Histological investigation 01 organisms with hard 
skeletons: a ca se study 01 siliceous sponges 
F HOffmann\ 0 Janussen2, W DfÖSe', G Arp', J Reilner' 
'l.WtwIJIyatGOrJiF'I'j""~oI~.~Grtq).~3,D-37'On~rio ....... _ 
,~ /nsIiIIAe _ NaIuI-. ~~. Matiw v_ ... Soot:Cim I. s..a.:~ 25. D-6CI32S 
FrriJutI *" .w.m. Getrnanr 
Abstract 
Silicrous ~nd c~lul'OO\ls spongH rommonIy an" lreared w,ll\ <Kid 10 ""''''''\' the spiculcs prior to 
t'mbf:dding and culti ng fot hlslologic~1 im-estig.>lions. H istology of "pkulatNl sponge ms .... 
"",,...,;mls a chaUenging problem in sponge histot«hl\Ology. Furthennore. Il~ in " Iu 
hybridiution (FISH).a key mrihod for studying spong~ zs:xiated lIIiocrobes, is not poesible alter 
add tre>tment. Fot a brood ra"se of sjliceous spon~ species, we dr-..,lop<'<i and ", .. Iuared 
mI'lhods fot" cmbedding in paraffin, methylmethacrylatc reins. LR IVhJle res'n and cryom~trix_ 
Diffurt.'f1! methods for roUing li581.1C blocb aS wdl as mounling and staining _I~ all'O W"te 
t~k'<l. Our aim was to l ..... blc histological im-e;tigations and flSH wilhou! prior removal of the 
spieul .... To obtain an ov'-'1"view of tissu" and skeleton arrangement. we re<:<>mmcod embedding 
, i55UC blocks with LR Whi'" l'11Sin combined wilh '" i1Ioc 5taining techniqu<'!l for largc spedmens 
with thkk and numcrous spiculcs, but pa,affin embcdding and subsequcol 5taining for whole 
smaU spedmcns. f or F1Sl-I on siliceous ~ponS<'!l' .... e remmmcnd Histocryl cmbcdding [f the 
spkule rollieni is high, bul paraffin embcdding If I1 is Iow. Classical hislologkal tc<:hniques Me 
u!l«l for d<tailed lissuc ('X.Omi""ti0n5. 
"~y word,: embedding.. fISH, histology, lR Whil .. ""in, methylmethacryLaIC ""in, paraffin, 
l'orife,a, spkuks 
Spongl"!l 3rt: ancient melaw.> wilh simple body 
pl.IU roIlsisting of only a fe .... sp«i;Jlite<l cell 
1)'1'('11. llIcy ha,'c Iong been used as model org;..n_ 
isrns for histologie.l studics for light and eJectron 
microlropy (Simpson 1~). fl«a~ thc Porikr. 
are or... of the richest phyl. for invesligating thc 
b;osynthe!lis of bioacth ... ""tu,al prodUCIS, they 
havc """i"ed increasing aU<:'I\lion frorn organic 
cll<."";515 .nd pharmacologisls (Faulkne, et .1. 
Im, Garson 1994, Munro "I ~I . 1999). Wh",.,a , 
the in~rcelluJar matrix of highe' animals is sterile, 
sponge tiss...., often conlains "Ut numbers of 
microbes, wlUch mayor ""'y not be in"oI",d in 
c.,, __ .... "': F I_,.", ~ '" GOIIio .... ~ 
O.p.'lmut cl G'G.ei.ne .. , G'.blctogy GI""P . 
-,.,_. 3. ~ G(Itt;oogoo., Gerrnony. Tol. +4!1-~1· 
:l87to15. Fa. H!l-SSI ·3oIrnIll.; E_ -""'.gwdog.<IO 
fl 8 .... _eoo .. ,· ' :1 
/JioIoocfIoolc & ~~, 7lj J 'I: 19 1 199. 
001:10.1 O8(W1 0520290310001613042 
Ihe produclion of the secondary metabolit..,; (Un' 
san CI a) Im, l)ebitus et al. 1998, F1",,""S ct 31. 
1998, Gal"!l()n ct al. 1998, Turon ct al. 2000, 
HCTlts<:hel et aJ. 2(01). A oombination 01 dassk,,1 
hlstology ",Hh modern mol«ula. bioIogica.l meth· 
ods hu berome n«esary to ~Iuddate the impact 
olspongl .... assodated ba.o:terl;oo on the metabolism of 
lhe hoost 'po<1ge. Re<:ently, f1UOf'eSCenO> in .itu 
hybridization (flSH) on ~ponge """tlons (e,g., 
Man~ l'l al. 2000, Wet>ste, CI al. 2001) bec~m~ 
popular fOT identificalion and localiz.alion of 
spongc·aS5OCiated b;.cleTia within sponge tiss...." 
Paraffin is lhe dassica.1 cmbedding lechnique for 
light mkroscopk ""amin.otion of any tissue (Ro-
mcis 198':1), ;an(! ;1 commonly is used for sponge 
hi~tology. Cryotechniquet, which im"ol, ... cutting 
frou ... rissoe blocks on • special CJyomicrotome, 
an! faSIeT and ca.ie •. For boIh tl'Chniques, s iliceou, 
or (.!cai"1!OUS Spi(u!es must be remo-'ed wHh hyd-
rofloork add (HF) or hydnxhloric .eid (HCC), 
'" 
re;pectively, to ob\.oin S<.'Chon, of good '1u.1lity. This 
procedure has ",veral disadvantoges. First, it ma k.,,; 
oi>s<;,,,.'ation 01 thc skc1ctal arrangement in lhe 
"""tions impossible . 5e<:ond, il ri~h in sili~oous 
.pirol"". aS most cold-waler sponges are (Barlhel 
1995), the lissue may ~ollap", alter .pieule rem""a! 
Furlhennore, afler HF Irealmeni, FlSH is no longer 
possible (personal <'>l>servation). F"" .pongc sp<.'Ci"" 
lacking a mineralized skeleton, HF treatment can 
be omitted and FISH is possiblc on both paraffin 
"""liorlS (Manz el al. 2(((1) and cryosections (Frie-
dlich d a1. 1999. Websler and Hill 2001. Webster el 
a1. 2(01), 
Ilmbcdding in resin is an alternative l<'Chnique. 
Some methylmelhacrylal~ (M MA) ",sins are uscd 
in hislolngy, usually in kits combined wilh benzoyl 
pero~ide (Romeis 1989). Trade names includc 
Techn""iI and Histocryl. MMA "",ins ha,'e 
b.:en uSC<! Im histolngy .nd FlSH on s ponges 
laeking a si)iceous skeleton (Wagner el al. 1998, 
Maru: ct .1. 2(((1. Böhm cl al. 2(01), Magnino et al. 
(1999) Irealed Ihe s iliceous spong~. T/u>t",.llQ 
8wi"I",.i. wilh HF prior 10 embcdding in Teehn""il 
8100, 
Resins originally designed for Iransmission elee-
IMn microscopy also h.-,ve been used for lighl 
mkroscopy, Cakified biofilms were .tained m 
Mx prior 10 cmbcdding with ei!her lipophilk 
dyes after dehydralion or walc,- soluble dy"" on 
hydrated sampI"", and cul wilh a cireular ,,",w 
using a diamond knife (Reitner 1993, Arp 1999). 
Bhauacharyya et 01. (1999) des.:ribtod a Similar 
methOO for undeealcified rat libia . Embedding in 
lR While combined wilh m biO( staining has OCcn 
applied sucees.fully on spong"" wilh a calcareous 
basal s keleton (Wörheide 1997) and on silierous 
sponges (Reilner 1992, 1993, Holfmann CI al. 20(3). 
An easy melhOO for oblaining """Iions wilhoul 
HF Irealmeni is . imply to Cul lhe fixed, unem-
bedded sponges by hand wilh a sharp sealpel inlo 
< I mm secti"". and k1 lhem air dry (Hoop'°r 
2(((1, Soesl et al. 2(((1), Sections oblain~>d in Ihis 
way are good lor cxaminotion of skclela l .rehile-c-
lure for taxonomie delermination, bul !issue SITUC-
!ures aTC d~'Slroyed. Margot cl .1. (2002), howe""r, 
uSC<! this melhod Inr FlSH On Medilerr.me.n 
axineWd demosponges, 
We 1"Sled differenI mclhod. lor emb<.>dding. 
culting and st.lining using a wide range 01 spong<"S 
from Ih~ Norwegian, Anl,1T'clk and Medilerranean 
Seas, The aim of lhe sludy reported here was 10 
~" 'alu"te mclhod, Ihal a110w preparation of lissue 
_Iions appli"able for histologieal inv<"Stigahon 
o.nd FlSH 01 siliceous .ponges Wilhoul remO\'ing 
Ihe .picules. Although SOme 01 the d~'SCrib<.>d 
embedding lechniques may also be use<! for 
electron mkroscopy, we focus on appliealion for 
light and fluores.:ence mi~TOS<:Opy. 
Materials and methods 
Sampllng lind fixlltlon 
The specimen. used lor Ihi. ",port were coll~"Ct~>d 
during many ye.rs from differenl loc.lities, Spedes 
from Iho.o Sula Ridgc region, Norwegian Sea (M' OS' 
N; 08' OS ' E), were sampled in May, 1999 on Ihe 
Norwegian reseaKh \'essel /01"," Hjor/ using a 
lriangular dredge, in July/Augu'l, 1999 on Ihe 
German research "essel P~dlYrl and the mann~>d 
submersible Jago. Sponges from lhe Kors/jord ""ar 
lhe city. 01 I.Iergen. Norwegian west c"",1 (60' 1)9' 
12- N; OS 08' 52" E) were sampled in Augusl and 
Sep lember, 2(((1, and in Oclober, 2001 on lhe 
Norwegian ~aKh """",Is Hans BraUs/rom and 
Au .... Ua using a lriangular dredge. Sponges Imm 
Sul. Ridge were growing in associalion wilh living 
and d<'Caying oor31 rws 01 Wp),f!iJ> p<'rluSQ , on drop 
.tones or in solt oottom sedim, ... ts nc.lr Ihc rcds 
al 200- 300 m deplh (Frei"'ald cl al. 2002). In 
Kors/jord, Ihe sponges were growing on a hard 
bottom slope 100- 300 m d""p. 
During Ihe deep sea expedition ANDEEp 11. 
28,02.- 1.04.2002, sponges ",ere co11e<:!t.>d from lhe 
Anto"'lic Weddell Sea by Agas. iz Irawl and ~pi. 
benlhic sledge Qanuss-en, in p",s.). In Ihe M~>diler' 
ranean deep "' •• we r«orded large popUI.llions of 
very small sponges. whieh ha"e bo", ... conected in 
the course of sc"eral expeditions, During Ihe 1993 
and 1998 expeditions wilh lhe ~aKh ,""s SEI 
Mr/ror (Me25 and Me40), many ~k>d iterranean 
deep sea slations in Ihe Sporades Basin, Sea 01 
Crete and Ihrough"uI lhe Le,'anl Ba,in we", 
sampled by Irawl. epibenlhic s k>dge and box co",r 
(Hiekc cl al . 1994, 1999), As parI 01 a deep "'. 
monitoring exp<.>dilion off Ihe coasl of l.raeL a few 
Irawl'le,led a new sampling lechnique in Ihe deep 
",a and relrieved very sma11, .pong"S « I cm). 
Spcci~'S used for Ihis ",porl are li.led in Table l. 
All sp''Cimen were fixed in 2% formaldehyde+ 
0.04% glularaldehyde in /ilter-slerilized sea w.ler, 
in 4% formaldehyde in sterilized sea waler, or ~,-. 
formaldehyde in pho!!;phate buffered saline (pBS) 
solution p.-epared wilh d istilled waler. Fi ,~>d ,,",m· 
pIes Subsequ~nlly were dehyd raled in .ln elh.lnol 
seri"" (15, 30, .nd 50'% EIOH in artificial sea water 
or I'BS) .• nd sto .... >d in 70'% elhanol. 
192 Bio/8CIlnic & Hislocllemislry 2003. 78(3 - 4): 191- 199 
Hexactio s"» 
I./a/acos.,.,.."~'" Tt:#'IUi"T 
1910 
c.w~ enl81C!icus Sc>o..o.n " 
K1"",,~-...co< 18\0 
~n.'P. 
-
............,.. 
PlBkoffis ~It ~ 1880 
T.tr~ 
~rI ~S0u.A8 \880 
Geoodiot ~ EIc.Jrrwto_ 1858 
Geodia lI\IIC8IIdnrIoi ~ \ 858 
'SCJ'S pliIIIy&fll Sou.A8 \8110 
1henea murlc8!8 E\owI;_M, 1858 
Thenea n, sp. 
Tantoti1;m sem/IIllberltes s.:-.oT 1870 
TiInklIUn n. 'P. 
Sd>BtiIM ~ RnLy " CkNDv 
"., 
5d>em!l!!i trIIicto$mtnus R ........ " 0.""" 
"" Rhizaxioellll n. 'P. 
C~rI";tio 000 '101 pi-.. 
Foocapi& Itwq:w 8owmoN<oo< 196e 
~ 6i1guII ~ 196e 
~ l.N<.of.Ull159 
PMkeIIi.tJ ....,li/abtt.m P~LU\S 1766 
PMkttllia IObuSt8 So __ M 1866 
~~,.ao....._ 11166 
 IWl/atCtica RIIlLn" Oaov 
,-
SM .... '" 
... 
"" 
SR. 1<0 
SR. KO 
SR. KO 
SR. KO 
SR. KO 
"' SFI . KO 
"' 
"" 
"' 
SR. KO 
SR.KO 
SR.KO 
SR. 1<0 
' 0 
'" 
"" 
C/looodooc:/lldil n, IIP. ANT 
ulnmo:'uli.l ~R'OI..EV " o.c...". ANT 
"" SR. SUt RiIlII*' 110 . KoosIjood; MT. W_ SeIo ~~ 
ME. _' __ I Seo 
Embeddlng, cutting and at.lnlng 
\'Je developed prolocols fO'r 1 2 cm
' 
tissuc blocks. 
For larger sampk'$. tim"" for d~hydration, staln;ns-
infiltration a,>cl hardening m .. SI be longe., For 
~JIer samplcs, the times should be the same, or 
.her experiJT\C1\tmS- may be. little Moner. Em~' 
ding kdmiqut'S e'-al .. ated in our i ludy an: li51('(1 in 
Toble 2. 
Paraffin 
TIS$~ blocks ... ere <'frIbedded in p.liraffin os d .... 
..rn~ ea..Jier (Romcis 1989). To n:rn()\'e air, blocks 
"",1«<1 ,,' ith paraffin ... en' pL1<:ed in . p;oralfin bath 
in a deslccator 10.- a few minules at 60" C. Paraffin 
blocks ... "re co"k-d on a ~ 5' C pb!" prio.- 10 
cutting. Paraffin _tions 3< lO )Im thick w"re cul 
.. sing a rotary mkrot~ HM 3-10 E (Microm, 
Walldo rf. G.-rmany) with single-us.e blades. 
Thick. !ioectiOll5 ca" be made by using I"" trim_ 
ming option on the rotary m;cmtorne and cutt;ng 
each S<.'Ction a i if trimming Ihe block, 1>,HtiruJarly 
fO'r Stud y 01 the skeletal .. chi te"' .. re, whi(h ;. 
imporlant 1O'r taxonomy, 30- 50 11m sectioru are 
re<juin.-..:I. n.e slic0:5 "',,re mounted on siJanized 
gl.>55 slides. dew.~oo by xyleno: tre.>t""",r and 
dried fO'r 3t least 1 h al 60" C prior to 5taining or 
FISH. Staining techniques acrording 10 Coldner 
and Gie~ (Rom~is 1989) ga'-e gaod resultl;. 
SKtioni wc"" mounl~'(\ wilh Biomount or Enteibn 
b;,lsam (Plano w. ''Iannel. Wdzl~r, Genruony; 
Merck. Dannsladt. Germany) and """,~ under a 
light microscope. For 4·.6-diamidino-2·phenylin· 
dole (DAI'I) staining, se<:tions ,,"cre COIWed wilh 
3 1- 3 )lg/ml DAPI So.llution fOT S 10 min in the 
dark. rinsed wilh d istilled watcr. mo unted with 
CitifluOl" anlifading agent (Plano W. Plannet), and 
"""'e<! under an epifluorecence miocl"05COp". 
Cryolechnlques 
TIss .... bloch wcre cmbedded in Cryornatrix (Shan. 
don Inc. J>ittsburgll. PA) as dcscribed by Ihe 
manufxw ...... Frozen bIoo::ks Wer<! stored al - 20 
C, sectioned with , cryomkrolo.ne (MIkrom) al 
- 2() C. Dnd placed on ro., ted g1ass slldos (Cryo-
Frost, "i~""r). SKtions were ai r dried al room 
temperar .. ", Im 20 min prior 10 5taining and FISH_ 
TIss"" blocks were cmbedd ed in TL'Chnovlt 7100 
(Plano W. Plannet) as d~'!ICribed eMli"r (Manz eI aJ, 
2000). s..'Ctions S- 10 11m thick w~'" cut uslng a 
ro!.uy mkrotome as deKribed aoo.~. 5edions 20 
11m and thkker ""ere cut usinga circulnsa ... (LeM:a 
1600) with diamond knife. 
Fm cmbedding with H;'tocryl (Plano W. Plan-
n~'1), dchydraled Ii ...... blocks we r<' infiltrat<!d with 
Histocryl and benzoyl peroxide as des.:.ibed by the 
manufactu_. SampIes ,,'er<! transferred inlO thc 
poi ymo::riution aolutlon (10 ml Infiltration 
soIution+ 15- 30 1'1 a«eLerator) an<! immediately 
cooled wlrh running I~b water for S min. S"mples 
Histology 01 siliceous sponges 193 
P.,allin CryarTlllrri. 
""'" """-
Crye.-nlUoty m~ 
-
"''*",. 
....... SiIanI • .., 1IicIH. ... ~. 
-............. 
''''' """'" dWlgmedi .... 
remained in the .... aie' baih for a few hours unli! 
hardening was rompi".... 5«ti0n5 S 10 11m Ihick 
.... "'" C1JI using a rot~ry mic:rotome, and thiel<er 
"""tiom w~rn cul using a circul~r ".,.... ....ilh 
diamood knik as deKribcd abo\~. 
5«tiOO1S "-ere fi~ed 001 glas. tlides by he.1 (60 C 
for at least I h) for su""-"lu .. ,,1 sla ining and FISH. 
lf staining is d""i"-><!, lissue blocks muSI be st~ined 
beio .... embo>dding in L~ Whlle. We dewlope<l 
prolocols for toluidinc bill" 0 (CI. 52().1(), (akein, 
and DAI'I as folIows. For slaining wilh to! uidin~ 
bluc 0 .nd Cak.,in, lissuc $o1mplt"J were d~hy­
draled in an aset.'llding EIOH series. "rom 99'}:. 
EIOH, sampIes were placed in IN: ;,Iaining solulion 
",emight (Ioluidin~ bl~: 1 g / lOO ml EIOH; Cal-
<cin: ".,Iur.t.><! 501u lioo In EIOH). Sampi" were 
w ashed 3 4 times in EIOH bcforc "'1I1.bo,·dding. Fm 
DAP! st3ining.. tis,ue blocks were plan'" in a 10 
I'g/ ml DAI'1 in 90% EIOH s taining 5OIulion ()\:e, · 
night. SampIes weN washed In 99% EIOH, and 
dehydrated in 99% EtOH fot a fo.. .... houl5. TIssue 
block1o fot all s\.1ining ttdmiqucs wern infiltratl'd 
.... ·ith 1:2. thcn 2:1 tR Whitc:EtOH far alle3$1 18 h, 
then embcdded in LR Whi'" and hardened For 20 h 
at 60 C. ßecause all d yel d~ribcd are soluble in 
both EIOH "nd LR \Vhite, infiltralioo solutions 
should ron\.1in a minor amount 01 lhe uwd d)'e. To 
rffiuct' uninll·ntiOll.lI t'Xtraetion of d~ during the 
embedding s teps, il i!I I1oo.lpfullo u~ EtOH from 11100. 
w~ng Siepoi to prepare infiltralioo soIutioos_ 
TlSSUe """tionI; from 20 11m to 1 mm were C1JI using 
a circular SO"" wllh a diamond knlfe. Sections were 
mounted 001 gJ~55 slid~ .... ilh Biomounl b.l l~lm . 
Fluores«"I\CC in silu hybr;d;7.~lion (fISH) a nd wide 
fleld de<;Q1 .... olulioo "P;nu~ mk.-oscop y 
w","" ""rformed on spou8c lii.iue WlClions as 
d<'S<ribed "arlier (Marlt ~I aL 20)(1). Sampk'S wer" 
....". T ecMoviI 7100 eR .... 
Rotaty microtome AoIary " ... '*",. CifaJIoI, aaw 
~~W c:ifaJIa, saw 
-, GIue. heili ...... 
..-HoI, __ 
"2'1' ... ~. 
viewed under a Zeiss axioplan micrusoope (bi"" 
überkochen, Genn.any). Micrographs " "re taken 
using a CCO Camera and processe<l wilh Meta-
Morph. 
Results and dlscusslon 
A romparison ot lhe metho,Js d"""ribed concening 
embedding.. section quality and slaining tl'Chniques 
is shown in Tab!e 3. 1he embcdding process for lhe 
cryol,ochniquc is q"ick, easy and ",I;obl". The othcr 
methods la k" long"r and a,.., m"", (umpi"" .... ith 
many pitfalls. which may caullC un~"'p'-octed trou-
ble. Most of th~ diffkullies, howe""r, Can b<o 
oven:ome by joOme Iraining and standardi2ali<m 01 
Ihe m<:thods. 
The besl spong" !issue """lioM induding s llic-
'-'Ous spi<:ull'5 arc oblained with LR Whit" block1o 
cu t on a cireillar saw; valuabl" inFormalion aboul 
sIo:eleloo slmelure and tissue-skeleton arra"8""">'" 
can be achle,-ed using Ihis me1hod. 5...:lions 20 
HXXl 11m aTC cul, and can be ground to appro~i­
mole1y 5 11m. hen uJlrarnicrotom" !ol'Cliotu for 
electron microscopy can be obtaincd from spieu· 
laled tissue blocks (Uri2 et a1. 20(0), bol the knift. it. 
usily <famoged if spicule!i are thiek and numerou,," 
Prior DAPI sldining allows "isualizatiOO1 of both 
differenI sponge cdJ typ<'!I;md asM>Ci.ot..'<l b;\Cteri."o 
(Fig. 1). 
The calcium binding fluorochrome Catc..in i!I 
oommonly used in gro","lh~ince labeling ('><peri-
ment/i 00 calcareous skeletons (Rahn 1976a.b, 
Rcilner an<! Gautrel 1996. 'M!rhcide 1997). Applio'd 
10 cakified biofilms. CaJMn grcatly enhal\Ct"S Ihc 
conlrasl of. for examp"'. ",",,11 """phototrophie 
b<Kteria, aod labels Ihc surf;>Ct.'S of carbonate 
cry<;lals (Reitne1" 1993, Re-ilner et al. 1995. Arp 
1999). In silk<,<>us sponges. lissue contrail is 
enhan<:ed and different cell types aOO li;,;,ue 
siruciures are d .. arly visibl .. (Fig. 20). Slaining 
with loluidi ..... blue 0 ..., /Neo: cannol bc rerom· 
m( ... ded fur sponges wilh d .. ",,1lC tissue and numero 
ous spong"'associal~>d Metcria, t"'<:aullC I~ 
1!14 Blorschnic & Hisrochemisrry 2003. 78(3 4): 191 199 
Tabla 3. Compa riSOO 01 embeddi ng methOds for siliceous Spo<1g<!S withool sphJte removal 
Paramn Cryomalrix Hislocry1 Techoov~ 7100 LR While 
Embeddir>g operat",n , - , , , 
Sec1ion q!lalily , 0 ++1+ +' +1++ ' H 
Mintmum SIlCtion Ihickr>ess (~m) , , ""'. 
, ,,",' 
'" FISH on section H -, H H 
DAPI Slaining on seclion H -, H H 
~, Giemsa sla inong on secloon H , 0 0 
.... very good ... good: 0 poot: 
Fig . I . DAPI SI!'ur>e<:t 20 I,m LR Whfte SIlCtions of PIlakellia ventilattrum (a) arocl PIilkO<1is simple~ (b), Choanocyte 
chambers (ce). canals (ca) . and ert<!opOnacocytes linong ca nal wa lls (~) are VISible. Thls lochoique clea ~y show!! 
diHere"""s in tinue slr""ture and clenS/ly of BSSOCiaied mocroorga,,;sms (tiny OOts in lhol ~ssue) in theS(! 1..0 sponge 
specles. Bars _ 30 I,m. 
liSSu~., MC " .'Sily 'I\cn.ta ined. Th" pruto""l e,m be 
~djust,od tu the sp.,<ics u' inlc rcst b)' in,,-',tin!; more 
,md lunW" w~shi ng stc'ps 0' b)' d ilution 01 Ihe 
sl.,i ning solution. High cont,.1S1 ligh t mie'O>iCopy 
in",!;~'S c.,n bc obl,lin",j using Ihis mdhod (Fig. 2b). 
Block st~ining. ho""cw,, incre""-,,, thc ~muunl of 
liss",- ",mpie' n,,,ode<l ",hclle"" sc\'Cr,,1 ,t"in illg 
t,,,,hniq"'" are In bc appli,>d on the ",me spo"g" . 
. \1.l tcri.,t a"ail"biHty may be a lim iting 'aeto" 
"'p<.<:i., lIy when d e.'li .. g with le'r sm., lI « 10 
mm) spon!;", such as jU\'cnilcs "nd S()me deep sea 
sp<.<:i,.,.. Oll'ing tn Ihe hydrophobie eharaete, and 
high d ensily o' the ,,·,in, FISH on LR White 
,,-"'tinn, i, impos..iblc, Olignnuclootidc prob.!" 
u,,->d I", FISI·j "re waler .;nluble and will nol 
penet,., te Ih,· ".,.in. The ",me is Ime lu' DAPI 
.md hislologie,,1 st"ins in "'lu<..:>us Whlli<lT1. 
Using a rotar}' mkrotomc I\'ilh sharp st,'C1 knile. 
tne Ix.,.t ..... ",tion quali ty w.lS ,*,taine<l with Hi>!(>-
n)'l . This I\'as tTU" alS()'M sp'-"ic,> "nd ti"uc p~rt, 
e""tainin;; many spieu l,·s, c.!; __ thc coTtex v' C .. ,Id,,, 
sp<"ic"' . Sc"li"ns d"",n 10 5 ~m " .... e suitable 'nT 
FISI·j (Fil\- 3), S,xti()T\S "I laryin;; Ihicknc'S> "nd 
'lua lit y wc'e obl., ;nc,<j 'TOm T,,,hnOlit bl""h, but 
p.1TIS """bl,· fo' FISH co"ld bc found on most 
..... "tions. Soxti"ns 01 beUeT q \l~lity "'<'TC obt.,in,'" 
" , ;ng .. eireut"r "'I\'. Th,,,,-' ""Iions " ·er..· rel"t il d ), 
Ihiek ( > 20 I,m), ",hieh m"k~'S in k 'pretat;on uf 
FISH .lifflc" 11, b" t the th iekn,_ I, .. <lI .'nl.'gl'O'" Im 
stud)'ing skelet .. 1 ,,,ehi l,,,I,,,,,, FOT OOscT1.,tion, uf 
thc skell'ton, "-,,,lion, 30 500 I, m are n,,,,<,,,,,,,y. 
Be",.,,,",, of the incomp.,tibil it)' 01 l- IJI,IA ,,-'Sin, with 
EtOH, onl)' 'quoous . t.l ins C.ln b<> used for Histo-
eryl .lnd T,,,,h"''''il "-,,tions. Unfnrt "n.,tely. wc 
nbSCf'\",j incompll'te ~nd irregu l," ,t.,ining wilh 
Gold,,,,r .lnd Giem", in both ).,Il-·IA ,,'Sins, c;.,rrits 
(1992) d,oscribed gond "",,,11, lor dill"Mn histolo-
gica l ,taining of soft ti<Sul' embedd,od in T~",hnO\ it 
7100. The ' ., il u .... o. nur attempl< m.,y be duc to thc 
"'ugh "nd "n~\t'n surl.,,,,,, 01 ,,"Iinns o' spieu l.,wd 
ti""c, In Hi,tocr)'1 """hon" wc ,..,metim", 
'*,"'T\~-.J ma",i,e "tain aceumutahon, bt:hH",n 
Ih" Spi"lk'S. ",hc'''", th,· su""unding tiss"c Tt.' -
m.1 in,-d 1.1rgdy un,tainc-d. F,n par.,!iin "-"'lion,, "',. 
Histe>logy 01 siliceous sponges 195 
Fig. 2. (a) FluorOChromo image (Ca lc<li~) and (b) lighl 
mlcroSC09Y image (lo1vidir>e tJloo 01 01 Pha~ellia venr~ 
IabllJm on 100 ~m LR White sections. Bundles 01 sp;cules 
(spl !ypical lo< lhis species witt1 accompanyir>g ceIIs (ac) 
are .isitJIe. Note choanocyte chamoor$ (ce) and <:aMis 
(ca) in tOO cOOaOOSOlTl8 and differenl ~I Iype$ in lhe 
ectosome (~) et the sponge su~ace. Bars _ 100 ~m. 
~n'ed that the time ,,~>eded for slain;ng ror"-b 
laled negalively wilh se<:lion thickn~'Ss. It ~ms 
thaI lhe spOllg" lissue in Ih ick 5<'.:lions binds lhe 
.Iain mon' n'adily Ihan il d ucs in Ihin ,;.;..:tion5. 
T'.Ha ffin 5<-..:lions wilh""t prior diSS.,)ut ion of the 
si !i~....,u. skeleton were of poor qualit)" in liss,,~ 
parIs Ihal conl.,in~><;l numerouS thick '" Inn,; 
spicult'S, c.g" Ihe cortex of Gt'OJ;a or Ihe lissue of 
",me Hexactindlida. ){l'Sults \wre bt,tter for lissue 
wnlaining few or small spicule. (Fig , 4), a nd 
allho"s" lissu<' S<.'Cl ions .1Iw.1)"s wc'" parti)" torn, 
usabl~ a",aS fnr FIS)'( and histologieal 51a ining 
could be found on mosl "",lions. Carcful C<Xl lin ;; 
of Ih~ par., lfin blocks prior to ~utting and the use of 
a new blad e /or each cutting ,,'mI enh"""e<l 
Fig. 3. Fluorescence in s~ u hybridizat"'" (FI 5H) 00 a 5 
~m Histocryl &action 01 Goodia barreW. Altho<Jgh this 
sect"'" was cut lrom a high'" sp;culaled Ussue part 
<lirectty I>e..eath 1he- CQrtex , tiS$OO Sl"-"'tures as canals 
(ca] and a large spor>ge eell (eeß wirh indusiens. cw i) are 
cl&arty visible. Tl1e oIi900L>CIeotide probe EUB3Ja 
IAmann et a l. 1990), co<Iing tor a ll eubacteria , shows 
n;gn dens;". 01 associated bacteria in 100 spange liss.ue, 
Bar _ 30~m. 
5<-'Chon 'lua lit)". Paraffin s<:ctions with the spicules 
in situ are p rekrable, esp<'Ci.' 11 y for \w)" sma ll 
spong<.,; where thc s.ample materia l i. limite<! , The 
<'xI,,-'mel)" sma ll spi,u le.,; ca u.., .,lmost "" d is ru?-
t;on 0/ Ihe tissue, so c'llli .... spongc'S can oc 
embeddcd. casily cut, .nd subsequently sta incd 
wit h minim.1 1loss of mater; al (Fi);. 5). 
\-'.,r)" p<JOr se<:ti<.mS"'" obtained from cryoblocks. 
The spon;;" tiss"" is enlirdy lorn, and no tissu," 
struaur .. -.,; are I'isible, Thi s probabl)" is due 10 
d iilcrcnc{'S in toughn(.'SS beiwcen Ihe spicules and 
Ihe emocdd .. "<l tiss"". H islo!ogi,a! Slaining 01 Ihl"'" 
.....:!ions was not ouempled, bllt applicalion of Fl5H 
p rebes a nd I)A»I sra inin g ga, ~ deM signals, 
Inform,uion gained from these "",tions, ho,,"<'\'CT. 
waS Cl'mp""ble to !hal from homOj;cnized sponge 
lissu", IV<' ""ume Ihal Ihc qualil)" of cr)""""lions 
ffiu ld be improR>d by \'arying culting lemp.,ralur<' 
a nd more pra,ti,c , 
For oll tc..:hniques described here "sing rola,y 
microtom .. 'S. ",-'Ction quahty was ne;-c, a. good a, 
that obtain~>d wilh p rior acid t,,-'~tment. Althou);h 
S(lme histol Oj;i~a l slaining cou ld oc appli<>J sue· 
ce»fully on spon);" S<. .:tions w;th sili''''''''5 spicuk'S, 
wc R..:omm~nd spicu l ~ rcmü\'al für hislulo6i(~1 
imeshW,tions IVh~"" "",!ions < 20 ~m are n<>ed~>J, 
<'.g .. for s tud)"ing fine hss,,,· slr"c!u"" . jI,·lelhods Im 
embedd ing an d cuuing si liceous spong~'S should 
bc chosen a"mding to thc aim 01 in\'esligation and 
196 Bioleclmic & Hislochemjslry 2003. 76(3 4) : 191 199 
FIg. 4. T""I_ 01 ~ SIl. from lhe AnIa,ClIC 
Weddell Sea. appt<>.Umate/y 4970 m <Ieep. _ in 
I*aftin . .-ioned at JO ""' _ atained """"""'11 10 
GoIdner (Ror\'I8q; 1969). ThS '-IOn .-. lhe ce"lIf. 
c:ore witI"I ~es (11yIes) errtw:lddo<f in spongin 
blMfl; andlhe surrct.n:IinV .'u paci<ed wiIII "OCI--'" 
(iIocf',oNe). Bar _ 250 ~m 
Ihc prop",li,.,. ollhc cuminl'd spt'Cies; the ... ;, no 
starod.,d melhod a\ailablc I"'~I fil5 all r>I'<XIs. 
Fot an ",."..,;"w 01 ri""" arod sli;o,k .. ton arran!;<"'" 
""-'111 we ~ LR Wh;l':: ernbedding rom-
b".ed ... ·'lh n> bI« 5e~,ning and culting with a 
drcular sa ... ·. For '-"ry small ~ponges. " 'e rewm-
mc."1Id p3laflin embedding of a cenlr.1 longiludi",,! 
CIlI IhrotJgh lhe ""li ... spong" wi thllut prior acid 
ln.'3tm~nt. .nd CI,t1 ing or lrimming wilh rotary 
mlerntome 10 oblain djflc,,-~u SI...:li'm Ihid",~sscs. 
I'or 1'ISH on siliceous spong~"'. we """mmend 
HlStocryl embedding ror sl"'Cio.'S or I~"" parts 
",llh high spieule conlenl, and para ffin embedding 
il ehe sp;eule conl"'" i, k>w. Sr<1ions should bc cul 
",Ih a roI3ry mkrotome 
ft), det.iled !iss .... "".mi""l;on. wo. ...,.,.,..,mend 
dassial hislologie.1 mCll\ods, Lc .• spieule ... moul 
byacid prior 10 embedding.. cu!ting .nd stain,ng. 
FIg . ~. Axial cut 01 TlIrlIOOum Ij). from the MeditelTalllNln 
SN. Easl,,", levanl Ba"",. "I'PfO.UMhI~ 1200 m oeep. 
.,_"hoJ in I*affin MCtionttd at 30 I""- and ........a 
-=oordihO 10 GoIdner tRomM 19691 TIw oectIOn Ihrougtl 
lIIe entlfe specrnen shows lIIe 5PQIIe orianIaoon and IN 
0fi3NC basal plahl '" ......-gen ...., wIIC:h sorne 01 Iht 
...articaf lj)ICt.lkts ~ (.ffOW). Ba, _ 250 ~m 
Acknowledgments 
I\'c Ih3n~ the c ....... 01 "-~3rch H$sds POS<'IolOII , 
Io/JIIII H,."I , HQm 8raIl5l"''''. and the I\.,.ri"" 
Biologie,,1 St.tion 01 ~.." Uniwrsily. Arreso 10 
ehe laller '"-0 insl.3llalion:l .. ·at ~upporkd by lhe 
1111' (I mp .. : ... ing Human Keso:.>an::h l'otenti.l!) Pr0-
grAmm" frum the Eurore •• n Union Ihmugh Con· 
tract No. HPRI-Cl"·I999-«1056, This pa"". 
r.::prt.'SCnts publkalion no. 30 01 Ihc ,,-"SCareh 
l''''IIram IIOSMr\N (03F0358 C). Financi.ll support 
.... ~! p,,,,'id <.'d by the Bundeolminist".ium /(iT B,I · 
dung und Forschung (B \IßF). Germany. Wc lh.n~ 
lhe organill'rS 01 ehe ANDEEP-Exp<.'dih<ln!l. IV" 
lh~nk Hans Tore Rapp lor a S6lS1ancr " 'im samphng 
and identification 01 ~ponS"'. Katj.. Schumm for 
,aluablc and in"",,-ali,e I~"Chnical assisl.3ncr dunng 
the c~per,mental .... 0.1<. Gabn.::la Schumann_Kindcl 
IOT introducrion to FISH "n sponge """Iions. and 
Histology ot siliceoUs sponges 197 
1111 M.1r<J"a,t lOT assistance with the cryomicr or 
t""'c_ 
References 
Am.". IU, Krumh.,l. L, St.hl DA (1990) F!uo"""""t· 
oIigo.mdCOlid e p..ooing 01 " 'hohl C<'lIs IOT determinath'e. 
phytogenelic, and em'i"",,,,,,,,.a l stud i"" in mi.crobiology, 
I. &u1 .... "I. In: 762-770. 
Arp C (1999) ClJdjia>riim of N""-""'n'" C!!"nobo<f,,"1 
Biofilrn. (USA, PR Chi"", Indo"",ia, Cm''',"y) - I",pl",,-
lion. for Iht! inl"P .. lolion of jos>il micO'()/NlIiI"" Ph.D. 
Thesis. Uni>tlSity 01 Götlinsen. 
8.rth~1 D (1995) TIssue romposition 01 spon!\'-,< f",rn Ih< 
lVeddell Sea, Ant.:lrctica : not much me.' on the bon..-s. 
Mn,. [00. Prog, Sn. 123 H 9 _ 153 
8h .... <haryy. T I(, S.,,~hu B, Tonurni D (1999) halua -
.ion of LR IVhite r"'Sin IOt histolog~ of the und,,,.kifitod 
.at tibia. Bioi«". "Hi,/ochnn, 75: 7 14 
Böhm M, lIen""".1 U, Friedrich A8, Fi.s.der L. Sldf.n 
K, C.m ul in V, MOlle , IM, Mlill er WEG (2001) Mole-
cu!. . ""pon!iC 01 ..... sponge S~""'i/ts do","'''"I. '0 
ba<teri.1 infr.'CIion . ,1.111" Biol_ 139: 1037 10l5_ 
D.bi .us C, Cuell. C , M.ncini t W.ikrd", J, Guem .. 1-
P, NIe"I • • )L. Pi . t ,. F (1996) Quinol""'-'S lrom " 
bacterium and Iyms ine mctabolitcs from its "ost spong~, 
Su/\' ..... rrm. Imm lhe Coral Sea_ I- Ma" lJioI«hnol. 6: 
136 141 
F.ulk"u )D. Uns"n MO, B .... lt)" CA (1994) 1he 
eherni.try 01 ..,rn~ .ponges and I .... i, symbiont" Pu,,' 
-"/'P1. CIIrm. 66: 1983- 1990. 
Flowen A E, C.""" MJ . Webb RI . Dumd.i EI. Ch ... n 
RD (1998) Cellulor origin 01 ~hlorinato;d dike!opipe'd-
, ines in lhe d iclyocerot id sponge Dy>idea herl>lmr (Kel-
le,)_ 011 n ...... R ... 292: "H7- YJ7. 
F .. iw.ld A, "Ühn~rt>.eh V. lindbe rs B, \'Vil..,n )8, 
C.mpb.n J (2002) 11>< Sllia Rod Complex, Norweg;an 
Sh.>U. FlIC'" 47: 179- 200. 
Fried ri<h AB, M .. k.rt lt F.nd.rt T. H..,k~, J, Prokll<h 
p. H~nlll<h.1 U (1999) Mi<robi.l di''''''ily in I .... marine 
' ponge Aplysino ru'<"I"niOOa (for"""1'ly Vmmgir> 
"",,,,..,iroI.» .rtaly''-'<l by Auo"-"",,,,,< in .ilu hybridiza. 
hon (F1SH). Mo,. Biol. 13-1: 461 - 470, 
G.",,,,, MJ (19'94 ) The bk"ynülI'Si. 01 sponge ""-""'\d'ry 
metabolit,,,, ",hy it is importa",_ In : SI""'S'" in Ti ...... nd 
S"",< , ; 'an Soe;. RWM . ,'an Kempen TMG. B,ack"",,, /. 
Eds_ & Ik"", •• R<:>\W,darn. pp. 427- .1-\(), 
G ...... " MJ, F1,. ...... AE. W.bb KJ, Chuan RD, M,C.I· 
I",y EJ (1996) A sponge/dinoflaggclatc aS5OM.ti"" in the 
haplosclomd dern ... ronge Holiclarw ' P: cellu la, origin of 
<ytoloxic alkaloids by Percoll density gradieni 1, .ctiOn.:J · 
'ion, Cdl Ti"u, Rn, 293: 365 373_ 
G. rrits PO (1992) V"fi.-l,"n ""' Fd,"'ms "''' c;..".-,./\'. da,;n 
2·HyJroxy<'t"~J-mrlO"'JI1>1 ,,;nstlxt/,I wird, He,..,.,. Kul· 
,~." Weh'''''im/Ts_ 17 pp, 
Henlsche1 U, Sch mid 10'1, W.gne, M. Fi . ... I.r L. Cem~rt 
C, !l.d .. ) (2001) Isolation aOO ph ytogene" c a nalysi, of 
b,,,,,,,,ia ",i!h . ntimicroblal octil'ities frurn It.e M'-'<lile'''' , 
""an sponges Apl!!,;"" a~ . nd IIplys;"" ",,,·,,,i<1Jlo. 
f~MS Mirrobiol. ~col_ 35::lOS 312_ 
!Ii .. k" W. lIalb. d. I'; rtirkay M. Wi.k~rt H, Ed., (1 99-1) 
Mittel"""" 1993 CTU i ... NO, 25, 12 M'y-20 August 1993_ 
Mrlror- f!<'<ichl' 9-\ - 3, Insli lu' lür Meereskunde. Uni,.., .. 
. ität Hamburg. 
Hieke W lieml,oOC'I1 C. U nke P, r';,kay M, Welker! H, 
Ed., (1999) Mittelrn..,.. 1997/98 C . uise No. 010, 2!! Ctctobo>. 
1997 10 Fd>ruary 1998. Mtttor-fkrkhir 99- 2, [ .. Iitul 
lü. M,,,,,,,,kuOOe, Universilät Hambu' g_ 
lIolfm.nn F, M. pp 1fT. Zöll" T, Rei tne , J (2003) G",wlh 
aOO regeneration in ",,1th'ated f,agments 0/ t .... boreal 
deep wate, sponge c;.",.JiQ bo"",li fIo;o.·,>rt>.nk. 1&58 
(Geodiid ..... T,1,..,tinellida. Demooportgiae), /_ 8!Ol«hno/ 
100 l!W- 118. 
Hoope' ) (2OOJ). "Spo".~ujar" - G"i<k /0 Spongr Co/leet"", 
and ta"'lificahon . Interne' hup:II ..... ·w_q mu""'"rn.qM.g<>-
v,a ulna tu ",wekome 
J.nu • ..,n D (in press)_ First report on the deep ""3 
"",il<:,. Imm .he Northem Wedell Se •• 00 the .Iore of 
South ",ndwich Trench . fkr, P'*rforscIt-
M.g nino C . S .. ~ A. hndoni T, G a in,. E (1999) 
End"bi,,"I. of the co,. l rccf sponge Throntl1> ,u'n!wri 
(Porife,., Demnspongi..,)_ In,m. 8W/, 118: 213- 220. 
Man. W, A, p G, Schumonn·"lnd el C , S • • wzyk U, 
R.itn • • I (2<)))) Widdield decon;-olulion epifl"",""""",, 
rnic""""'f'Y c"mbincod w;lh ßuo",""",,,,,, in situ hybridi-
za~on ,ewal, .t.e ",atial ...... ngement ui bacteria in 
sponge ti ...... /, M O:roltiol. A-lRthod, 40: 125- 13-1. 
M' rs"l 11, A<eb. 1 C. T"ri[ E, Amil. R, Pu~nt •• JLF 
(2002) Consi' tenl aSS<:riioti"n of "",,,,,,,,haeal A"'h ..... 
wilh .portg'" of .he genus Axintll. Mlr, lliol , HO: 739 -
'" MURro MIIG, Blu nl JW, Dumd .. i EI, lfi eklo,d 5111, Lill 
RE, Li S, B.tt.rshill CN, Duek worth AR (1999) The 
dis<O\'ery .nd d.,,'dopmen. 01 mari"" rompounds ,,·i .h 
ph,,'rn.:><'Cu' ical po'ent i.I , /. BioI","",>/, 70: 15- 25. 
R.hn BA (197M) Die pol),<" """" F[UO!'ffi>:enzma.kie,... 
ung des Knochenbau •. Zri" I"fo,",olilm 22: )6- 39, 
R.hn BA (19761» Die polychrome Sequenzmor"ierung 
des " """""ns. No", lI<la Lro)>O!Ji"" 4-1: 249 255_ 
R.ilne, J (1992) Corraline sponges: An .tt~rnpl "I • 
phylog"""'i< taxonomie analy>i •. &,1. Grou'so. !lbh 
(Ei I : 1- 352, 
R.i tne , J (1993) Modem "ryp'ic microbiaJi'e/m".":wa,, 
lacies fmrn IJ7.ard Island (G,eat EI.lrrier !Wel, Aus.ra li.) 
formation and ~ts. For;.,s 29: 3 40. 
Reiln~, /. Go"lm 1'. M,rin F. N. uw.il" F (1995) 
Au tomicrites in a modem ma,ine mic,ooiali,c, Forma· 
ti"" model ;'i. "'gank malri<'es (UZdrd Is[and, Creal 
EI.lrrie, Rod. Au",.lia), 8"111'1;" .u 1'I,,"ilut 0.:"'''''8"",. 
piliqu< M,m"", 1~: 237 263. 
Reiln. , I, C.utret P (l'i%) Skelel.,1 formaHon in .he 
modem but ull,,,,,,,,,,,,,,,-.I;'-.- choetetid sPOOl>" Sp'""," 
Im1> (A"wlho.:'-tcl".! wcllsi (DernIJSptrn!5i • ..." Pod ien ), 
rod,. 3-1: 193 206. 
Rom.i. B (\989) M;krosl:opi,d.r T,c/mu;. Uro..n u 
5cl1w.""" .... ,g. München, Pf" 120 13-1, -IM, 499 500. 
Sim p..,n TL (1'iS-l) T/o, &11 Bi%gy cf Sp.ms". Springe,. 
N~,," Yo,k. 66-1 p p .. 
Soe.t MWM •• ri't"n B. Mom>w C (2OOJ) S/",n!>", of /~ 
NOrlh fA;t Atlaillie. Expe,1 Ce",,,, 10, Taxonomi< Id,",ti-
flCa.ion, Uncsro l'ubli.hins. I'aris. 
Turon X, BK "'ro MA, Uri. MJ (:!OOO) Dist,ibution 01 
b'om;n.'ted compoun d< ... it hin the 'portge Aplysi'lII 
198 Biotechnic & Histochemistry2003. 78(3- 4): 19 1 199 
...,..".noo.: """pling cl X·ray microo.""lysis with 0'JI"r. .... 
lion lechniqueI, Cr/I r", ... R.,o. XII : 31l _ 322. 
U"""" MD, lIollond ND, r."I~n er DJ (199-1) '" bn"";· 
""Ir<! ~ry mo>IiOboIi ... oyn,t..,.;>ed b)" tho! cyano-
bKtrnol0Y"'bion. 01. 0 mari...,!f'O'W' _ ~mua.tion 
cl Ihr cry>Iallinc- ~i.., in .... Sponge ........ A!.., 
800/, 12~: 1 10, 
Uriz MI, 1\ ... "" X, Se.,.rro MA (2000) Silk. d~1I1on In 
Demosponll"" .plculoj;cn .... ' in C .. mbt c .. mbt . Cr/I 
Ti;.; ... Rn. 301 , 299 309. 
WOS""" C, SI.ff .... Il, Ko .... ioJ C. htel R. t.com .1.1, 
S •• illh.lrt H, Sim •• T, Müll .. WtG (1_) Apop ... io In 
m.o~ >ponSJ'S' a bioma,""", for "".'i""""",,l;oI . . ..... 
I<admium and b.oc1.:n..), Mar, 800/. J)l: U 1- ~21, 
W.bs ... NS. lI i1l RT (ZOOI) Thc culluroblc micr\bl.1 
comrnunity 01 tM C",. , I:Iarri<!r f( • ..,( .ponge RIoJrIoI""iJ .. 
odctQ!otk iS dominah.'<l by an ~·prolcobac.crium, Mar, 
Bi<II, I:JS: g.o 85l. 
Webs •• • NS, W.U. J EM, 11111 KT (ZOOI) o..'I""Iion_ 
pIIylogenctic ..... I)'5i3 01. _-..I C""",tchooo4e and Eu ... 
pn:"- HiS ~ RI'':A S""" oeq~ from • 
GtN. Borrie< RM..,.,..ge. AI •• , 800ttr10 ..... 3: 600 608. 
W. bOl • • NS, \\-1100 .. KI . BI..,kall U . 111 11 RT (ZOOIl 
Ph)'logencllc di.'ersity of b"cteria ",oocia.od wi'h 'M 
m •• i"" 'I""W' RloJrIolorilh odOfilbil •. .... ,,,,,, fn,-,,,," 
M..-rt!l>ooI. 61: -lJ.I -1-1-1. 
WM,.I ... G (1997) Tht RLtf c.-. DwfI,"8" U/ ... 
"" ...... tI ... CDmoI, ... ~gt "" ..... /roao Il'iIU'O ... 
,_ 1900 """'" u.. I .... ,'cifi<. Ph.D . • h<:tif.. Uni. ersity 
04 G6t,i"llCfl 
HislolOgy 01 s.iliceous spot>ges 199