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ASM CUE 2007 Try Something New Workshop

Microbial Ecology: Opportunities for Inquiry-Based Learning

Presenters:

Hank Een!orn" Ph#$#%eos&ien&es $i'isionNationa( Energy Te&hno(ogy )a!oratoryU#S# $epartment o* Energy" Pitts!+rgh" PA ,-2./Harry#Een!ornNET)#$1E#%1

Hank Een!orn is an en'ironmenta( mi&ro!io(ogist with a Ph#$# *rom 3+tgers Uni'ersity# Heworks at the U#S# $epartment o* Energy4s Nationa( Energy Te&hno(ogy )a!oratory in Pitts!+rgh" PA# H&+rrent resear&h interests in&(+e the mi&ro!ia( e&o(ogy an !ioremeiation o* in+stria((y5&ontaminate

soi(s an seiments6 the ro(e o* (i&hens" !ryophytes" an higher p(ants as ha!itats *or !a&teria6 an themi&ro!ia( i'ersity o* +n+s+a( nat+ra( en'ironments *o+n within the state o* Pennsy('ania#

Mary M+(&ahy" Ph#$#io(ogy Program 8 A((egheny 9nstit+te o* Nat+ra( HistoryUni'ersity o* Pitts!+rgh at ra*or" ra*or" PA ,/70,mnp,e&hange#+p!#pitt#e+1r!io(ogy+p!#pitt#e+;./2502-?

Mary M+(&ahy is an asso&iate pro*essor o* !io(ogy at Pitt5ra*or ;(o&ate a!o+t a ?0 min+teri'e so+th o* +**a(o># She re&ei'e her Ph#$# *rom the Uni'ersity o* Misso+ri at Co(+m!ia" an her

resear&h interests are in the area o* p(ant5anima( intera&tions an p(ant mating systems# Mary +ses this eer&ise in a sophomore (e'e( e&o(ogy an e'o(+tion &o+rse# Mary is parti&+(ar(y intereste in e'e(op(a!s that en&o+rage st+ent &reati'ity an &riti&a( thinking ski((s#

ELECTRONIC COIE! O" T#E #$N%O&T! $N%

O" OT#ER M$TERI$L! $RE $'$IL$BLE $T T#E "OLLO(IN) (EB!ITE:

http:@@www#pitt#e+@mnp,@mi&ro!ia(Be&o(ogy#htm
mailto:[email protected]:[email protected]:[email protected]:[email protected]://www.pitt.edu/~mnp1/microbial_ecology.htmmailto:[email protected]:[email protected]:[email protected]://www.pitt.edu/~mnp1/microbial_ecology.htm


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3e(ease on: Sat+ray" +n .r ,??-#9mages &opyright i(( Watterson an Uni'ersa( Press Syni&ate#

Taken *rom: http://www.ifmb-a.uni-bonn.de/extremophile.jpg
http://www.ifmb-a.uni-bonn.de/extremophile.jpghttp://www.ifmb-a.uni-bonn.de/extremophile.jpg


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Table of Contents age

Workshop 9ntro+&tion .

9ntro+&tion *or St+entsThis introduction is for the students with little exposure to microbiology, and

we recommend a more sophisticated introduction for more advanced classes.

=

S+ggeste Pre5(a! Homework Assignment *or St+ents:

Pra&ti&e S&oring E&oP(ates -

S+ggeste Dormat *or St+ent )a! Eer&ise:

$esign yo+r own Eperiment

$ata S+mmary Sheet

?

,0

ey to St+ent Homework ,,

Notes *or 9nstr+&tors

Materia(s Neee *or )a! +sing %en+ine E&oP(ates

3e&ipe *or Da+ E&oP(ates

9nstr+&tions *or St+ents on How to 9no&+(ate %en+ine E&oP(ates

,.

,.

,=

Samp(e $ata an Ana(ysis ,7

Use*+( 3e*eren&es an )inks 2,

(or*s+op Introduction

Mi&ro!ia( e&o(ogy st+ies pro'ie e&e((ent opport+nities *or open5ene" inF+iry5!ase (earnin

in the &(assroom# 9n this workshop" we wi(( emonstrate a (a!oratory eer&ise in whi&h st+ents generat

hypotheses a!o+t mi&ro!ia( e&o(ogy an then test them +sing GE&oP(ates"G m+(tiwe(( test p(ates that a((o

rapi etermination o* the meta!o(i& &apa!i(ities o* a !a&teria( pop+(ation witho+t teio+s an time5

&ons+ming reagent preparation !y the instr+&tor# A*ter ino&+(ation with a s+spension o* !a&teria washe

*rom a soi( or p(ant samp(e" the in&+!ate p(ate ret+rns a +niF+e set o* positi'e ;p+rp(e> an negati'e

;&(ear> rea&tions that a((ows st+ents to assess whether or not two samp(es &ontain simi(ar or issimi(ar

!a&teria( pop+(ations# 1+r eperien&e is that st+ents enoy e'e(oping their own eperiments +sing the

p(ates" an that the eer&ise is parti&+(ar(y +se*+( in i((+strating physio(ogi&a( i'ersity o* mi&ro!ia(

pop+(ations whi(e a(so in'o('ing st+ents in the e&iting pro&ess o* e'e(oping their own s&ienti*i&

hypotheses#


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*This introduction is designed for students who have never taken a microbiology class, and although I

have not provided one here, I recommend a more sophisticated introduction for students in moreadvanced classes. Mary Mulcahy

Introduction for Beginning !tudents,

%istinguis+ing $ong Bacteria &sing Metabolic CapabilitiesHow o yo+ te(( one !a&teria( spe&ies *rom anotherI How &an yo+ te(( whether a person is si&k

with Streptococcus pneumoniae or Staphylococcus aureusI Most o* +s are *ami(iar with ienti*ying'erte!rate spe&ies an p(ants !ase on morpho(ogi&a( *eat+res# Howe'er" !a&teria 'ary re(ati'e(y (itt(e their morpho(ogi&a( *eat+res# a&teria( spe&ies &an !e (+mpe into gro+ps !ase on the shape o* the &e;ro5shape" &o&&oi" &hains o* &e((s" et>" sometimes &o(ony &o(or" an sometimes &o(ony appearan&e the ege o* the !a&teria( &o(ony wa'y or smooth" *or eamp(e>" !+t these physi&a( *eat+res are rare(ys+**i&ient to *+((y &(assi*y !a&teria to spe&ies#

$espite the (a&k o* morpho(ogi&a((y 'aria!(e *eat+res" !a&teria( spe&ies &ertain(y o 'ary *rom oanother6 rather than morpho(ogi&a( *eat+res" the wea(th o* *eat+res that isting+ish !a&teria are!io&hemi&a(# Some !io&hemi&a( *eat+res that isting+ish !a&teria are &ontaine within the str+&t+re o*

&e((s themse('es# 9n the , a(so ha'e a spe&i*i& set o* other &ar!on so+r&es that &an an &an not !e +se *or energy# $es&ri!ithat pattern o* &ar!on5+sage is &a((e L&omm+nity5(e'e( physio(ogi&a( pro*i(ing ;C)PP>#

Ea&h gro+p has !een pro'ie with 7 E&oP(ates# o+r gro+p is aske to e'e(op a F+estion a!o+!a&teria( &omm+nities *o+n in i**erent nat+ra( en'ironments +sing these E&oP(ates# o+r eperiment


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may !e F+antitati'e an take samp(es at si (e'e(s o* a known *a&tor ;an there*ore +se regression or&orre(ation ana(ysis> or yo+r eperiment may !e F+a(itati'e an &ompare two or three is&rete gro+ps#o+ wi(( +se the 7thp(ate as a &ontro(# e*ore yo+ !egin e'e(oping yo+r F+estion" p(ease pra&ti&e s&orthe E&oP(ates#

!uggested #oe1or* for !tudents: ractice !coring Ecolates

Learning to !core $n Ecolate

Dig# , shows the (ayo+t o* &ar!on s+!strates on o+r E&oP(ates# Ea&h s+!strate ;&ar!on so+r&e> has !eengi'en a +niF+e n+m!er# 3e*erring to Ta!(e 2" yo+ &an see whi&h s+!strate ;&ar!on so+r&e> is *o+n in eo* the we((s# Dor eamp(e" s+!strate O,0 is i5Erythrito(# )ike a(( the s+!strates here" i5Erythrito( is *o+nthree times on the E&oP(ate# 9t is *o+n in the *o((owing we((s: C2" C/" an C,0#

"ig2 32 A rawing o* an E&oP(ate in whi&h the &ir&(es are we((s# A sing(e s+!strate an a ye is presenea&h we((# The s+!strate is a +niF+e &ar!on so+r&e that &an !e +se as energy !y the !a&teria The

n+m!ers insie the &ir&(es ea&h &orrespon to a parti&+(ar s+!strate# P(ease re*er to Ta!(e 2 to eterminwhat s+!strate &orrespons to what n+m!er#

Prior to !eginning yo+r eperiment" p(ease pra&ti&e s&oring the E&oP(ates# There are three main&a(&+(ations that yo+ sho+( !e a!(e to o andto +nerstan:

4 "unctional %i.ersity ,00 Q n+m!er o* positi'e ;p+rp(e@pink> &ar!on so+r&e we((s Tota( n+m!er o* &ar!on so+r&e we((s ;.,>

This 'a(+e 'aries *rom 0 to ,00R with 0 !eing (ow i'ersity an ,00R !eing high(y i'erse#

o+ wi(( nee to report the in&onsisten&y *o+n within ea&h samp(e#

4 'ariation of Results 1it+in !aple ,00Q i@.,i the n+m!er o* &ar!on so+r&es in whi&h the three rep(i&ates were not A))positi'e or A)) negati'e# 1n Ta!(e 2" i s+m o* e'erywhere there is a , or 2 ;not . or 0>


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4 !iilarity;SSM> ,00Q a BBBBBBB a ! &

QP(ease note that water is not a &ar!on so+r&e#a N+m!er o* &ar!on so+r&es +se !y !oth samp(e A an samp(e ! N+m!er o* &ar!on so+r&es +se !y Samp(e !+t not !y Samp(e A

& N+m!er o* &ar!on so+r&es +se !y Samp(e A !+t not !y Samp(e N+m!er o* &ar!on so+r&es not +se !y !a&teria in either samp(e9* the two samp(es gi'e ienti&a( L*ingerprints on the E&oP(ates" Ssmwi(( !e ,00# 9* thetwo samp(es gi'e ea&t(y opposite L*ingerprints" the 'a(+e wi(( !e 0#

lease score Ecolates 35 65 7 8 by filling in table 3 on page 92

The shae we((s are the ones that t+rne pink or p+rp(e#

Samp(e E&oP(ate O,

Samp(e E&oP(ate O2

Samp(e E&oP(ate O.


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Table 32 Samp(e P(ate O, is a(reay s&ore *or yo+ as an eamp(e# o+ nee to s&ore samp(e p(ates 2 a.# P(a&e a Kero in the &e(( ta!(e i* none o* the three rep(i&ates t+rne pink or p+rp(e# P(a&e a , in the tai* 1NE o* the three rep(i&ates t+rne pink or p+rp(e# P(a&e a 2 in the ta!(e i* TW1 o* the three rep(i&atet+rn pink or p+rp(e" an p(a&e a . in i* a(( three rep(i&ates are positi'e ;pink or p+rp(e>#

Samp(e Samp(e Samp(e

S+!strate Name S+!strate O P(ate O, P(ate O2 P(ate O.Water , 0

5Methy(5$5%(+&osie 2 0

$5%a(a&toni& A&i 5)a&tone . 2

)5Arginine = 0

Pyr+'i& A&i Methy( Ester - 0

$5Vy(ose / 0

$5%a(a&t+roni& A&i 7 0

)5Asparagine < 0

Tween =0 ? 0

i5Erythrito( ,0 025Hyroy enKoi& A&i ,, 0

)5Pheny(a(anine ,2 0

Tween


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lease $ns1er t+e "ollo1ing >uestions:

;, pt> X+estion ,: How manydifferent &ar!on s+!strates are there on the E&oP(ateI BBBBBB

;, pt> X+estion 2: What is the name o* the &ar!on s+!strate esignate n+m!er ,-I

BBBBBBBBBBBBBBBBBB ;, pt>

X+estion .: Using the (etter to esignate the row an n+m!ers to esignate &o(+mn" (ist the we((s&ontaining s+!strate O,-# ;Dor eamp(e" i5Erythrito( is *o+n in C2" C/" an C,0#>BBBBBBBBBBBBBBBBB

;, pt> X+estion =: What is the &ontro( s+!strate in this p(ateI BBBBBBBBBBBBBBBSho+( the &ontro( we(

t+rn pinkI BBBBBBBBBBBBBBBBBBBBBBBBBBBB

;, pt> X+estion -: Using the (etter to esignate the row an n+m!ers to esignate &o(+mn" (ist the we((s

&ontaining the &ontro(# BBBBBBBBBBBBBBBBBB

/3 pt0 Table 62 P(ease *i(( in the in*ormation in this ta!(e !ase on yo+r &a(&+(ations

!aple late "unctional

%i.ersity

4 'ariation

of Results

6

8

/3 pt0 Table 82 P(ease *i(( in the in*ormation on this ta!(e !ase on yo+r &a(&+(ations#

ercent !iilarity

!aple late 3 !aple late 6 !aple late 8

!aple late 3

!aple late 6

!aple late 8

;, pt> X+estion /: P(ease +se Ta!(e . to answer this F+estion# These three p(ates &ame *rom the *o((owso+r&es:a&teria( wash *rom the +nwashe (e*t han o* oe Smitha&teria( wash *rom the +nwashe right han o* oe Smitha&teria( wash *rom the +nwashe (e*t han o* ane $oe#

ase on Ta!(e ." propose a hypothesis *or whi&h p(ate &ontains the !a&teria( &omm+nity o* ane $oeJshan# P(ease ep(ain yo+r reasoning#????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????

??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????

?????????????????????????????????????????????????????????????????????????????

!uggested "orat for !tudent Lab E@ercise: %esign your o1n E@perient

o+ ha'e !een pro'ie with 7 E&oP(ates# P(ease e'e(op a F+estion a!o+t !a&teria( &omm+nities *o+ni**erent nat+ra( en'ironments that yo+ &o+( in'estigate +sing these p(ates# The E&oP(ate p(ates wi(( g


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yo+ a Lmeta!o(i& *ingerprint o* a parti&+(ar &omm+nity an the F+estion that yo+ &hoose to ask wi((pro!a!(y !e one o* the *o((owing three types:

,> !patial coparison: L9s the !a&teria( &omm+nity *o+n here the same as the one *o+n thereI

2> Teporal coparison: L9s the !a&teria( &omm+nity the same now as it wi(( !e (aterI

.> Manipulati.e e@perient: L$oes the !a&teria( &omm+nity &hange when something is ae to ortaken away *rom its ha!itatI or L$oes the !a&teria( &omm+nity &hange when this en'ironmenta(&onition is a(tereI

o+r eperiment may !e F+antitati'e an take samp(es at si (e'e(s o* a known *a&tor ;anthere*ore +se regression or &orre(ation ana(ysis> or yo+r eperiment may !e F+a(itati'e an &ompare twthree is&rete gro+ps#

repare a Brief roposal* In my classes I do not use this form, but rather provide students to turn in a proposal written in a

particular format. The answers below make up much of the content of my proposal but the format of thproposal. Mary MulcahyP(ease *i(( in the *o((owing in*ormation:

,# Whatspecific F+estion has yo+r gro+p e&ie to askI

2# rie*(y" in a *ew senten&es" p(ease state why this is an important F+estion#

.# P(ease state yo+r n+(( hypothesis#

=# rie*(y es&ri!e the methos yo+ wi(( +se to o!tain yo+r samp(es# ;Note there is no nee to es&ri!ehow the E&oP(ates wi(( !e ino&+(ate# 3ather" i* yo+ are &o((e&ting soi(" *or eamp(e" how wi(( yo+ gaththat soi( an *rom whereI Wi(( yo+ +se ranomiKation at a((I How many samp(es wi(( yo+ &o((e&tI>

-# What res+(ts o yo+ epe&t" an whyI


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%ata !uary !+eet

P(ease *i(( in the in*ormation on yo+r si E&oP(ates ;yo+r 7 thp(ate sho+( !e +se as a &ontro(>#E&oP(ate O, $es&ription: BBBBBBBBBBBBBBBBB E&oP(ate O2 $es&ription: BBBBBBBBBBBBBBB

E&oP(ate O. $es&ription: BBBBBBBBBBBBBBBBB E&oP(ate O= $es&ription: BBBBBBBBBBBBBBB

E&oP(ate O- $es&ription: BBBBBBBBBBBBBBBBB E&oP(ate O/ $es&ription: BBBBBBBBBBBBBBB

Contro( P(ate O7:


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lease $ns1er t+e "ollo1ing >uestions:

;, pt> X+estion ,: How manydifferent &ar!on s+!strates are there on the E&oP(ateI BB.,BBBB

;, pt> X+estion 2: What is the name o* the &ar!on s+!strate esignate n+m!er ,-I B=5Hyroy enKo

A&iB

;, pt> X+estion .: Using the (etter to esignate the row an n+m!ers to esignate &o(+mn" (ist the we((s&ontaining s+!strate O,-# ;Dor eamp(e" i5Erythrito( is *o+n in C2" C/" an C,0#> $." $7" $,,B

;, pt> X+estion =: What is the &ontro( s+!strate in this p(ateI BBwaterBBBBBB Sho+( the &ontro( we((s

t+rn pinkI BBBNo ;organisms &an not +se water *or energy 8 the tetraKo(i+m ye wi(( on(y &hange &o(o

the !a&teria &an meta!o(iKe the s+!strate#>BBBBB

;, pt> X+estion -: Using the (etter to esignate the row an n+m!ers to esignate &o(+mn" (ist the we((s

&ontaining the &ontro(# BBA," A-" A?BB

/3 pt0 Table 62 P(ease *i(( in the in*ormation in this ta!(e !ase on yo+r &a(&+(ations

!aple late "unctional

%i.ersity

4 'ariation of

Results

6 862694 ;294

8 =


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Notes for Instructors

Materials Needed for Lab &sing )enuine Ecolates

,# M+(ti&hanne( any !ran -0 m( with gra+ations marke or steri(iKe (ieg(assware# These are going to !e 'ortee so sho+( !e siKe appropriate *or 'orteing# Ea&h gro+p prowi(( reF+ire a!o+t = steri(iKe t+!es@'ia(s per E&oP(ate ;8 =Q/2= per gro+p6 'ia( is not rea((y ne&essary*or the &ontro( p(ate> !+t etras may !e hany# Si o* these t+!es@'ia(s wi(( !e +se to &o((e&t the soi( o(ea'es# The remaining t+!es are +se to &omp(ete the seria( i(+tion# Dor the &ontro( p(ate" p+re PS !+is neee" an it may !e hany to ha'e an aitiona( t+!e a'ai(a!(e to ispense it into#

=# io(og E&oP(ates ;approimate(y Y,0@ p(ate>

-# Steri(e PS so(+tion ;see re&ipe in methos se&tion># Ea&h st+ent gro+p may nee ,00 m( o* !+**erper samp(e &o((e&te an E&oP(ate ino&+(ate" an there*ore" *or / E&oP(ates p(+s a &ontro( p(ate" it is n+nreasona!(e to ha'e , ) o* PS a'ai(a!(e *or ea&h gro+p# This so(+tion &an !e mae in a 2 ) 'o(+me*(ask" a+to&(a'e an (e*t to &oo( o'ernight# Again" s&hoo(s witho+t a+to&(a'es &an sti(( o this (a!# Th&an +se whate'er steri(iKation te&hniF+es are a'ai(a!(e to them" an the &ontro( p(ate wi(( a((ow st+entssee how *ai(+re to steri(iKe the !+**er might a**e&t their res+(ts

3e&ipe *or PS:# 9* esire" thespat+(as &an !e steri(iKe !e*ore5han#

8 paper towe(s to p+tinto the !ags to keep the p(ates h+mi whi(e they in&+!ate#

Recipe for "au@ Ecolates

Dake E&oP(ates ;s+&h as +se in this workshop> are mae with ,R pheno(phtha(ein ;w@'> in ethano(# P2- +( per we(( an a((owe to ry# TheGPSG is 0#, M Na2C1. in eioniKe water ;,0#/ g@)>#


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Instructions for !tudents on #o1 Inoculate )enuine Ecolates

,# Co((e&t yo+r samp(e# 9t is essentia( that yo+ o not &ontaminate yo+r samp(e with !a&teria *rom yo+rhans or other p(a&es# Steri(e test t+!es with (is are pro'ie" an i* at a(( possi!(e" yo+ sho+( &o((e&tyo+r samp(es ire&t(y into these steri(e t+!es witho+t any +tensi(s# To a'oi &ontamination !y +nwante!a&teria here are a *ew tips:

$o not to+&h the soi( or p(ant that yo+ p(an to samp(e with yo+r hans or *ingers#$o not &hoose samp(es *rom soi( or p(ants that yo+ think someone has to+&he#$o not !reathe or sneeKe on yo+r samp(es or into yo+r t+!es#$o not (ea'e yo+r steri(e t+!es +n&appe an open *or air!orne !a&teria to enter#Contin+e to +se steri(e te&hniF+es when i(+ting yo+r samp(e in step O2#

9* yo+ wi(( nee to take yo+r samp(es with *or&eps or other too(s in orer to get the samp(e into the sterit+!es" p(ease ta(k to yo+r instr+&tor a!o+t a s+ita!(e isin*e&tion pro&e+re *or yo+r too(s# Se'era( gramper samp(e sho+( !e more than eno+gh *or the net step#

2# A*ter yo+ ha'e taken yo+r samp(es to the (a!" yo+ wi(( nee to i(+te them in steri(e Gphosphate

!+**ere sa(ineG or PS# Mi yo+r samp(e in steri(e PS in a set i(+tion ;wt@'o(> so that yo+ is(oge t!a&teria into the so(+tion ;Dig# 2># To get goo res+(ts *rom yo+r E&oP(ate" yo+ on4t want too many ortoo *ew !a&teria" +st the GrightG n+m!er# o+ wi(( ha'e to g+ess to a &ertain etent at yo+r i(+tion"epening on what is !eing samp(e# 9n o+r eperien&e" yo+ want to i(+te the samp(e +nti( it is !are(yt+r!i ;!are(y &(o+y># This is o*ten a ,05.weightweighti(+tion *or soi(;, g o* soi(@,000 g *ina( so(+tiwhere we ass+me that ea&h m( o* PS is approimate(y , g># Dor p(ant matter" a i(+tion o* ,052;, g o*p(ant matter@,00 g *ina( so(+tion> is pro!a!(y !etter# Dor ea&h samp(e" yo+ p+t the steri(e t+!e or the &apthe steri(e t+!e on the s&a(e an tare it ;or +se &(ean weigh !oats or weigh paper wi(( work as we((># Thyo+ a samp(e to the t+!e ;or to the &ap> +sing a steri(iKe +tensi( so that yo+ ha'e (ess than = g o* soi(Di(( yo+r t+!e with ? as m+&h m(# Sin&e ea&h m( is approimate(y ,g" yo+ ha'e +st mae ,@,0 or ,05,

i(+tion# orte this origina( i(+tion at high spee ;or shake thoro+gh(y> *or , min+te# Using .m( o* t

,0

5,

i(+tion an 27m( o* the p+re PS" &reate .0 m( o* a ,0

52

so(+tion in a new steri(e t+!e# To o!tain t*ina( i(+tion" take .m( o* the ,052so(+tion an a 27m( o* p+re PS# See Dig+re 2 *or a s+mmary o* ti(+tion pro&e+re# o+ wi(( +(timate(y nee ,00 +( o* so(+tion per we(( anat least,0 m( o* i(+tesamp(e per p(ate" an .0 m( is a hany amo+nt# 9* yo+ *in that yo+r so(+tion is too &(o+y a*ter miingyo+ wi(( want to i(+te the samp(e *+rther with the !+**er# eep tra&k o* how i(+te yo+r *ina( samp(ean in&(+e this in*ormation in yo+r (a!oratory report#


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Dig# 2# S+mmary o* steps to &reate the appropriate &on&entration# The i(+tion series is &reate in orea&hie'e a *ina( &on&entration o* ,g o* soi( @ ,000 g o* *ina( so(+tion# The weight@weight i(+tion *a&tors&ommon(y +se in mi&ro!io(ogy#

.# Dor ea&h 'ia(@*(ask o* !+**er i(+te samp(e" po+r the *(+i into either the !ottom ha(* or the top ha(* steri(e Petri ish# Using the m+(ti&hanne( pipette" a ,00 +( o* the p(ate wash s+spension to ino&+(ateea&h we(( in the E&oP(ate# 9n orer to (earn the te&hniF+e" the entire &(ass wi(( wat&h a emonstration othe ino&+(ation o* a p(ate# Try to a'oi to+&hing the sies or !ottom o* the we((s with the pipette tips# 9possi!(e" ho( the tips +st a!o'e the we(( to re(ease the (iF+i an o not rest the pipette tips on any parthe E&oP(ate#

=# P(a&e yo+r p(ates in a p(asti& !ag an in&+!ate them at room temperat+re# A sma(( pie&e o* moistenepaper towe( insie the !ag wi(( he(p to maintain a &onstant high (e'e( o* h+miity an a'oi e'aporatio*rom the we((s# o+ wi(( nee to &he&k yo+r p(ate ai(y an *i(( o+t a ata sheet showing whi&h we((s gi

a positi'e rea&tion ;p+rp(e &o(or> ai(y# 9t +s+a((y takes a!o+t = ays *or most o* the positi'e we((s to&hange &o(or# St+ents sho+( !e &a+tio+s to keep the p(ates horiKonta( ;*(at> an not tip the p(ates whieamining them toay or s+!seF+ent ays#


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-# $ata Co((e&tionDor eperiments that are not eamining a mi&ro!ia( &omm+nity o'er time" yo+ sho+( ino&+(ate

yo+r E&oP(ates in the same (a! perio an then eamine them e'ery ay +nti( the &o(or !egins to &hang3e&or the Lmeta!o(i& *ingerprint *or ea&h p(ate on the *irst ay o* &o(or &hange an *or three s+!seF+ays# A samp(e ata sheet is pro'ie at the en o* this hano+t that yo+ &an photo&opy# o+ sho+(mark own whi&h we((s were p+rp(e an whi&h we((s were not p+rp(e on the atasheet#

A*ter re&oring the ata" go !a&k an (ook thro+gh the ata sheets# Was there a ay on whi&h tp(ates appeare to stop &hanging &o(or an" i* so" were there some positi'e an some negati'e we((s onthose ays or were a(( the we((s positi'eI Use on(y the ata *rom the parti&+(ar ay on whi&h the &o(or&hanges appeare to ha'e &ease# Dor eamp(e" i* ay = showe the *ewest Qaitiona(Q p+rp(e we((s" tyo+ sho+( +se the ata *or ay = *or A)) yo+r p(ates# 9* a(( yo+r we((s +(timate(y showe positi'e p+rres+(ts" speak to yo+r instr+&tor#

X+estions to ConsierA(( te&hno(ogy has its (imitations" an E&oP(ates are no e&eption# As yo+ ana(yKe yo+r res+(ts

p(ease &onsier the *o((owing F+estions:,# How might temperat+re a**e&t yo+r res+(tsI $o a(( !a&teria grow eF+a((y we(( at room temperat+reI

How are we in*(+en&ing the res+(ts !y in&+!ating the E&oP(ate at room temperat+reI2# How might the pH o* the PS !+**er a**e&t yo+r res+(tsI $o yo+ think yo+ might get a i**erent*ingerprint *or a &omm+nity epening on the pH o* the PS !+**er yo+ +seI $o yo+ think yo+ might i**erent res+(ts *or a sing(e p+re !a&teria( spe&ies epening on the pH o* the !+**er +seI.# Co+( antagonisti& intera&tions !etween !a&teria in yo+r samp(e a**e&t the pattern on E&oP(atesI=# What i* protists eist in yo+r samp(e6 &o+( they a**e&t the E&oP(ate patternsI What a!o+t *+ngiI-# 9* yo+ a&&ienta((y to+&he the we((s +ring pipetting into the *irst < we((s an then &ontin+e to pipinto the remaining we((s with the same tips" how might this a**e&t yo+r res+(tsI/# How &o+( yo+ test whether yo+r PS !+**er was steri(eI7# What is wrong with ass+ming that the n+m!er o* p+rp(e we((s on the E&oP(ate &orrespons to spe&iei'ersityI


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&ntreated !aple 6

, 2 . = - / 7 < ? ,0 ,, ,2

A 0 0 , 0 0 0 , , 0 0 , 0

, 0 0 , , 0 , 0 , 0 , ,

C , 0 0 0 , 0 0 0 0 0 0 0$ , , 0 , , , 0 0 , , , ,

E 0 , 0 0 0 , 0 0 0 0 0 0

D 0 0 0 0 0 , 0 0 0 , 0 0

% , 0 0 0 , 0 0 0 , 0 0 0

H , 0 0 0 0 0 0 , , 0 0 ,Tota( &ar!on so+r&es that were &onsistent(y positi'e *or a(( three rep(i&ates: -Tota( &ar!on so+r&es that were &onsistent(y negati'e *or a(( three rep(i&ates: ,/Tota( &ar!on so+r&es positi'e *or 2 o* the . rep(i&ates:


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&seful References 7 Lin*s

)eneral References

a+oin" E#" E# eniKri" an A# %+&kert# 2002# 9mpa&t o* growth stage on the !a&teria( &omm+nitystr+&t+re a(ong maiKe roots" as etermine !y meta!o(i& an geneti& *ingerprinting# App(ie Soi(

E&o(ogy ,?:,.-5,=-#Camp!e((" C# $#" S# # %rayston" an $# # Hirst# ,??7# Use o* rhiKosphere &ar!on so+r&es in so(e &ar!on

so+r&e tests to is&riminate soi( mi&ro!ia( &omm+nities# o+rna( o* Mi&ro!io(ogi&a( Methos .0:.=,#

C+rtis" T# an W# T# S(oan# 200-# Ep(oring mi&ro!ia( i'ersity\a 'ast !e(ow# S&ien&e .0?: ,..,5,.Disk" M#C#" #D# 3+ether" an ## a'itt# 200.# Mi&ro!ia( a&ti'ity an *+n&tiona( &omposition among

northern peat(an e&osystems# Soi( io(ogy an io&hemistry .-:-?,5/02#%ans" #" M# Wo(insky" an # $+n!ar# 200-# Comp+tationa( impro'ements re'ea( great !a&teria( i'er

an high meta( toi&ity in soi(# S&ien&e .0?: ,.

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