-$ =.-4 g

0>%$> 5.


20/62

(Weller and Anderson unpu!l.U modi1ed

from 2e#man 3485)

Source Dn :ournal of "#colog, 3448,

supplement to ol. >>, No. 5, CCM" @

"rovasoli@6uillard National Center for

Culture of Marine "#toplankton

Stock Solutions 'er $$m: distilled

water d;# 3.>> g

+9NaN$> -.9 g9Na- glcero"$7 9.= g=90>%$> =.7 g>9Na-'*A 7.= g89NaSi$>. 40-$ 8.9 g*9KeCl> .50-$ 3.>9 g,$9CaCl- .-0-$ >8.9 g,,9 f/- vitamins (see

!elow)

3 m2

,#9 trace metals (see

!elow)

3 m2

f/# vitamin Stock Solutionorking Stock

to one liter of distilled water, add t#e following%iotin 3=.= m2 primar stockitamin %3- 3.= m2 primar stock*#iamine 0C2 -==.= mg

"rimar Stocks%iotin =.3 mg. m2@

itamin %3- 3.= mg. m2@3

D trace metals mg per 9== m2 distilled waterMnCl-. 70-$ 3==

mg

Add eac# constituent

to O7==ml d0-$, fullJnS$7.80-$ -= mg


21/62

dissolving !etween

additions. *#en make

Na-Mo$7 . -0-$ 3= mgCoCl- . 50-$ 7 mg0-Se$> - mgNa>$7 .n0-$ 3 mg

!o prepare ,: of medium

,9 *o 4== m2 of d0-$ add -== mg of

M'S !u&er

#9 Add 3 m2 of t#e stock solutions (,-

,#).

79 After all additions, !ring to 3 2 and

adust t#e p0 to 5.< (it will pro!a!l !e

ver acidic).

\D\ (fres#water) Medium @ CSD+$

Modi1cation

+eferences (Weller and Anderson

unpu!l.U modi1ed from 2e#man 3485)

Source Dn :ournal of "#colog, 3448,

supplement to ol. >>, No. 5, CCM" @"rovasoli@6uillard National Center for

Culture of Marine "#toplankton

*o 4== ml of dd0-$ add -== mg of

M'S !u&er and dissolve. Add t#e

following stock solutions. After all

additions, make up to 3 2. Adust t#e

p0 to 5.


22/62

WCl 3.9 g / 9== m2 \D\stock use 3.= m2

N07N$> 3.>> g / 9== m2 \D\ stock use 3.= m2

NaN$> -.9 g / 9== m2 \D\ stock use 3.= m2

Na- glcero"$7 3.5- g / 9== m2 W stock use >.= m2

0>%$> 3.-7 g / 9== m2 M2A stock use =.> m2

Na-'*A -.3< g / 9== m2 M2A stock use 3.< m2

NaSi$>.90-$ 33.>9 g / 9== m2 fstock use =.55 m2

KeCl>.50-$ =.84 g / 9== m2 M2A stock use 3.8 m2

CaCl-.-0-$ 37.8 g / 9== m2 M2A stock use -.5 m2

f/- vitamins (see !elow) use 3.= m2

trace metals (see !elow) use 3.= m2

f/# vitamin Stock Solution

orking Stock

to 3==ml of distilled water, add t#e following

%iotin 3.= m2 primar stock

itamin %3- 3.= m2 primar stock

*#iamine 0C2 -==.= mg

"rimar Stocksitamin %3- =.3 mg / m2

%iotin =.3 mg / m2

D trace metals

Add to 9== m2 distilled water, 1rst dissolving seperatel

MnCl-.70-$ 3== mg

JnS$7.80-$ -= mg

Na-Mo$7.-0-$ 3= mg

CoCl-.50-$ 7 mg

Na>$7 .n0-$ 3 mg

Add as t#e following stock solution

0-Se$> =.579 mg 6Se stock use >m2
http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#DYIYhttp://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#Fhttp://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#DYIYhttp://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#F


23/62

Medium f and f4& - CS0.90-$ --.8 g+9 Ke citrate g / 2 add !ot#

constituents to

32 of d0-$ and

autoclave to

dissolve

Kerric citrate 4.= gCitric acid 4.= g

9 itamins(i)orking Stock

Solution (make fres# solution

ever > mont#s)

to 3== m2 of distilled

water, add t#e following

%iotin 3.= m2 primar stockitamin %3- 3.= m2 primar stock*#iamine 0C2 -=.= mg

(ii) "rimar

Stocksitamin

%3-

3=.= mg /3== m2 d0-$

%iotin 3=.= mg /3== m2 d0-$=9 Na0-"$7.-0-$ 33.> g>9 Na-'*A.-0-$ >=.= gStore all stock solutions in t#e refrigerator.

'reparation Methods

,9 !o 'repare Medium f

Add 3 m2 of eac# stock solution (, ?

) to 3 litre seawater. ispense to

asks and autoclave at 3-3[C (39"SD,

39 mins).


25/62

'hosphate (see Stock 5.@ Na0-"$7.-0-$ ). *#is must !e sterilised

separatel from seawater to prevent precipitation. ilute original

p#osp#ate stock wit# distilled water suc# t#at 3 m2 added to eac# ask of

sterile medium will give t#e required concentration of p#osp#ate in t#e

medium. Autoclave dilute p#osp#ate stock at 3-3[C (39"SD, 39 mins). After

cooling, dispense asepticall wit# sterilised automatic dispenser.

or e5ample1

Kor 3== 3-9 m2 'rlenmeer asks, eac# containing 89 m2 medium,

prepare dilute p#osp#ate stock as follows

f and f4 media1

*ake 8.9 m2 of original p#osp#ate stock and make up to 3== m2 wit#

distilled water.

"our into a -9= m2 Sc#ott !ottle and autoclave to steriliBe. ispense 3 m2

per ask asepticall.

f# and f4# media1

*ake >.89 m2 of original p#osp#ate stock and make up to 3== m2 wit#

distilled water.


per ask asepicall.

Scale up in t#e same proportion for larger volumes.

!o 'repare Medium f4

"repare as medium f, !ut also add 3 m2 of Na-'*A.-0-$ stock solution (>).

!o 'repare Medium f#


26/62

"repare as medium f, !ut using =.9 m2 of eac# stock solution instead of 3.=

m2 of eac#.

!o 'repare Medium f4#

"repare as medium f-, !ut also add =.9m2 of Na-'*A.-0-$ stock solution.

#9 !o 'repare Medium f# concentrated nutrients

*ake 9m2 of eac# stock solution (, ? =) and make up to 3== m2 wit# distilled

water.

"our into a -9= m2 Sc#ott !ottle.

Autoclave at 3-3[C (39"SD, 39 mins).

Alternativel, 1lter sterilise using a =.-- m 1lter into a sterile -9= m2

Sc#ott !ottle.

Pse 3 m2 per 3== m2 sterile seawater adding correct amount of nutrient

asepticall.

$ee notes or Aquaculturists i a "reater #olu!e o concentrated nutrients is

needed

29 '9 medium CMA uses the abbreviation 2 for 2' medium&

+eference 2oe!lic#, A. +. and Smit#, . '. (345.

Note a!out Salinit Dn medium 6 and derivatives t#e 1nal preparation steps

require miing of nutrients wit# seawater and distilled water in a >3 or 73

ratio. *#e full marine seawater (O>>@>5practical salinit units) used inCMA+ means t#e resulting media salinit is O-


27/62

d;# 3==.= g#9 W-0"$7 >7.< g79 itamins

orking Stock Solution to 3== m2 of distilled water,

add t#e following%iotin -.= m2 primar stockitamin %3- 3.= m2 primar stock*#iamine 0C2 3==.= mg (fres# solution

ever > mont#s)"rimar Stocksitamin %3- 3=.= mg /3== m2d0-$%iotin 3=.= mg /3== m2d0-$

+9 "DD Metal Mi g / 2

Add t#e Na-'*A

to O89=m2 of d0-$

in a volumetric

ask and stir over

low #eat to

dissolve. Add eac#

of t#e ot#er

constituents

separatel to

O-==m2 of d0-$

and full dissolve

!etween additions.

*#en add t#is

second solution to

t#e Na-'*A and

make up to 32.

Na-'*A 5.= gKeCl>.50-$ =.-4 g0>%$> 5.


28/62

,9 !o 'repare 2 medium

*o 89= m2 seawater add

istilled water -9= m2

Nitrate stock - m2

itamin stock 3 m2

"DD Metal Mi 9 m2

Soil 'tract 9 m2

ispense to asks and autoclave at

3-3[C (39"SD, 39 mins).

'hosphate must !e sterilised separatel from seawater to prevent

precipitation.

ilute original p#osp#ate stock wit# distilled water suc# t#at 3 m2 added to

eac# ask of sterile medium will give t#e required concentration of

p#osp#ate in t#e medium. Autoclave dilute p#osp#ate stock at 3-3[C

(39"SD, 39 mins). After cooling, dispense asepticall wit# sterilised automatic

dispenser.

or e5ample1

Kor 3== 3-9 m2 'rlenmeer asks, eac# containing 89 m2 medium,prepare dilute p#osp#ate stock as follows

2 medium

*ake 8.9 m2 of original p#osp#ate stock and make up to 3== m2 wit#

distilled water.

"our into a -9= m2 Sc#ott !ottle and autoclave to sterilse. ispense 3 m2

per ask asepticall.

2#medium1

*ake >.89 m2 of original p#osp#ate stock and make up to 3== m2 wit#

distilled water.


per ask asepticall.

Scale up in t#e same proportion for larger volumes.


29/62

!o 'repare Medium 2#

Make up dilutant, consisting of seawater and distilled water (>3 ratio)

ilute 6 medium ! adding an appropriate volume of dilutant suc# t#at

@ medium is #alf of its original concentration (6-medium).

#9 !o 'repare Medium 2 concentrated nutrients

Add Nitrate stock -= m2

itamin stock 3= m2

"DD Metal Mi 9= m2

Soil 'tract 9= m2

"#osp#ate stock 3= m2

Make up to -== m2 wit# distilled water.

"our into a -9= m2 Sc#ott !ottle.



Sc#ott !ottle.

Pse -m2 /3==m2 sterile seawater and distilled water (>3 ratio).

Add correct amount of nutrients asepticall.

!o 'repare Medium 2# concentrated nutrients

Pse 6 concentrated nutrients suc# t#at

6- use 3 m2 /3==m2 sterile seawater and distilled water (>3 ratio).

69 use =.7 m2 /3==m2 sterile seawater and distilled water (>3 ratio).

2Se medium ? Modi@cation of 29 '9 medium

+eference %lack!urn, S. D.U %olc#, C. :. S.U 0askard, W. A., and 0allegrae&, 6.

M. +eproductive Compati!ilit Among Kour 6lo!al "opulations of t#e *oic


30/62

inoagellate 6mnodinium Catenatum (inop#ceae). "#cologia. -==3U

7=(3)8>@>5 practical salinit units) used in

CMA+ means t#e resulting media salinit is O-< psu. Dn our culturing

eperience t#is is an optimal salinit for estuarine and coastal agellate

species, particularl dinoagellates.

Stock Solutions 'er :itre distilled water

d;# 3==.= g#9 W-0"$7 >7.< g79 itamins

orking Stock Solution to 3== m2 of distilled water,

add t#e following%iotin -.= m2 primar stockitamin %3- 3.= m2 primar stock

*#iamine 0C2 3==.= mg (fres# solution ever> mont#s)

"rimar Stocksitamin %3- 3=.= mg /3== m2d0-$%iotin 3=.= mg / 3== m2d0-$

+9 "DD Metal Mi g / 2

Add t#e Na-'*A to

O89= m2 of d0-$ in

a volumetric ask

and stir over low

#eat to dissolve.

Add eac# of t#e

ot#er constituents

separatel to O-==

m2 of d0-$ and

Na-'*A 5.= gKeCl>.50-$ =.-4 g0>%$> 5.


31/62

full dissolve

!etween additions.

*#en add t#is

second solution to (adust p0 to 8.< @


32/62


Sc#ott !ottle.

Pse - m2 /3==m2 sterile seawater and distilled water (>3 ratio).

Add correct amount of nutrients asepticall.

+9 Soil 45tract Solution

See soil etract protocol for details.

Pse soil etract at a concentration of =.9 m2 per 3== m2 medium.

!o 'repare 2Se medium

Kor eample to make 9=== m2 Medium 6Se

Dn a sterile 9=== m2 Sc#ott !ottle add asepticall

sterile seawater (,)

7=== m2

sterile distilled water (#) 3=== m2

sterile 6Se concentrated nutrients (7) 3== m2

sterile soil etract solution (+) -9 m2

Mi. *#is medium is now read to !e decanted asepticall into sterile

culture asks.

Soil 45tract

Soil 'tract is an adaptation of '.6. "rings#eims !ip#asic soil@water medium

and is a component of some of our media. As t#e c#emical composition is

not well@de1ned and ma var from !atc# to !atc# its use in eperimental

situations is not recommended, #owever in our eperience certain specieswill not grow well wit#out it and soil etract is added for !ot# culture

maintenance and eperimental studies. An etensive list of modi1ed Soil

ater media developed for a range of microalgae from speci1c

environments can !e found on t#e P*'V we!site (*#e Culture Collection of

Algae at t#e Pniversit of *eas).


33/62

#ttp//www.ute.org/

CMA protocol

Soil must !e collected from a natural uncultivated environment or a ric#

garden loam ma !e suita!le. No fungicides, insecticides, garden fertiliBers

or fres# manure s#ould !e present. At CSD+$, topsoil from a local sand

!us#land environment #as proved to #ave particularl !ene1cial growt#

promoting properties. Soil from cla or ot#er soil tpes are less suita!le in

our eperience.

Soil s#ould not !e stored or processed in t#e algal culture la!orator, since

it is a potent source of unwanted microorganisms. Soil s#ould !e aged under

moist conditions (prefera!l for 5 mont#s or more) and t#en kept dr and

awa from lig#t.

!o 'repare Soil 45tract

,9 Sift dr soil (not recentl treated wit# fertiliBer or #er!icide) once t#roug#

a coarse sieve and twice t#roug# a 1ne sieve (3mm mes#).

#9 Mi 3 kg of soil into - litres of distilled water.

79 Autoclave for 5= minutes at 3-3[C and cool overnig#t.+9 Kilter t#roug# a!sor!ant cotton wool packed into t#e stem of a glass 1lter

funnel.

9Centrifuge at 9=== rpm for -= minutes in -9= ml polet#lene centrifuge

tu!es and collect t#e deep !rown supernatant.

=9 Kilter again t#roug# a!sor!ant cotton wool.

>9 ispense t#e supernatant (9= m2 aliquots) into 3== m2 Sc#ott !ottles or

3== m2 media !ottles.

89 Autoclave for 39 minutes at 3-3[C.

*9 After cooling, wrap caps wit# para1lm to prevent air!orne contamination

from fungal spores or !acteria.

,$9 Store sterile soil etract at 7[C (cold room or refrigerator).
http://www.utex.org/http://www.utex.org/


34/62

EM EaworskiFs Medium&

+eference Culture Collection of Algae and "rotoBoa (CCA") G Catalogue of

Strains 34 39.4 g9 '*A KeNa -.-9 g'*A Na- -.-9 g=9 0>%$> -.7< gMnCl-.70-$ 3.>4 g(N07)5M$8$-7.70-$ 3.== g>9itamins

Canoco!alamin

(itamin %3-)

=.=7 g

*#iamine 0Cl (itamin

%3)

=.=7 g

%iotin =.=7 g89 NaN$> @5>


35/62

Note Speci1c W stock solutions and stock solutions from ot#er media are

used in CMA+ to prepare t#is media. A concentrated working stock solution

is made up and used at a concentration of -m2 per 3==ml of seawater..

!o prepare ,$$ ml working stock 1 use at # m: per ,$$ m:

seawater&

Stock Solutions primar3 stock source 6olume

,9NaN$> 39= g / 2 0-$ f stock solution, use -.9

m2

#9 Na-glcero"$7 >.-7 g / 2 0-$ W stock solution, use 9 m2

79 Na-Si$>.40-$ --.8 g / 2 0-$ f stock solution, use -.9

m2

+9itamins f stock solution, use 9 m2

9*race Metals

KeNa'*A 7.> g / 2 0-$ W stock solution, use 9 m2

W@"rimar *race mi (see !elow) W stock solution, use 9 m2

Na-'*A.-0-$ >= g / 2 0-$ f' stock solution, use 5.-m2

=. *+DS 3== g / 2 0-$ W stock solution, use 5.=9

m2

>9 N07Cl -.5< g / 2 0-$ W stock solution, use 9.=

m2

89 0-Se$> 3.-4 mg / 2 0-$ 6Se stock solution, use 9.= m2

*9 istilled water 9-.89 m2

Kilter@sterilise t#roug# =.-- m sterile 1lter under aseptic conditions.

W@"rimar *race mi


36/62

CuS$7.90-$ 7.4 mg / 2 0-$

JnS$7.80-$ --.= mg / 2 0-$

CoCl-.50-$ 33.= mg / 2 0-$

MnCl-.70-$ 3 mg / 2 0-$

Add eac# ingredient to O89= m2 of distilled water, miing

t#ouroug#l !etween additions and t#en make up to 3 2.

All stock solutions are made up in distilled water.

Store all stock solutions in t#e refrigerator.

M(: Medium - Boods ;ole

+eference Nic#ols, 0. . (348>) in

&andbook o Phycolo"ical Methods,

'd. :. +. Stein, pp. 35@38. Cam!. Pniv.

"ress. (+. +. 2. 6uillard, personal

communication).

Adapted for fres#water Algae

Stock solutions "er 2itre distilled water

(d0-$),9 CaCl-.-0-$ >5.85 g#9 MgS$7.80-$ >5.48 g79 Na0C$> 3-.5= g+9 W-0"$7 .40-$ -9 Na-'*A 7.>5 g89 KeCl>.50-$ >.39 g*9 Metal Mi Add eac#

constituent

separatel to

CuS$7.90-$ =.=3 gJnS$7.80-$ =.=-- gCoCl-.50-$ =.=3 gMnCl-.70-$ =.3< g


37/62

O89=m2 of

d0-$, full

dissolving

!etween

aditions.

Na-Mo$7.-0-$ =.==5 g

,$9 itamin stockCanoco!alamin

(itamin %3-)

=.===9 g / 2d0-$

*#iamine 0Cl (itamin

%3)

=.3= g / 2d0-$

%iotin =.===9 g / 2d0-$,,9*ris stock -9=.= g / 2d0-$Store all stock solutions in t#e

refrigerator.

!o 'repare M(: Medium

Add 3m2 of eac# stock solution (, ? ,,) to 3 litre distilled water.

(Kor species w#ic# cannot use nitrate su!stitute 3m2 of N07Cl made up to

9.7 g /2 0-$)

Adust p0 to 8.- wit# 0Cl.


!o 'repare M(:/N(# Medium

Make M%2 medium as a!ove and add -.9 g $iod nutrient !rot# No. -!efore autoclaving.

M(: Medium - Boods ;ole1 CS0< working modi@cation


38/62

*#is recipe closel resem!les t#e oods 0ole M%2 Medium listed

previousl. 0owever it is made up using stocks from ot#er media used in

CMA+.

Stock Solution Source Add 6

,9 CaCl-.-0-$ stock solution =.< m2

#9 MgS$7.80-$ MMDD stock solution =.9 m2

79 Na0C$> 3-.5= g / 2@30-$ 3.= m2

+9 W-0"$7 6 stock solution =.- m2

9 NaN$> f stock solution =.9 m2

=9 Na-Si$>.90-$ f stock solution 7.9 m2

>9 Na-'*A f' stock solution =.39 m2

89 Ke citrate

Kerric citrate

Citric acid f stock solution 3.= m2

*9*race Metals f stock solution =.9 m2

,$9 itamin stock f stock solution 3.= m2

,,9*ris stock stock solution -.= m2

Store all stock solutions in t#e refrigerator.

Kor species w#ic# cannot use nitrate su!stitute 3m2 of N07Cl made up to

9.7 g / 2 0-$

Adust p0 to 8.- wit# 0Cl.

!o 'repare M(: Medium

Add eac# stock solution (3 G 33) in t#e volumes indicated to 3 litre distilled

water.


!o 'repare M(:/N(# Medium


39/62

Make M%2 Medium as a!ove and add -.9g $iod nutrient !rot# No. - !efore

autoclaving.

ME Medium Modi@ed EorgensenFs media for diatoms&

%ased on recipe o!tained from Pniversit of *asmania G 0armful Algae

Culture Collection

*#is media #as !een used

predominantl for t#e culture of Pseudonit*schia species and also in trials

of some 'haetocerosspecies w#ere !iomass and maintenance of normal

morp#olog seems to !e !etter t#an in ot#er media. "reparation of t#e

media is !ased on t#e utiliBation of M: and f@stock solutions.

Stock Solution Source Add 6 m:9:-,&

(a) Normal (!) concentrated

,9 NaN$> f stock solution (39=.= g / 2 ) -.= m2 -=.=

#9 W-0"$> M: stock solution

(make up at -.= g / 2 ) 9.= m2 9=

7. *race Metals D M: stock solution (see !elow) =.3 m2 3.=7. *race Metals DD M: stock solution (see !elow) 3.= m2 3=.=

9 itamin %3- f stock solution (3.= mg / 2 ) =.9 m2 9.=

=9 Na-Si$>.90-$ f stock solution (--.8 g / 2 ) 7.9 m2

79

>9 '*A M: stock solution (3= g / 2 ) 3.= m2 3=

Autoclave eac# stock solution ecept t#e itamin %3- w#ic# s#ould !e

=.-um 1lter sterilised.

(a) lists t#e normal stock volumes needed to prepare 32 of media. (!)

lists t#e volumes to prepare a concentrated nutrient solution as

descri!ed under "reparation !elow.


40/62

79 !race Metals 0

Na-'*A -.9 g / 20-$ Add t#e Na-'*A to

O89=m2 of d0-$ in a

volumetric ask and stir

over low #eat to dissolve.

Add eac# of t#e ot#er

constituents separatel to

O-== m2 of d0-$ and full

dissolve !etween

additions. *#en add t#is

second solution to t#e

Na-'*A and make up to 3

2.

CoCl-.50-$ 39= mg / 2

0-$CuS$7.90-$ -9= mg / 2

0-$JnS$7.80-$ -9= mg / 2

0-$

+9 !race Metals 00

KeS$7.80-$ >.= g / 2 Make up eac# constituent

separatel in O-== m2 of d0-$

and full dissolve. *#en

com!ine eac# solution and

make up to 3 2.

Citric acid >.= g / 20>%o> 3.9 g / 2MnCl-.70-$ 3.= g / 2

!o 'repare concentrated nutrient stock enough for ,$: media&

Add asepticall eac# of t#e prepared sterile stock solutions to 94 m2 of

sterile distilled water in a -== m2 Sc#ott !ottle (making total volume -==

m2).


41/62

*o make media, add -= ml of t#is 1nal nutrient solution per 3itre of

seawater.

M:A Medium

+eference %olc#, C. :. S. and %lack!urn S. D. (3445). Dsolation and

puri1cation of Australian isolates of t#e toic cano!acterium Microcystis

aeru"inosa W]tB.Journal o Applied Phycolo"y 8, 9@3>

M2A is derived from ASM@3 medium reported in 6or#am etal, (3457).

Dsolation and culture of toic strains of Anabaena fos-aquae (2ng!.) de

%r^!. +erh. int. +er. )i!nol ," 845@5 g (add 1rst _ stir on

low #eat to full dissolve)KeCl>.50-$ 3.9< gNa0C$> =.5= gMnCl-.70-$ =.>5 gt#en add 3=m2 of t#e following primar stocks (eac# made


42/62

up separatel)"rimar Stocks (per 2itre d0-=)CuS$7.90-$ 3.= g.JnS$7.80-$ -.- g.CoCl-.50-$ 3.= g.

Na-Mo$7.-0-$ =.5 g.

Kinall, make up t#e micronutrient stock to 3 litre wit#

distilled water

Df precipitate forms increase p0 up to 8.

(Df precipitation !ecomes an issue t#en replacing t#e two

sulp#ate stocks wit# equimolar amounts of t#e trace metal in

t#e c#loride form #as proven usefulU %en 2ong, pers comm)

89 Na0C$> 35.4 g*9 CaCl-.-0-$ -4.7 gStore all stock solutions in t#e refrigerator.

M:A Medium 'reparation Methods

!here are + components as

follows1

,9 istilled Bater

Autoclave to sterilise

#9 !o 'repare M:A Medium 5+$

concentrated nutrients #$m:

volume&

*o 3>=m2 distilled water add

MgS$7.80-$ 3= m2NaN$> -= m2

W-0"$7 9= m2

0>%$> 3= m2

0-Se$> 3= m2

itamin stock 3= m2


43/62

Micronutrient stock 3= m2

Kilter sterilise using a =.-- m 1lter

into a sterile -9= m2 Sc#ott !ottle.

79 Na0C$> 35.4 g / 2 0-$

Autoclave to sterilise.

+9 CaCl-.-0-$ -4.7 g / 2 0-$

Autoclave to sterilse.

!o 'repare M:A Medium

Kor eample to make 3=== m2 M2A Medium

Dn a sterile 3=== m2 Sc#ott !ottle add asepticall

sterile distilled water (,)

457 m2

sterile M2A 7= concentrated nutrients (#)

-9 m2

sterile Na0C$> (7)

3= m2

sterile CaCl-.-0-$ (+)

3 m2

Mi well after eac# addition.

*#is medium is now read to !e decanted asepticall into sterile culture

asks.

!o 'repare M:A Medium ull3 Autoclaved&

Kor aenic cultures. *#e media is essentiall t#e same !ut due to t#e

autoclaving process t#e Na0C$> concentration is adusted.

Kor eample to make 3=== m2 autoclaved M2A Medium add


44/62

distilled water ,& 48> m2

sterile M2A 7= concentrated nutrients #&

-9 m2

sterile Na0C$> 7& 3 m2

sterile CaCl-.-0-$ +& 3 m2

Adust p0 to 8.9 to , as t#is can

improve survival of cano!acteria on solid media ("arker, 34(3-.5 g/2)is sterilised ! autoclaving. *#is is t#en added

asepticall ust prior to pouring to give a 1nal concentration =.3 mM Na-S$>

(i.e. 3 ml / 2 of Na-S$>stock).

Agar @ 1nal concentration of 3= g / 2(3R)

@ preferred media for Nodularia, !ut alwas use #ig#l puri1ed agar e.g.

ifco %acto puri1ed agar.

Agarose @ Kinal concentration of 9 g / 2(=.9R)

@ preferred media for Microcystis andAnabaena. (alt#oug# t#e latter grows

onl ver slowl, or not at all on solid media). *o prepare 9== ml of non@saline solid M2A medium

two components are mied, eac# -9= mls and dou!le@strengt# @

Add t#e gelling agent (9 g agar or -.9 g agarose) to -9= ml M?

water in a 9== ml Sc#ott !ottle wit# a magnetic stirrer.


45/62

Add 3-.9 mls of 1lter@sterile 7= concentrate to -9= ml sterile M?

water in a -9= ml Sc#ott !ottle. (full strengt# I -9ml/2 in liquid

media)

Autoclave gelling agent, and dou!le@strengt# nutrients.

Kinal steps detailed !elow

*o prepare 9== ml of saline solid M2A medium

t#ree components are mied, eac# 358 mls and triple@strengt# @

Add t#e gelling agent (9 g agar or -.9 g agarose) to 358 ml

M? water in a 9== ml Sc#ott !ottle wit# a magnetic stirrer.

Add 3-.9 mls of 1lter@sterile 7= concentrate to 358 mls M?water.

Autoclave t#e gelling agent, t#e triple@strengt# nutrients and

358 mls of triple strengt# saline

@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@

Kinal steps in preparation of solid media

Cool to 79 =C in a water !at#, and in t#e same !at#, warm t#e CaCl-and

Na0C$>and if appropriate Na-S$>. $n a magnetic stirrer plate in t#e laminar ow, place t#e !ottle

containing t#e gel. Stir gentl w#ile asepticall adding nutrients (and for

saline media, adding seawater also).

Asepticall add 3 ml eac# of vitamins and CaCl- and if appropriate

Na-S$>, and 3= ml of Na0C$>.

"our media into sterile petri plates or McCartne !ottles```.

r agar/agarose plates in t#e laminar ow for -= minutes prior tostoringU and/or place McCartnes in a !asket on a s#arp angle until slopes

are solid.

@@@@@@@@@@@@@@@@@@@@@@@@@@@
http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#MLAx40http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#MLAx40http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#MLAx40http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#MLAx40


46/62

```Df using a dispenser, and in case pro!lems arise during dispensing t#e

solid media, it is advised to keep some #ot sterile water for rinsing t#e

dispenser asepticall.

Mineral Medium 00

+eference 0ug#es, '. $., 6or#am, ".

+. and Je#nder, A. (349.50-$ =.-4 g0>%$> 5.


47/62

second solution to

t#e Na-'*A andAdust p0 to 8.< @


48/62

!o 'repare Mineral Medium 00

Add eac# stock solution (, ? *) to 49=

m2 distilled water.


'orph3ridium Medium

Medium for the heterotrophic

dinoHagellate Crypthecodinium

cohnii9

+eference Starr, +. C. and Jeikus, :. A. (344>). P*'V @ *#e Culture

Collection of Algae at t#e Pniversit of *eas at Austin.J. Phycol. #* (-)

p47. ('.6. "rings#eim, pers. comm.)

!o prepare medium1

Kor eac# 9==ml of medium required com!ine

distilled water -== ml

itered seawater -9= mleast etract =.9 g

trptone =.9 g

autoclace to sterilise

add asepticall 9= ml sterile soil etract.


49/62

3 lagU - eponentialU > declining growt# rateU 7 stationarU 9 deat#. 'ac# of

t#e p#ases is descri!ed !elow and in Kig 3 (goto 1g). Alternativel :ump

straig#t to growt# rate equation

:ag phase

*#e condition of t#e innoculum #as a strong !earing on t#e duration of t#e

lag p#ase (Spencer, 3497). An innoculum taken from a #ealt# eponentiall

growing culture is unlikel to #ave an lag p#ase w#en transferred to fres#

medium under similar growt# conditions of lig#t, temperature and salinit. Dn

general t#e lengt# of t#e lag p#ase will !e proportional to t#e lengt# of time

t#e innoculum #as !een in p#ases >@9. A lag p#ase ma also occur if t#e

innoculum is transferred from one set of growt# conditions to anot#er.

45ponential phase and calculating growth rates

*#e growt# rate of a microalgal population is a measure of t#e increase in

!iomass over time and it is determined from t#e eponential p#ase. 6rowt#

rate is oneimportant wa of epressing t#e relative ecological success of a

species or strain in adapting to its natural environment or t#e eperimental

environment imposed upon it. *#e duration of eponential p#ase in culturesdepends upon t#e siBe of t#e innoculum, t#e growt# rate and t#e capacit of

t#e medium and culturing conditions to support algal growt#. %iomass

estimates need to !e plotted over time, and logistical constraints determine

t#eir frequenc !ut once ever one to two das is generall accepta!le. Cell

count and dr weig#t are common units of !iomass determination. n-#i#o

uorescence and tur!idit can !e used as surrogate measures w#ic# ena!le

#ig#er temporal resolution due to t#e logistical ease of measurement

(correlations !etween uorescence or tur!idit and cell count can !e

esta!lis#ed !ut t#e will !ecome less accurate as eperimental conditions

are varied. Kor eample cell uorescence ma var wit# temperature so an

eperiment wit# several test temperatures ma need correlations to !e

determined for eac# temperature. Correlations also !ecome innacurate as
http://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#batchgrowthfighttp://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#equationhttp://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#equationhttp://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#batchgrowthfighttp://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#equationhttp://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm#equation


50/62

cultures move into stationar p#ase so uorescence can not !e used as a

su!stitute for cell counts w#ere an estimate of 1nal cell ield is needed).

$nce t#e growt# p#ase #as !een plotted (time on @ais and !iomass on

logart#mic @ais) careful determination of t#e eponential (straig#tline)

p#ase of growt# is needed. *wo points, N3 and N-, at t#e etremes of t#is

linear p#ase (see 1g !elow) are taken and su!stituted into t#e equation

2rowth rate ) GI J :n N# / N,& / t# - t,&

#ere N3 and N- I !iomass at time3 (t3) and time- (t-) respectivelU

2evasseur et al(344>).

ivisions per da and t#e generation or dou!ling time can also !e calculated

once t#e speci1c growt# rate is known.

ivisions per da3 ) iv9da3-,J GI / :n#

2eneration time ) 2enI t J , / iv9da3-,

Kor #ealt# cells of a ro!ust species, small innoculums equal to =.9 R of t#e

volume of t#e new culture will normall generate new #ealt# cultures. Df

t#e species is delicate or t#e culture less #ealt# t#en a larger innoculum of

O 3=R ma !e needed to support a new culture. (Man of t#e stock culturesin CMA+C are transferred wit# a =.9 to 3 m2 innoculum into 7= m2 fres#

medium representing a 3.-9 R to -.9R innoculum).

eclining growth

eclining growt# normall occurs in cultures w#en eit#er a speci1c

requirement for cell division is limiting or somet#ing else is in#i!iting

reproduction. Dn t#is p#ase of growt# !iomass is often ver #ig# and

e#austion of a nutrient salt, limiting car!on dioide or lig#t limitation

!ecome t#e primar causes of declining growt#. #en !iomass is increasing

eponentiall a constant suppl of air (or air plus C$-) will onl !e in !alance

wit# growt# at one point during eponential p#ase. At low cell densities too

muc# C$-ma lower t#e p0 and depress growt#. C$-limitation at #ig# cell


51/62

densities causes an furt#er !iomass increase to !e linear rat#er t#an

eponential (wit# respect to time) and proportional to t#e input of C$-.

2ig#t limitation at #ig# !iomass occurs w#en t#e cells a!sor! most of

t#e incoming irradiation and individual cells s#ade eac# ot#er (#ence t#e

often quoted term Eself@s#adingF). 6rowt# in most p#toplankton is

saturated at relativel low irradiances of 9=@-== Zmol. p#otons m @- s G3 (cf

noontime irradiance at t#e water surface in t#e tropics of -=== Zmol.

p#otons m@- s G3 ). Microalgae are t#erefore generall well adapted to

surviving conditions of low incident lig#t and ma survive for etended

periods under t#ese conditions.

Stationar3 phase

Cultures enter stationar p#ase w#en net growt# is Bero, and wit#in a matter

of #ours cells ma undergo dramatic !ioc#emical c#anges. *#e nature of t#e

c#anges depends upon t#e growt# limiting factor. Nitrogen limitation ma

result in t#e reduction in protein content and relative or a!solute c#anges in

lipid and car!o#drate content. 2ig#t limitation will result in increasing

pigment content of most species and s#ifts in fatt acid composition. 2ig#t

intensities t#at were adequate or optimal for growt# in t#e 1rst > p#ases cannow !ecome stressful and lead to a conditon known as p#otoin#i!ition. Dt is

important t#at w#ile t#e measured lig#t intensit wit#in t#e culture will

decrease wit# increasing !iomass if t#e incident illumination is maintained

relativel #ig# t#en a large proportion of cells ma !ecome stressed,

p#otoin#i!it and t#e culture can !e pus#ed into t#e deat# p#ase. *#is is

especiall t#e case if t#e culture is also nutrient stressed. Dt is prefera!le for

man species to #alve or furt#er reduce t#e incident lig#t intensit w#en

cultures enter stationar p#ase to avoid p#otoin#i!ition. Some green algae

and cano!acteria ma survive in t#e vegetative state (ie not as csts) for

over 5 G 3- mont#s under ver low illumination. Kor man species lower

temperature com!ined wit# lower irradiance can furt#er reduce stress.

Survival is inversel proportional to temperature !ut onl in darkness. Some


52/62

algal species ma form long lived csts or temporar resting csts wit#

greatl reduced meta!olism under di&erent conditions of stress. *#e s#ut

down of man !ioc#emical pat#was as stationar p#ase proceeds means

t#at t#e longer t#e cells are #eld in t#is condition t#e longer t#e lag p#ase

will !e w#en cells are returned to good growt# conditions.

eath phase

#en vegetative cell meta!olism can no longer !e maintained t#e deat#

p#ase of a culture is generall ver rapid, #ence t#e term Eculture cras#F is

often used. *#e steepness of t#e decline is often more marked t#an t#at

represented in t#e accompaning growt# 1gure. Cultures of some species

will lose t#eir pigmentation and appear was#ed out or cloud, w#ereas cells

of ot#er species ma lse (no recogniBa!le cells) !ut t#e culture colour will

!e maintained. *#e latter is an important consideration and one reason w#

colour s#ould not !e relied upon to guage culture #ealt#. %acteria w#ic#

ma #ave !een kept in c#eck during eponential and earl stationar p#ase

ma EeplodeF as cell mem!rane integrit !ecome progressivle

compromised or leak and a ric# car!on source for !acterial growt# is

released. Kree pigment and !acterial growt# are furt#er reasons w#measures of tur!idit or uorescence s#ould not !e used !eond earl

stationar p#ase as surrogate !iomass indicators, or especiall as indicators

of culture #ealt#. $ccassionall cell growt# of some species can reoccur

after a culture #as apparentl died. Dn t#is instance most vegetative cells will

#ave died, and possi!l most of t#e !acteria, releasing nutrients !ack into

t#e media. *#en eit#er t#e ver few remaining vegetative cells or more

likel germination of csts or temporar csts will !e a!le to fund t#is

secondar growt#.


53/62

Kig 3 6eneral pattern of microalgal growt# in !atc# cultures


54/62

Speci1c

6rowt#

rate

ivisions

per da

6eneration time I

ou!ling time

Speci1c

6rowt#

rate

ivisions

per da

6eneration time

I ou!ling timeW iv.da@3 das #ours W iv.da@3 das #ours=.3= =.377 5.4>3 355.>5 3.=9 3.939 =.55= 39. 37.78=.-9 =.>53 -.88> 55.97 3.-= 3.8>3 =.98< 3>.= =.7>> -.>3= 99.79 3.-9 3. =.999 3>.>3=.>9 =.9=9 3.4 3.>= 3.> 3-.> 73.94 3.>9 3.47< =.93> 3-.>-=.79 =.574 3.97= >5.48 3.7= -.=-= =.749 33.>.-8 3.79 -.=4- =.78< 33.78=.99 =.84> 3.-5= >=.-9 3.9= -.357 =.75- 33.=4=.5= =. 3.99 -.->5 =.778 3=.8>=.54 3.=== 3.=== -7.== 3.5= -.>=< =.7>> 3=.7==.59 =.4>< 3.=55 -9.94 3.59 -.>.88 3.8= -.79> =.7=< 4.84=.89 3.=45 4.93=.


55/62

Cell counting using a ;aemac3tometer

eference S. Sc#oen ECell CountingF in C.S. 2o!!an et al (34


56/62

consists of two c#am!ers, eac# wit# a volume of =.3mm>, containing a

marked counting grid 3mm- in area .

(e e5tremel3 careful when handling haemac3tometers as the3 are

fragile if dropped and ver3 e5pensive items of equipment costing up

to KAus #$$9$$ each

Method

,9 Algal Sample G Non@motile cells w#ic# do not need 1ing can !e

counted as soon as t#e sample is collected. 0owever, if t#ere will !e a dela

!etween sample collection and counting, or if t#e cells are motile t#en t#e

sample will need to !e preserved. *#e most common 1ative used for

marine microalgae is 2ugolbs Solution. *#e recipe for t#e acidic form is given

#ere !ut note in t#e case of microalgae wit# calcium car!onate scales, suc#

as t#e coccolit#op#orids, t#e acid will destro t#e organisms and a !asic

solution s#ould !e prepared instead.

Kor cultures add 3 drop of 3@-R 2ugols solution to 3 ml sample, for 1eld

samples 3= drops per -== m2 of sample or until t#e colour of weak tea.$veruse of 2ugolbs will cause some delicate agellate species to overstain,

lose agella or !low up entirel.

2ugolbs is made ! dissolving 3== g "otassium Dodide (WD) in 32 of

distilled water, t#en 9= g crstalline iodine (D-) is dissolved in t#is solution

and t#en3== ml glacial acetic acid is added. 2ugolbs s#ould !e stored in t#e

dark as t#e iodine is lig#t sensitive and will degrade. Dt s#ould also !e stored

wit# a tig#t 1tting lid and kept awa from t#e general culture environment.

Note1 Sample dilution or concentration *#e #aemactometer can !e

used w#ere cell densities are in t#e range 9 3=7@ 3=8cells / m2. Dt is more

likel t#at cultures will !e less rat#er t#an more dense t#an t#is range !ut

occasionall ver dense cultures, suc# as nanoplanktonic agellates and


57/62

some cano!acteria, ma need to !e counted. Dt is !ot# ine;cient and ver

di;cult to accuratel count t#ese cultures wit#out 1rst diluting t#e sample

(contrast wit# t#e Sedgwick@+after Cellw#ere cell densit range is >= G 3=7

cells / m2). *#erefore dilute wit# a known volume of culture media and t#en

1. Alternativel a 2ugols ilution Solution (2S) ma !e used w#ere 3== m2

of =.-m 1ltered seawater or distilled water (depending on w#et#er t#e

culture is marine or fres#water) is prestained wit# concentrated 2ugols until

it is a weak tea colour. *#en a known volume of culture can !e added to a

known volume of t#e 2S. Psing 2S also means t#at all cells are eposed

to t#e optimum concentration of 2ugolbs w#ereas adding concentrated

2ugolbs could destro some cell tpes w#en it mies into t#e sample. Kor

eample a culture wit# a densit a!ove 3 3=8cells / m2 will #ave L3===

cells in t#e counting region of a #eamoctometer (see detailed eplanation

!elow) and a 3 in 9 dilution (3 m2 of culture 7 m2 of 2S) will allow t#e

sample to !e counted more readil.

Df t#e culture is dilute, concentrate ! centrifuging or settling in a at

!ottom measuring clinder (allow 3 #our of sinking for eac# 3=mm of

clinder #eig#tU t#erefore overnig#t is a practical solution). Kor eit#er met#od

once concentrated, remove and discard up to 4=R of t#e clear supernatant

(upper portion of t#e liquid) wit#out distur!ing t#e settled !iomass.

0omogeniBe t#e remaining sample and count, !earing in mind t#e need to

integrate t#e concentration factor I 1nal count (settled volume / initial

volume).

#9 *o 1ll #aemactometer c#am!ers, place t#e t#ick coverglass over !ot#

grids and take a "asteur pipette and 1ll its tip ! capillar action wit#sample. 0old t#e pipette at an angle of O79=(#ig#er or lower to control ow

rate) and place t#e tip at t#e leading edge of t#e coverslip. it# ver gentle

pressure, allow t#e sample to ow quickl and evenl into t#e c#am!er,

eactl 1lling it. *#e c#am!er surface in t#e Neu!auer !rand is a at mirror@

like rectangle and t#e sample must cover t#is rectangle !ut not ow over its
http://www.marine.csiro.au/microalgae/methods/Sedgwick%20Rafter%20counting.htmhttp://www.marine.csiro.au/microalgae/methods/Sedgwick%20Rafter%20counting.htm


58/62

edges. Dt is useful to rest our #and on a !enc# and stead t#e pipette tip

wit# a 1nger.

Df ooding occurs, rinse #aemactometer and coverslip wit# distilled

water, and repeat procedure.

+e1ll t#e pipette for eac# c#am!er. *#e time taken to 1ll t#e c#am!er

s#ould !e s#ort, to minimiBe setting of cells in t#e pipette.

79 Allow cells to settle (O3min) and c#eck grid under t#e microscope (

-= o!ective) for satisfactor distri!ution of cells, i.e. evenl spread.

+9 !he ;aemac3tometer grid in detail

*#e grid is divided into 4 large squares, eac# 3mm 3mm, ! triple

lines.

'ac# large square is divided into -9 medium squares, eac# =.->mm on a

side, and eac# medium square is furt#er divided into 35 small squares, eac#

=.=9mm on a side.


59/62


60/62

Kor all #aemactometers, t#e fundamental measurement is t#e average

num!er of cells per 3mm square, so t#e centre large square is usuall

counted. *o o!tain t#e total num!er of cells in t#is large square, t#e num!er

of cells in eac# of t#e -9 medium squares are counted, recorded t#en added

(see sample cell count)

Note #en counting cells !ordering on triple rulings, t#e convention is to

count onl t#ose cells touc#ing t#e top and left@#and side rulings of eac#

square.

9 After counting eac# of t#e two #aemactometer c#am!ers, t#e

#aemactometer and coverslip are rinsed wit# distilled water. Psuall t#e

procedure is repeated twice more to give a total of 5 counts.

=9 *o o!tain t#e cell densit, calculate t#e average cell

count and multipl ! t#e conversion factor (for Neu!auer I 3=7)

Sample Cell Count

sochrysis sp. (*a#itian) CS@388AgeU < das

6rowt# conditionsU -9=C, lig#t intensit 9=Zmol. p#otons m@-s G3, 3-3- lig#t

dark ccle

, > 9 3- < 5 # 7 8 < 3= 5 5 9 5 < 9 3- 5 7 < 7 5 < 7 < 9 < 8 7 7 9 37 5 < 9 4 3= 4 5 3> 7 7 3= 8 < 4 8 33 3> 5 3> *otal no. I 389 *otal no. I 3 7 8 7 > 5 < 3= 3= > > 9 5 5 9 4 5 4 < > 4 5 3= 5 4 8 7 > > 8 4 3= 4 39


61/62

9 3- 3- < < 3= - < > 8 *otal no. I 3 = 9 9 5 5 9 < 9 9 7 5 9 3> 9 - 4

4 8 7 4 33 < < < 5 > 7 5 8 3= 9 < 5 5 8 - 4 3= 9 5 4 9 5 3= 5 39 *otal no. I 387 *otal no. I 359

'ac# !lock 3@5 represents t#e total num!er of cells in t#e large centre

square.


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Mean I

#ere I total no.

I sum of all totals

_ nI no. of counts

Cell CountI ccf (c#am!er conversion factor for Neu!auer I 3=7)

Standard deviation SI w#ere variance I

I

R 'rror I

from a!ove eample

cell count UI 387.> 3=7 ccf

I 3.87 3=5 cells/m2

standard deviation II

I4.44

Rerror I 3== I 9.8R

Note Rerror s#ould !e !elow 3=R

australian national algae culture collection-making media

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