Phytoplankton Measuring and Culture Techniques

Phytoplankton Ecology; 24 April 2007; K!

"# $ield !a%pling
A. How do we collect phytoplankton?
1. Water sampling bottles at discrete depths
a. Examples of gear: Nansen, Nisken, Van Dorn, Kimmerer
b. Advantages
(1 !olle"ts all p#$toplankton (in"l%ding nanoplankton
(& !an determine detailed verti"al profiles if take samples at different dept#s
(' (%antitative ("an sample kno)n vol%me
". Disadvantages
(1 Need man$ samples to get a verti"al profile
(& *a$ miss p#$toplankton in bet)een sampling points (and t#in la$ers of
p#$toplankton are fo%nd in man$ lakes
(' +amples often need to be "on"entrated for "o%nting (settling
d. !aveats,variations - "an %se verti"al profilers (e.g., meas%res of in vivo
fl%ores"en"e on samplers to determine )#ere to "olle"t dis"rete samples so don.t
miss p#$toplankton la$ers.
2. Integrated water column sampling
a. Examples of gear: !oli)asa ("ol%mn integrated )ater sample, t%be )it# pl%g
(/snake., p%mp and t%be
b. Advantages
(1 !olle"ts all p#$toplankton (in"l%ding nanoplankton
(& !olle"ts p#$toplankton from all dept#s
(' (%antitative (sample kno)n vol%me
(0 Need fe)er samples to represent p#$toplankton at man$ dept#s
". Disadvantages
(1 1ose verti"al resol%tion
(& 2ften onl$ "an sample top 13 m at most )it# t#is met#od (good for s#allo)
lakes or epilimnion samples in man$ lakes
(' +amples often need to be "on"entrated for "o%nting
3. Phytoplankton nets
a. 4ear: fine mes#ed (often 13 m p#$toplankton nets
b. Advantages
(1 !on"entrates large algae, so often settling isn.t needed to identif$ samples
(& Easil$ deplo$ed from s#ore (tossing or boats
". Disadvantages
(1 A large 5 of p#$toplankton is small eno%g# to pass t#ro%g# even t#e finest
mes# nets (more t#an 635 b$ biomass and often more b$ prod%"tivit$ - so
t#is is not a good 7%alitative meas%re of p#$toplankton
(& 8#e vol%me of )ater passing t#ro%g# t#e net is #ard to meas%re - it.s #ard to
get good flo) meter reading and net "logs, "#anging effi"ien"$ - so is not
possible to get a 7%antitative meas%re
(' 9or"e of to)ing "an break %p and destro$ some larger algae
(0 2n"e t#e standard for p#$toplankton "olle"tion (before 1:63 p#$toplankton
nets are rarel$ %sed no) ex"ept to "olle"t /net. plankton for demonstration
1
p%rposes or to isolate for "%lt%ring (not t#e best met#od for t#at eit#er d%e to
"ell damage
. How do we preser!e phytoplankton "or counting?
1. #on$t preser!e% count "resh
a. Advantages
(1 Don.t lose "olor, a good diagnosti" for division level identifi"ation
(& Don.t lose motilit$ - "an also #elp )it# identifi"ation
b. Disadvantages
(1 Need to do rig#t a)a$ or algae )ill die or gro), so "o%nts )ill be ina""%rate
&. &ugol$s solution (13 g ;
&
and &3 g K; in &33 m1 distilled )ater and &3 m1 gla"ial
a"eti" a"id< %se = 1 drop - 3.36 m1 per 6 m1 sample
a. Advantages
(1 Adds )eig#t to "ells and #elps p#$toplankton "ells sink to bottom of settling
"#amber (see belo)
(& ;s not ver$ toxi"
(' ;s a relativel$ good preservative for bot# /soft. and lori"ate algae
b. Disadvantages
(1 >reaks do)n in s%nlig#t - need to keep samples and sto"k sol%tion in t#e
dark< if storing long term need to replenis# l%gols in samples periodi"all$
(& 8%rns t#e "ells bro)n, making it #arder to identif$ t#em to division (no "olor
"%es. 8#is is not a problem )it# algal "%lt%res, )#ere $o% kno) )#at algae
are t#ere and are ?%st en%merating, so is good for t#at %se. !an also en#an"e
"ontrast %nder lig#t mi"ros"op$, )#i"# "an aid in ;mage Anal$sis.
3. u""ered glutaraldehyde
a. Advantages
(1 @reserves m%"# of t#e "olor
b. Disadvantages
(1 ;s 7%ite toxi"
'. How do we concentrate algae "or counting?
1. Phytoplankton nets (see above ;.A.'
2. (edimentation chambers
a. Examples: Utermhl chambers< modified sedimenting "#ambers
b. Ao) t#e$ )ork
(1 +ettle all algae in a kno)n vol%me of )ater (settle b$ gravit$ - need to settle '
#o%rs for ever$ 1 "m #eig#t to allo) sinking of all "ells
(& Eit#er "o%nt on t#e "over slip (on bottom of BtermC#l "#amber t#ro%g# an
inverted s"ope - )ater "an limit magnifi"ation to =633D< or )it# spe"ial
sedimenting "#amber "an "o%nt on eit#er inverted or "ompo%nd s"ope
". Advantages
(1 Eelativel$ eas$ to %se
(& +tandard met#od sin"e 1:63s. 1ots of "omparable data )it# t#e same or
similar proto"ols
d. Disadvantages
(1 !an be #ard to find large vol%me "ells (e.g., 63 ml ne"essar$ to "on"entrate
t#e lo)er n%mber of "ells per vol%me from oligotrop#i" lakes
&
(& 2ften "#ambers are expensive and $o% "an onl$ settle a fe) samples at a time
(' +ome set%ps don.t allo) $o% to make permanent slides for re"ord keeping
(t#is is less important )it# t#e advent of digital "ameras t#at "an be %sed to
keep a permanent re"ord of fields of vie)
'. (ettling chambers with remo!al o" water - "an keep preserved samples
a. Advantages
(1 !an "on"entrate #ig#er vol%mes of )ater
(& !an preserve t#e "on"entrated "ells for f%t%re %se
(' !an %se alternate "o%nting tra$s (not ?%st BtermC#l "#ambers for "o%nting
"ells (not restri"ted to inverted mi"ros"opes
(0 2ften is "#eap to "on"entrate man$ "ells at on"e
b. Disadvantages
(1 Need to remove s%rfa"e )ater "aref%ll$ (gentle aspiration to avoid
res%spending "ells.
(& A fe) "ells ma$ be lost in transfer from t#e settling "#amber if not "aref%l
". 8ip: Add a drop of s%rfa"tant (soap to s%rfa"e of material to be settled, so as to
release an$ p#$toplankton st%"k in t#e s%rfa"e film
0. 'oncentration by water displacement - remove )ater from sample and leave algae
b$ gl%ing a fine mes# over a plasti" t%be, and s%bmerging t#e t%be gentl$ into t#e
sample. !an remove )ater in t%be b$ aspiration (as above. Determine vol%me of
"on"entrate after removal of )ater to "al"%late t#e "on"entration fa"tor.
a. Advantages
(1 !an "on"entrate #ig#er vol%mes of )ater t#an BtermC#l
(& !an preserve t#e "on"entrated "ells for f%t%re %se
(' !an %se alternate "o%nting tra$s (not ?%st BtermC#l "#ambers for "o%nting
"ells
b. Disadvantages
(1 Need to remove s%rfa"e )ater "aref%ll$ (gentle aspiration to avoid
res%spending "ells.
(& +ome small p#$toplankton ma$ pass t#ro%g# mes#, biasing sample
(' *ore time "ons%ming t#an met#od '
(0 A fe) "ells ma$ be lost in transfer from t#e settling "#amber if not "aref%l
). 'entri"ugation
a. Advantages
(1 (%i"k
(& !#eap if $o% #ave a "entrif%ge
b. Disadvantages
(1 2ften doesn.t "entrif%ge o%t all algae - not a good 7%antitative te"#ni7%e
(& +ome fragile algae ma$ l$se - not a good 7%alitative te"#ni7%e
(' +ometimes #ard to get all algae o%t of "entrif%ge t%be so t#at t#e sample
remains 7%antitative
(0 Earel$ %sed no) as a te"#ni7%e for pro"essing field samples
#. How do we estimate phytoplankton biomass "rom "ield samples?
1. 'hlorophyll a
a. F#at it tells $o%
(1 a ro%g# estimate of algal biomass
'
b. Advantages
(1 relativel$ 7%i"k
(& eas$
(' relativel$ inexpensive
(0 long re"ords of "#lorop#$ll a val%es for "omparison
(6 %nlike seston "arbon, doesn.t in"l%de nonGp#$toplankton "omponents of
seston (e.g., Hooplankton or soilGderived detrit%s, inorgani" "arbon
". Disadvantages
(1 "an.t tell t$pe of p#$toplankton (all #ave "#l. a
(& "#lorop#$ll per "ell and per "arbon varies )it# man$ ot#er fa"tors in"l%ding
lig#t "limate, t$pe of p#$toplankton(Ee$nolds dis"%sses t#is
d. *et#ods for meas%ring "#lorop#$ll a
(1 9l%orometr$
(a in vivo fl%ores"en"e -
i. Advantages - "an be done 7%i"kl$ and "an get "ontin%o%s profiles
bot# verti"all$ )it# lo)ered instr%ments and #oriHontall$ )it# p%mped
samples )#ile "r%ising
ii. Disadvantages GG often not a ver$ good estimator of extra"ted
"#lorop#$ll a be"a%se t#e relations#ip bet)een in vivo "#lorop#$ll a
and biomass varies )it# algal taxon, "ell "ondition (#o) m%"# energ$
a"t%all$ gets "apt%red b$ "ell vs. fl%ores"ing, lig#t "onditions, et".
iii. !aveats: Need to "alibrate ea"# sampling date,site )it# some
extra"ted "#lorop#$ll estimates
(b Extra"ted "#lorop#$ll - filter )ater sample onto membrane (often 3.& or
3.06 m or glass fiber (I3.J m filters< extra"t into solvent (varies
depending on met#od %sed - :35 b%ffered a"etone is most "ommon no)<
some met#ods re7%ire grinding of filters
i. Advantages - ver$ a""%rate means of determining "#lorop#$ll a in
seston< relativel$ eas$< "an dete"t lo) levels of "#lorop#$ll )#en
sample is dil%te< before extra"tion filtered samples "an be stored for a
fe) )eeks in %ltraGfreeHer (G&3 to GK3
o
! or a fe) da$s in freeHer
ii. Disadvantage - is some time involved in extra"tion and "ost in
b%$ing filters - "an.t get as m%"# spatial,temporal detail as )it# in
vivo fl%ores"en"e
(& +pe"trop#otometr$
(a Advantages
i. A""%rate
ii. !an estimate p#aeop#$tins (degraded "#lorop#$ll and ot#er
pigments (see belo)
iii. before extra"tion filtered samples "an be stored for a fe) )eeks in
%ltraGfreeHer (G&3 to GK3
o
! or a fe) da$s in freeHer
(b Disadvantage
i. Ee7%ires more "on"entrated sample (ma$ need to "olle"t and filter
lots of )ater in an oligotrop#i" lake
ii. ;s some time involved in extra"tion and "ost in b%$ing filters -
"an.t get as m%"# spatial,temporal detail as )it# in vivo fl%ores"en"e
(' +atellite and ot#er remote sensing
0
(a Advantages -
i. !an get #%ge spatial s"ales (#oriHontal
(b Disadvantages
i. +till need gro%nd tr%t#ing )it# season and lake
ii. Expense and availabilit$ of images
iii. 1ose verti"al resol%tion
iv. Need large )ater bod$
&. (eston 'arbon
a. F#at it tells $o% - amo%nt of "arbon in seston
b. Advantages
(1 !an be relativel$ 7%i"k and eas$
(& !arbon is often a basi" "%rren"$ for food )eb and biogeo"#emi"al st%dies
". Disadvantages
(1 Need to "olle"t and filter )ater samples for met#od (1
(& !an in"l%de detrital "arbon or inorgani" "arbon depending on met#od
(sometimes not a problem, sometimes s%bstantial - e.g., in small e%trop#i"
lakes
d. *et#ods
(1 Elemental anal$Her (EA - take filter and p%t in tin boat. EA "omb%sts and
red%"es to "arbon and nitrogen gas - get ! and N "ontent of seston
(a Advantages
i. +imple
ii. 4et "arbon and nitrogen "ontent
iii. Dire"t meas%re of seston
(b Disadvantages
i. !an in"l%de detrital "arbon or inorgani" "arbon (if treat )it# a"id
f%mes $o% "an eliminate inorgani" ! "ontamination
ii. Need an elemental anal$Her
iii. !ost of filters and r%nning samples
iv. @ro"essing time of filtering, preparing and r%nning samples
(& Estimate from: ("ell "o%nts for ea"# taxon D ("ell vol%me per taxon D ("ell
densit$ for ea"# taxon
(a Advantages
i. Estimates "arbon in algae onl$ (not detrit%s
ii. 4et spe"iesGspe"ifi" "arbon estimates
(b Disadvantages
i. ;n"redibl$ time "ons%ming
ii. Errors involved in "ell "o%nts, biovol%me estimates and "ell
densit$ "ompo%nd in t#is "al"%lation - $o% are making lots of
ass%mptions #ere (see met#ods belo) for "ell "o%nts and for
biovol%me
iii. 4enerall$ "ell densities are taken from literat%re, not meas%red for
t#at st%d$
E# &o' do 'e esti%ate phytoplankton co%position (ro% (ield sa%ples)
1. *icroscope counts+ identi"ication+ and biomass
a. Advantages
6
(1 8ells $o% exa"tl$ )#o.s t#ere
(& Lo% "an de"ide on level of taxonomi" resol%tion - an$t#ing from division to
spe"ies (#ardM
(' !an make permanent slides or digital images to #ave a longGterm re"ord of
"o%nts
b. Disadvantages
(1 Ee7%ires a lot of training
(& Ver$ time "ons%ming
(' Depending on desired taxonomi" resol%tion ma$ re7%ire #ig# 7%alit$ #ig#
po)ered "ompo%nd s"opes )it# spe"ialiHed feat%res (D;!, p#ase, inverted
s"opes, epifl%ores"en"e
". !aveats - need to make s%re $o% "o%nt eno%g# random fields or strips so t#at $o%
get a good sample (N133 "ells of ea"# ma?or taxon - often "ells are not
distrib%ted evenl$ on slides or ot#er "o%nting devi"es.
d. *et#ods
(1 BtermC#l "#amber or modified settling "#ambers
(a !on"entration of sample o""%rs on "#amber< sample is "o%nted )it# an
inverted s"ope< "an %se a whipple grid (grid in o"%lar or "o%nt random
fields or strips to determine n%mber of "ells per area of "over slip and
"onvert to "ells per vol%me
(b Advantages
i. No need to transfer sample from anot#er "on"entrating "#amber
ii. 1ong a standard met#od
(" Disadvantages
i. +ome diminis#ment of resol%tion if "ell #as large vol%me of )ater
overl$ing t#e sample
ii. 1imit to #o) m%"# "an "on"entrate samples
(& !o%nting "ells t#at "an be %sed )it# "on"entrated samples (or lab "%lt%res -
often more "on"entrated t#an in t#e field
(a Sedgwick-Rafter (S-R) cell - onl$ "an %se on lo) po)er (too deep< good
for large algae and pro"essing lots of sample
(b Hemocytometer - %sed for "o%nting blood "ells< #as a grid< "ontains a
kno)n vol%me of )ater (generall$ small< allo)s $o% to "#oose random
s7%ares of different siHes for "o%nting< need to #ave "on"entrated sample<
"an %se )it# relativel$ #ig# po)er
(" Palmer-aloney (P-) cell - "ir"%lar "#amber )it# & narro) "#annels<
#olds 3.1 ml< "an be %sed on #ig#er po)er t#an +GE (is t#inner< need
"on"entrated sample
(' 9iltration onto membrane filter (often 3.06 m and "learing of membrane
()it# immersion oil
(a Advantages
i. "an %se v. #ig# magnifi"ation
ii. "an make permanent slides
(b b%t even lo) va"%%m (never %se N3.6 atm "an r%pt%re and deform "ells
(e.g. flagellates
O
(" distrib%tion of "ells on filter is nonGrandom (often are "on"entrated at
edge - again, need to "o%nt strips a"ross t#e membrane or man$ fields for
an a""%rate "o%nt
(0 +taining )it# a fl%ores"ent d$e (e.g., A2 or DA@;, filtration and examination
%nder epiGfl%ores"ent s"ope (need & filter sets - one for stain and one for
a%tofl%ores"en"e
(a Advantages
i. reall$ onl$ )a$ to "o%nt tin$ p#$toplankton (nano and
pi"oplankton
ii. "an "o%nt #eterotrop#i" ba"teria at same time
iii. pro"ess "an be sped %p )it# image anal$sis (take digital images of
fields - also get permanent re"ords
(b Disadvantages
i. 8ime "ons%ming - need to stain and "o%nt "ells
ii. +tain and epifl%ores"en"e s"ope and b%lbs "an be expensive - need
at least & filter sets
2. ,low cytometry-particle counting
a. Advantages
(1 A%tomates t#e "o%nting pro"ess
(& 9aster t#an opti"al "o%nting
(' +ometimes "an give lots of %sef%l information - "ell vol%mes, DNA "ontent
b. Disadvantages
(1 !an be ver$ expensive
(& !an get signals from detrit%s and ot#er parti"les
(' Don.t #ave t#e same taxonomi" resol%tion as "o%nts (b%t t#at.s ok for some
t#ings
". Examples
(1 !o"lter co"nter - parti"les pass t#ro%g# opening bet)een & ele"trodes,
breaking t#e ele"tri" field< "al"%lates n%mber and ass%mes ob?e"ts are sp#eres
to "al"%late estimated vol%mes. !an.t "o%nt ver$ small "ells (I& m<
interferen"e problems )it# nonGalgal parti"les. Don.t get taxonomi"
information (onl$ sorts b$ siHe< good for "%lt%res.
(& #ptical beam co"nters (similar problems and advantages< some ne) ones
allo) imaging
(' $low cytometers - "ells pass in fl%id in a t#in laminar stream past lasers or
mer"%r$ lamps - lig#t is s"attered based on p#$si"al properties of "ell (e.g.,
siHe, s#ape, refra"tive index< "ells ma$ also emit fl%ores"en"e. 8#ese signals
are dete"ted and anal$Hed.
(a Advantages
i. !an tell n%mber, siHe, pigments of ma?or divisions
ii. !an "o%nt pi"oplankton
iii. +ome reall$ expensive ones "an sort o%t parti"%lar "ells to "%lt%re
or anal$HeM
iv. !an "ombine )it# fl%ores"ent probes to look at ba"teria and
vir%ses or at spe"ifi" "$to"#emi"al "ompo%nds - e.g., proteins, lipids,
n%"lei" a"ids
J
v. !an also %se fl%ores"entl$ labeled antibodies to tag "ertain taxa -
man$ of t#ese are being developed
(b Disadvantages
i. 4enerall$ need fres# samples (I1& #o%rs or deep froHen or
spe"iall$ preserved samples
ii. Ver$ expensive
iii. !an re7%ire lots of t)eaking and "alibration
iv. 2ften still not #ig# taxonomi" resol%tion %nless %sing ot#er probes
or te"#ni7%es
3. Pigment analysis
a. Advantages
(1 >e"a%se different algal divisions #ave different a""essor$ pigments, anal$Hing
seston for t#ese pigments "an #elp determine broad taxonomi" "omposition of
a p#$toplankton "omm%nit$
(& Easier and faster t#an "o%nting - re7%ires less time and skill,training
(' +amples "an be stored at GK3
o
! for mont#s
b. Disadvantages
(1 4ives $o% data at a broad taxonomi" level
(& @igment expression varies )it# environmental "onditions, so t#is reall$ is
more often like a presen"e,absen"e of a taxon rat#er t#an 7%antitative
(' !an re7%ire lots of sample
(0 !an be expensive and timeG"ons%ming (A@1!
(6 @eople still debate t#e "orre"t,standard met#ods for bot# spe"trop#otometri"
e7%ations and appropriate A@1! te"#ni7%es
". *et#ods
(1 +pe"trop#otometri" (as in "#lorop#$ll a - extra"t pigments from filtered
algae< %se a s"anning spe"trop#otometer to get absorban"e at different
)avelengt#s. Empiri"all$ derived e7%ations "an be %sed to separate
"#lorop#$lls a, b and ", as )ell as to estimate p#aeop#$tin ()it# an
a"idifi"ation step.
(a Advantages - faster and "#eaper t#an A@1!
(b Disadvantages - re7%ires lots of sample< fe)er pigments determined t#an
A@1!< "an #ave "ompli"ations from sample t%rbidit$
(& A@1! (#ig# performan"e li7%id "#romatograp#$ - extra"t pigments from
filtered algae< pigment separation is obtained b$ %sing different "ol%mns<
pigments are identified b$ "omparing retention time )it# standards, b$
BV,V;+ spe"tros"op$, or b$ li7%id "#romatograp#$,mass spe"trometr$
(1!,*+ and "omparing t#e mass spe"tra )it# t#ose of pigment standards or
kno)n libraries.
(a Advantages - "an identif$ man$ a""essor$ pigments< "an provide opti"al
and str%"t%ral data abo%t %nkno)n pigments
(b Disadvantages - met#ods need to be "alibrated and perfe"ted for ea"# set
of pigments )it# internal standards and extensive "alibration and "ontrols<
re7%ires expensive e7%ipment< 7%antifi"ation "an re7%ire lots of (A,(!.
(' Vario%s t$pes of field /fl%oroprobes.
(a Advantages GG Allo)s %se in field and ma$ sample "ontin%o%sl$ over
broad spatial s"ales
K
(b Disadvantages GG ;s in vivo meas%rement and ma$ be altered b$ )ater
"onditions, algal "omm%nit$ "omposition and "ondition< need lots of
"alibrations )it# extra"ted pigments and,or "ell "o%nts
$# &o' do 'e esti%ate cell *io+olu%e)
,# Microscopically
a. Bsing o"%lar mi"rometer meas%re "ell dimensions and approximate "ell s#apes
for ea"# t$pe of "ell (%s%all$ estimate for ='3 "ells per taxon per sample
(1 Advantages
(a !ell siHe varies greatl$ season to season and lake to lake - t#ink abo%t
diatom reprod%"tion, for example, so %sing tables of standard vol%mes for
ea"# spe"ies,gen%s )ill introd%"e a lot of error into biovol%me
"al"%lations< dire"t meas%rement is s%perior
(& Disadvantages
(a Ver$ time "ons%ming
(b Are still estimating vol%me b$ approximating "ell s#ape (not reall$
meas%ring it in 'D
b. A%tomated image anal$sis of digital images from mi"ros"ope fields (e.g.,
;mage@ro, ;mage P
(1 Advantages
(a @ermanent digital image
(b +aves time (a%tomated meas%ring f%n"tions or ma"ros
(" As above, is more a""%rate t#an ass%ming all "ells of spe"ies D are al)a$s
t#e same siHe
(& Disadvantages
(a ;s still time "ons%ming
(b +till not 'D - "an get area b%t not vol%me of "ells dire"tl$
&. Particle counters-(lo' cyto%etry - provide e7%ivalent sp#eri"al vol%mes
a. Advantages
(1 4et data along )it# "o%nts - 7%i"k
b. Disadvantage
(1 2ften not taxonGspe"ifi" vol%mes
(& ;s still an estimation of vol%me (not all "ells are sp#eres
.. How do we estimate phytoplankton producti!ity in the "ield?
1. !ell n%mber "#anges - problems )it# losses of "ells d%e to graHers and ot#er
mortalit$ - generall$ not a good indi"ator of gro)t# rate
&. !#lorop#$ll "#anges - as above
'. F#ole lake,stream ox$gen "#anges - di%rnal pattern or #$polimneti" ox$gen debt
give reasonable estimates of "omm%nit$ metabolism ("omm%nit$ primar$
prod%"tivit$ and respiration< need to make ass%mptions abo%t ex"#anges of gas )it#
t#e atmosp#ere (not a "losed s$stem
0. 1ig#tGdark bottle in"%bation - "losed s$stem so avoids atmosp#eri" ex"#ange< bottle
effe"ts ma$ be a problem )it# long in"%bations
a. ox$gen "#ange - "an do an$)#ere< simple< mi"rote"#ni7%es allo) for some lo)G
level meas%rements, b%t lo)er limit of dete"tion still relativel$ #ig#
:
b.
10
! %ptake - permit in"reasingl$ #ard to obtain for field )ork< allo)s
meas%rements of lo) levels of primar$ prod%"tion< "an.t easil$ separate gross and
net prod%"tion
6. !ell "ondition - met#ods being %sed more< e.g., fl%orometr$
""# Phytoplankton culturing
A. How do I ac/uire phytoplankton cultures?
1. !an order kno)n strains from c"lt"re collections (or trade )it# friendsM
a. Advantages
(1 Kno)n so%r"e (avoid "#eap so%r"es like )ards %nless for "lass onl$
(& 2ften is ot#er )ork done on t#at strain (geneti", bio"#emi"al, e"ologi"al
(' !an get same "lone if $o% #appen to kill $o%r "%lt%re
(0 ;s sele"ted to gro) )ell in lab
(6 2ften "omes )it# good ideas abo%t )#i"# "%lt%re media it )ill gro) on best
b. Disadvantages
(1 *a$ be a /lab rat. not representative of /)ild. algae - man$, man$ generations
for f%rt#er sele"tion for traits t#at are benefi"ial in lab
(& *an$ kinds of algae lose /)ild. traits in "%lt%re "olle"tions - e.g., gro)t#
pattern be"omes %ni"ell%lar instead of "olonial
%& 'solate yo"r own algae
a. +ome algae are sensitive to "olle"tion te"#ni7%es and do not do )ell if "apt%red
b$ net
b. +ome algae die 7%i"kl$ in a sample (even in 1 #o%r. !olle"t )ater and isolate
immediatel$ for best res%lts
". Al)a$s re"ord site and date of sample
d. Need to %se sterile te"#ni7%e and #ave "lean, nonGtoxi" glass)are (see belo)
e. Bse a disse"ting or inverted mi"ros"ope - easier to move #and,pipette in "orre"t
dire"tion to isolate "ells
f. *i"ropipette isolation of algae -
(1 Bse eit#er steriliHed glass (borosili"ate or preGsteriliHed plasti" mi"ropipettes
or "apillar$ t%bes. Lo% "an /p%ll. glass pipettes after #eating on a >%nsen
b%rner to make $o%r o)n mi"ropipettes
(& @i"k %p a "ell $o% )ant to "%lt%re b$ s%"king it %p into t#e pipette ()#ile
looking t#ro%g# t#e mi"ros"ope
(' @la"e "ell into sterile lake )ater or "%lt%re medi%m droplet
(0 Eepeat transfer into ne) separate droplets at least &D - need to be s%re $o%
#ave onl$ t#at "ell and #ave rinsed an$ "ontaminants
(6 !an eit#er s%"k %p "ells )it# "apillar$ a"tion or b$ gentle mo%t# s%"tioning
on t%be atta"#ed to pipette< blo) o%t to move to next droplet. ;f %sing
"apillar$ a"tion $o% )ant to fill t#e pipette )it# sterile )ater so t#at it doesn.t
s%"k %p too m%"# of t#e sample from )#i"# $o% are tr$ing to isolate a single
"ell - t#ere )ill still be eno%g# "apillar$ a"tion )#en $o% pla"e it into t#e
sample to s%"k %p a "ell
(O F#en #ave a single "ell isolated, add it to t#e "%lt%re t%be in )#i"# $o% )ill
gro) it
g. @lating on agar
(1 +treak a sample a"ross t#e agar and let gro)
13
(& Eemove "ells from individ%al "olonies )it# mi"ropipette or transfer loop
(' ;dentif$ and verif$ $o% #ave %niGalgal "%lt%res
#. *is"ellaneo%s te"#ni7%es
(1 *%ltiple serial dil%tions %ntil $o% #ave on average 1 "ell per t%be - gro) and
see )#at $o% #ave< make %niGalgal if not alread$
(& !an %se p#ototaxis or "entrif%gation as an initial "on"entrating step before
isolation
(' 9lo) "$tometr$ (see above
i. 2ften get greater s%""ess b$ first isolating into /enri"#ment. media (see belo)
and t#en transferring to defined media on"e "%lt%re is s%""essf%l
. How do I culture my algae once I ha!e them?
(& !"lt"re media
a. Agar - ma$ need to )as# to remove imp%rities (ordinar$ agar "an l$se some
"$anoba"teria or b%$ mole"%lar biolog$ grade agarose
(1 Advantages - "an often maintain slo) gro)ing "ells on agar - good for
sto"ks,ba"k%ps
(& Disadvantages - often algae don.t gro) like /)ild. algae on agar< ma$ need to
add n%trients to promote gro)t#
b. +oil media GG Needs to be gro%nd and dried. Distilled )ater is t#en added to t#e
soil (& parts )ater to 1 part soil and t#en t#e mixt%re is a%to"laved (often & #o%rs
and filtered. 8#e filtrate is a%to"laved again.
(1 Advantages - %sef%l )#en re7%irements of algae are not f%ll$ kno)n (also
ma$ add D2! for mixotrop#s< algae often gro) more like /)ild. algae on
t#ese media
(& Disadvantages - not defined< need to avoid soils )it# fertiliHer and pesti"ides<
generall$ loam$ and not "la$ soil is best. 8#e medi%m ma$ "#ange as so%r"e
of soil "#anges
". Enri"#ed media - add n%trients to nat%ral lake,stream )ater
(1 Advantages - algae often gro) more like /)ild. algae on t#ese media
(& Disadvantages - all "omponents not defined
d. Defined,s$nt#eti" media - %sing a %ltrap%re distilled )ater, vario%s
ma"ron%trients, tra"e elements and vitamins are added from sto"k sol%tions.
(1 Advantages - kno) all "omponents of media< "onstant t#ro%g# time
(& Disadvantages - time "ons%ming to prod%"e< man$ options - need to find
rig#t medi%m for $o%r alga< "an be toxi" to Hooplankton
%& Sterile techni)"e
a. Vital for maintaining %niGalgal "%lt%res - ot#er)ise "%lt%res )ill be "ontaminated
- "an never expose "ontainers or medi%m to air - )ill be "ontaminated )it# d%st,
spores and mi"roorganisms (in"l%ding algae. Never to%"# or expose open
s%rfa"esM
b. *et#ods
(1 Fipe all s%rfa"es and gloved #ands )it# et#anol before #andling "%lt%res or
steriliHed e7%ipment
(& A"id )as#, rinse and t#en steriliHe all glass)are in an a%to"lave
(' A%to"lave or #eat in a flame all transfer pipettes or loops
11
(0 Do "%lt%re transfers %nder a laminar flo) #ood, preferabl$ )it# BV lig#t to
preGsteriliHe s%rfa"es
(6 +teriliHe "%lt%re medi%m
(a A%to"lave on /li7%id. setting - t#is )ill #eat %nder press%re to
temperat%res over boiling
i. >e s%re to let ex#a%st slo)l$ (don.t end "$"le earl$ or $o% )ill
boil t#e li7%id and pre"ipitate ingredients (often bo%nd )it# iron or
sili"ate
ii. Don.t a%to"lave temperat%re sensitive materials - e.g., vitamins.
Add t#ese later and steriliHe b$ filtering
(b +terile filter
i. @ore siHe I3.& m
ii. !an get a%to"lavable large vol%me filters
iii. A%to"lave,steriliHe filter apparat%s
iv. @%mp (%s%all$ peristalti" or va"%%m p%mp medi%m t#ro%g# filter
into a%to"laved "ontainer - do t#is %nder a laminar flo) #ood if
possible. 2t#er)ise make s%re all "omponents and table top are )iped
off )it# et#anol and ever$t#ing is "overed and not exposed to air
v. Fon.t kill vir%ses
'. *+enic c"lt"re - gro)ing algae )it# no ba"teria (or vir%ses< take %nialgal "%lt%re and
eit#er s"reen filter gentl$ (allo)ing ba"teria to pass, "entrif%ge gentl$ ("olle"ting
#eav$ fra"tion, or %se repeated dil%tions into sterile medi%m and antibioti"s to kill
ba"teria
a. Advantages
(1 Lo% #ave p%re algae
b. Disadvantages
(1 Ver$ diffi"%lt and time "ons%ming
(& Need to "#e"k repeatedl$ t#at $o% don.t #ave ba"terial "ontaminants
(' Not all algae #ave been s%""essf%ll$ gro)n axeni"all$
,& -atch vs& contin"o"s c"lt"re
a. !ontin%o%s ("#emostat G medi%m p%mped into algal "%lt%re at slo), "ontin%o%s
rate
(1 Advantages
(a !%lt%res "an remain in exponential gro)t# for long periods
(b !an "ontrol gro)t# rate (m%st mat"# t#e dil%tion rate on"e t#e "ells are at
e7%ilibri%m
(& Disadvantage
(a Ee7%ires more expensive e7%ipment (e.g., p%mps
(b Ee7%ires dail$ "#e"king
b. >at"#
(1 (semiG"ontin%o%s - remove and add a per"entage of t#e medi%m at kno)n
intervals. 8#e faster t#e interval, t#e more t#is approximates "ontin%o%s
"%lt%re.
(a Advantages
i. !#eaper t#an "#emostats
(b Disadvantages
1&
i. >alan"ed gro)t# o""%rs onl$ d%ring exponential gro)t# (in
"#emostats is t#e standard "ondition so "ell "ondition )ill "#ange
bet)een fres# medi%m additions
ii. Need to keep good tra"k of fl%ores"en"e and ot#er parameters to
make s%re $o% are in exponential gro)t# p#ase
(& >at"# - transfer exponentiall$ gro)ing "ells to ne) medi%m to keep gro)ing
exponentiall$< re7%ires transfer often eno%g# t#at gro)t# is %nlimited
.& /eneral advice
a. Need to be "aref%l to avoid lig#t limitation - "ommon in dense "%lt%res< most
"ommon problem. Bse f%ll spe"tr%m fl%ores"ent b%lbs to mat"# nat%ral lig#t (old
standard )as /"oolG)#ite, b%t not as nat%ral a spe"tr%m. *ake s%re $o% meas%re
lig#t (s#o%ld #ave N13 moles 7%anta m
G1
se"
G1
, b%t probabl$ I&33 moles 7%anta
m
G1
se"
G1
to avoid p#otoGin#ibition
b. *a$ need to b%bble dense "%lt%res to avoid ! limitation, b%t be s%re to #ave
sterile filter on air line to avoid "ontamination
". !an gro) in Erlenme$er (or similar flasks, glass t%bes, et". 8ops m%st allo) gas
ex"#ange, b%t prevent "ontamination
d. Bse a lig#t: dark "$"le
e. Need to /feed. algae ne) medi%m at "onstant intervals to maintain "%lt%res in
same p#ase of gro)t# and same general bio"#emi"al "omposition (pigments,
n%trient "ontent
f. *%st preG)as# all ne) glass)are in dil%te a"id and rinse "opio%sl$
g. Avoid %sing any glass)are t#at #as been %sed for poisons (e.g., p#enol,
formalde#$de, mer"%r$
#. !olle"t and feed algae at same time of da$ (di%rnal r#$t#ms of "ell division
.se(ul /e(erences
Ameri"an @%bli" Aealt# Asso"iation. &336. +tandard *et#ods for t#e Examination of Fater and
Faste)ater. &1
st
edition. Ameri"an @%bli" Aealt# Asso"iation, Fas#ington, D.!. 1'KO
pages.
Anderson0 /#A#0 editor# 2001# Algal Culturing Techniques# Else+ier#
2 This is the current %ethods *ook sponsored *y the Phycological !ociety o( A%erica
9ogg, 4.E. and >. 8#ake. 1:KJ. Algal !%lt%res and @#$toplankton E"olog$. '
rd
edition. 8#e
Bniversit$ of Fis"onsin @ress, *adison.
Kemp, @.E., >.9. +#eer, E.>. +#eer and P.P. !ole, editors. 1::'. Aandbook of *et#ods in
A7%ati" *i"robial E"olog$. 1e)is @%blis#ers.
+tein, P.E., editor. 1:J'. Aandbook of @#$"ologi"al *et#ods: !%lt%re *et#ods and 4ro)t#
*eas%rements. !ambridge Bniversit$ @ress, !ambridge.
+tri"kland, P.D.A. and 8.E. @arsons. 1:JK. A @ra"ti"al Aandbook of +ea)ater Anal$sis. &
nd

edition. 9is#eries Eesear"# >oard of !anada.
FetHel, E.4. and 4.E. 1ikens. &333. 1imnologi"al Anal$ses. '
rd
edition. +pringerGVerlag, Ne)
Lork.
1'