Embed Size (px)
Citation preview
ths Lirz c%m»B m m AQvmio
• Professor U 2'ajor £>ro£«*sor
Minor rof«3aor /
1A Director of of Biology
^ean of the Gradaat# school
TKB LIFE CYCLS 05* A» AQUAffXC ACTZIfQIfVCKZS
2HSSXS
Pre®«itna to the Graduat# Council of the
North T«xa» Univeraity in Partial
Fuimimaat of the $«qpilir«mftt»
for the Degree of
MASTER OF SCXlfflCi
%
Miahael L@® Biggins, B.s,
Dmtar*, X«Ka»
June, 1964
TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS.. iv
Chapter
I. INTRODUCTION. 1
History Statement of Problem
II. METHODS AND MATERIALS. 7
III. RESULTS 15
IV. DISCUSSION.. 29
V. SUMMARY AND CONCLUSIONS 38
BIBLIOGRAPHY 40
ill
LIST OF XLLUSSRAXXOMS
Figure Pmg®
1. Design of a &lide Culture ........... 9
Plat©
1* Hyphal Attraction Between Two Simular Spores 16
2m Production of Secondary Hyigfom From on® Spore 20
3. Hyphal Attraction Between Two Dissimilar Spores#., 22
4« Hyphal Attraction Between Three simular Spores.. • • 23
iv
CHAPTER I
INTRODUCTION
The germs [email protected].. has bean described as being a sa-
prophytic group of the Actincxnycetales growing in the form
of a much-branched mycelium which terminates by forming chains
of spores an aerial hyphae (21)• Until 1950# members of this
genus had not been reported in American waters* This discov-
ery was made by Silvey et al,(19) in their studies which link-
ed these aquatic organism to the production of unpalatable
tastes and odors in water supplies of the southwestern United
States • In later work Roach and Silvey (17) proposed that
these aquatic streptanycetes possessed bisporulative charac-
teristics, Which were regenerated in an lsogamous pattern#
and on this basis separated them from the other genera found
in the Family contrast, terrestrial
streptanycetes have received a great deal more attention
over a much longer period of time. This interest has been
stimulated largely as a result of the antibiotics produced
by this group.
As a point of reference and possible contrast# a brief
review of the major contributions that have been made in the
developmental morphology of these terrestrial forms will follow.
Orskov (16) i n 1923 was the f i r s t to notice that nnn-
b@rs of the genus Strm&mmsm. prodaced two separata growth
phases - the vegetative, substrate mycelium, and the sporo~
genous, aerial mycelium. He believed that the aerial mycel-
ium mm produced m a resu l t of the simple branching of sub-
s tra te mycelium* K1 ienberger-Jtobel (14) in 1947 was the
first to propose that the aerial mycelium did not arise as
simple fermclM* of the substrate mycelium. Rather, she con~
tended that the sporogenous phases were produced from the
formation and geminat ion of bodies produced by tha close
contact of vegetative hypihae. These bodies foro-acl by hyjihal
contact were ca l l ed initial cells* Also# she renaned the
substrate and aerial hypbae previously described by Orskov
as feh© primary and secondary mycelium, respectively.
Srltoon challenged SCLlenberger-&Jobel • 3 interpretations
on tha basis that the initial cells w r t resul t s of cultural
and staining artifacts rather than actual developmental
stages (8)• while workers such as Carvajal (5), Jones (13),
Diakenson and McDonald (7), Gregory (9) and Oaves (6) observ-
ed primary hypihae being closely appressed if not actually
fused# they were not able to demonstrate the direct origin
of secondary mycelium from such contacts or fusions as Kiien*
berger-Nobel had asserted. Of the above investigations#
Gregory's evidence for the fusion or anastamosis of primary
hyphae lias been construed as toeing the most convincing (15,11)
for he seemed to demonstrate the disappearance of cell wall#
or membranes at points of contact.
In I960, Hopwood (11) , observing living unstained
specimens reported, as had Orskov (16) that the secondary
or serial mycelium was a branching product of the primary
or substrate mycelium* He further observed that the second-
ary mycelium could be produced without the previews fusion
of primary hyphae. These findings of Hopwood, when contrast-
ed with the great amount of nutritional mutant data support-
ing heterokaryosis (2,3), or genetic reccmbinatlon (13,10,4,1,),
has led to the perplexing problem of how nuclear material
could be transferred in these groups of organisms•
The mechanism of hyphal fusion, or anaatwosis, is a
convenient answer; however, because of cultural and staining
artifacts involved in this work, current workers (20,12)
have found cytological evidence lacking. The purpose of
this investigation was an attempt to clarify the relative
taxoncmic position of and to formulate seme of the basic
cyclic morphological and physiological processes occurring
in an aquatic actincroycete possessing streptoraycetal charac-
teristics. To further these aims, the following experimenta-
tion was conducted.
1) The entire life history of the organism was r w i w -
ed through an unprecedented slide culture technique Which
allowed continual observation of a growing organism without
aid of stains or deleterious factors which would terminate
or alter growth.
2) Ba.sic physiological phenomena concerning the initla-
tic® and propagation of the »«veral observed morphological
phase® were investigated by manipulating acme of the physi-
cal <ind chemical factors of the environment surrounding the
organisms housed in slide cultures, tubes, and flasks.
CHAPTER BIBLIOGRAPHY
1. Alikhanian, S. I., and Borisovo, L. N., "Recombination in Actin-omyces " Journal o£. General Microbiology, XXVI (1961), 19-28.
2. Bradley, S. G., and Lederberg, J., "Heterokaryosis in Streptomv-ces," Journal of Bacteriology. UCXII (1956), 219-225.
3. Braendle, D. H., and Szybalski, W», "Genetic Interaction among Streptcraycetes: heterokaryosis and synkaryosis," Proceedings of national Academy of Science# IXII (1957), 947-955.
4. Braendle, D. H., and Szybalski, W., " Heterokaryotic Compatibil-ity/ Metabolic Cooperation, and Genetic Recombination in Strep-tomvces," Annals o£ Mew York Academy of Science# LXXXII (1959),
5. Carvajal, F., "Studies on the Structure of Strectomvcea crriseus, " Mvcolooia, XXXIX (1947), 426-440.
6. Davis, G. H. G., "Morphological Appearances in Streretomyces BP " Journal of General Microbiology, XXII (I960), 740-743.
* t
7. Dickenson, P. B., and MacDonald, X. D., "An Electron Microscope Examination of the Initial Cell Stage in Streptomvces BPP. " Journal a|. General Microbiology, XIII (1955), 84-90.
8. Erikson, D., "The Morphology, cytology, and Taxonomy of the Ac-tincmycetes," Annual Review of Microbiology, III (1949), 23-54.
9. Gregory, K. F., "Hyphal Anastamosis and Cytological Aspects of Stxeptomwces scabies," Canadian Journal of Microbiology, II (1956) ,649—655.
10. Hopwood, D.A., "Genetic Recombination in Strentomvces coelicolor." Journal of General Microbiology, XVI (1957), ii-iii.
11. Hopwood, D. A., "Phase-contrast Observations on Stre-ptorovces coelicolor," Journal of General Microbiology, XXII (1960), 295-302.
12. Hopwood, D. A., Serraonti, G., and Spada-Serraonti, Isabella, "Het-erozygous Clones in Streptomvces coelicolor," Journal of Gener-al Microbiology, XXX (1963), 249-260.
13. Jones# K. L«, "The Development ©f the Young Vegetative My-celium in " Michigan Academy Q£ Science Arts and Letters, XXXVI (1950), 13-26
14* Klienberger-Nobel, E., "The Life Cycle of Sparing Actinomy-ces as Revealed by a Study of their Structure and Septation,"
&£ Microbiology, I (1949), 22-32.
15. IfcClung, H. M., "Variability in Streptomytjetes," Annals of I3£ Acaderov, LXXXI (1959), 879-886. '
16. Orstoov, J., "Investigations into the Morphology of the Ray Fungi," Copenhagen: Levin and Munksquard.
17. Roach, A. W., and Silvey, J. K. G., "The Morphology and Life Cycle of Fresh Water Actinoraycetes," Transac^on of the ^nerican Microscopical Society, L5QCVTI (1958), 36-47.
18. Sertnonti, G. , and Spada-Sermonti, L., "Genetic Recombination i n Streptomvceg," Mature, CLXXVI (1955), 121
19. Silvey, J. K. G., Russell, J. C., Redden, D.R., and MCCOCTI-ick, W. C., "Aetinomyeetee and Consnon Tastes and Odors,"
fit j¥i6.rican. Water. Works Association, XLII (1950) 1018»»1026«
20. Stuart, D. D., Jr., "Fine Structure of the Nucleoid and In-ternal Merabrane System of Streptamyces," Journal of Bac-teriology, LXXVIII (1960), 271-281.
21. Waksman, S. A., and Henrlci, A. T., "The Nomenclature and Classification of the Actinoraycetes," Journal of Bacter-iology, XLVI (1943), 337-341.
CHAPTER 11
MATERIALS MID METHODS
One of the first attempts to Hollow the growth of one
spore throughout its complete development employing a slide
culture technique was made by Hojwood (2). In that study the
coverslip was coated with a liquified solid medium and, upon
resolidi fication, the medium layer was inoculated with a spec-
ific spore suspension. The coverslip was then inverted and
placed over a microscope slide with lens paper strips as dis-
tance-spacers . In order to prevent contamination, wax was
employed to complete a sealed chamber. In using this tech -
nique, only the earliest stages of growth could be studied so
that observations on the development of a sporulating mycel-
ium had, in most instances, to be made from mature colonies^
Certain aspects of Hopwood's basic culture methods were
used after extensive modification. These modifications were
not only necessary in order to be able to continually observe
the origin, development, and sporulation of the secondary
mycelium, but were also necessary in order that metabolic
and physiologic studies could be conducted during each stage
of the life cycle. Specifically, these modifications allowed
i f l s
the ©nviromssant surrounding the organism to b® ehanged While
performing a minimal amount of physical aggravation to the
speeiaantf•
The construction of tta* ndcro-^owth ofhanibars is ©torn
clearly in Figure I. In preparation for construction of cm©
of thane chambers, all parts to be used were first placed
under ultraviolet radiation. They wrt permitted to stand
for a period of tin* until tests mtmmd that there wur® no
contaminant® available m the equipment. A regular glass
slide <175 ran x 25 rat) was used for the basic structure of
construction. h rounci sterile coverslip (32 iroi diameter)
was then coated with a layer of solid mediua approximately
100-150 microns in tMetea§a» The spotw wars inoculated
from a spore suspension onto the layer of solid medium Which
was than fixed with the coated surface down and was sealed
with Kroenlg wax to a vinyl cylinder (4 rat height, ©.!>» 25 ram,
I «o. 17 mm) • The cylinder was transferred to a glass slide
where tot© Kroenig wuc was used to fix the growth chamber
••curtly to the slide. T m nioro«cosyat«m « s regulated in
regard to humidity and atmoepihare. The agar slant in the
bottom of th® growth chwtoer was of low agar concentration but
was of sufficient hydration so as to supply adequate moisture
to the nutrient substrate. Immediate saturation of the mi-
croafcraoaphere was accomplished by injecting a small quantity
of water CO.2 ml) into the area opposing the slant (Figure II.
5?T"
7\J"
CO tr UJ > o o
o: liJ ffl
9
UJ o
\
o — <0 _J
cr o < l&J
UJ (0 m 35
_J >-•9 X
3 z < Z > £ o
1%
13
Gaseous exchange as well as dedication was controlled by use
of two air portals. These portals were produced by passing
a "heated 20-gauge needle halfway into the under surface of
the vinyl disc at two points • After the disc had been per-
forated, the portals were temporarily closed vrith silicone
grease. The grease plugs could be easily pierced at any time
to open the system so that either liquids or vapors could be
added for metabolic studies•
The above modifications of Hopwood's procedure provided
a system of high reliability for the observation of the com-
plete life history of the organism and control of several
physiologically vital factors.
This investigation involved the study of two organisms
from the North Texas State collection* namely number sixty-two
and the Cooper Strain. Strain Sixty-two received most of the
attention devoted to this study, while the Cooper Strain was
utilised only when spores of different size and parental origin
ware required. Both isolates had the following common character-
istics* 1) Bach strain was isolated from a reservoir of the
Southwest, 2) Bach produced metabolites that exhibited an
earthy odor similar to that found in water supplies, and 3)
Each had streptcmycete-like characteristics, i.e., each devel-
oped chain spores.
The spore suspensions employed in this study were prepared
by flooding slants with sterile distilled water. After a period
of gentle agitation, the resulting mmpmmim wm filtered
through sterile filter paper (Watson number one) in order to
remove clumps of vegetative rayceliura, and the resulting fil-
trate was diluted to appropriate cof entrafclons for micro-
charnber inoculations.
Two types of media was* utilized throughout this investi-
gation. A minimal xmMvm - after aavis <1) - was used
in instances where alow and limited growth was desirable,
where nutritional factor® were studied, or when pigtaent pro-
duction v h to fee evaluated. The enriched ise&lm, an the
other hand* was the gmmtal tool for the investigation or mor-
phological cfclee and the affects of jfcyvical agents. The
media were formul&ted as follows t
M1S2 - sodium citrate lO.Og,, gluooee lO.Og,, sodiura ill-
trate 2,0g.« potassium nitrate 2.0g., oalciura chloride O.lg.,
magnesium sulfate O.OSg., dipot&ssium phosphate 2,0g«, dis-
solved in distilled water to roalce one liter*
Enriched - Tryptooas© soy broth 5 g., nutrient broth 5 g.,
SSraerso s broth 5 g., brown sugar 5 g,, aamonium nitrate 2 g,»
and soil water extrat to make one liter,, adjusted to « pri of
6.5. •i.'can'.perature ranges of 0°b-50° o, and a pH range of 0.5-
12,0 ware studiedi however, normal observations were conducted
at 24° - 25° C, pH 6.5-7.0, and with or without light depend-
ent upon l fccttatoey conditions sine® no control# war® consid-
ered.
1 n
In considering the metabolic pathways existing within
the organism, anaerobic conditions were established. Anae-
robiosis was achieved through three divergent procedures•
Brewer jars were filled with slides, agar slants, and flasks
and ware examined after periods of 1) gas replacement with
nitrogen and helium, or 2) ignition of hydrogen. The jars
containing the slides were hydrated by enclosing a water
saturated towel. The second method utilized a further po-
tential of the slide culture technique. The portals were
fitted with glass tubes drawn to capillary size, and wore
sealed into place with additional cement. The glass tubes
were connected to rubber tubing that composed part of a
gas train so that hydrated helium, nitrogen, filtered lab-
oratory air of pure oxygen could be passed through the micro-
chamber under slight positive pressure. This permitted con-
tinous microscopic observation of the organism under a
variety of micro-atmospheres for a determined period of time.
Thirdly 300 parts per million iralonate (fixed at pK 6.0)
was used to study its effect on the major aerobic pathway.
The last facet of the investigation explored the origin
of nutrients used in the synthesis of secondary hypae. The
experimental design consisted of removing nutrients from
the primary growth phase and determining whether the second-
ary phase could be formed without additional nutrients.
This was accomplished by washing the primary mycelium four
times with % balanced salt solution (containing catione
in til© aarne ratio as found in the minimal raediusm), and
allowing the washed mycelium to stand in the salt solution
for periods up to ttsre® weeks.
Morphological observations were recorded with an A«0*
iipmmmr Model @33 Microscope having carora m4 phase con-
trast attachments • Kodak Plua«~x 35 mx Film mm developed
normally in Kodak i-iicrodol £w®l©j»r.
A. t
GiAPTER BIBLIOGRAPHY
1. Davis* Ernst M. / "Assimilation of Inorganic Hitrogen toy Actinomycetes," an unpublished Master's thesis, Depart-ment of Biology, North Texas State University# Denton, Texas, 1962.
2. Hopwood, D. A*, "Phase-contrast observations on Strepto-mvces coelicolor," Journal of General. Microbiology* XXII (1960), 295-302,
CHAPTER 1X1
The 1*1 tm Cycle of an Aquatic streptocoycete
The following results have been obtained front a study
of over two thousand different Mloro-olwrtbar culture slides,
llnlss® otherwise designated these descriptions ware ba»©d
on isolate number sixty-two NTSU cultural cm enriched me-
dium, For reasons of clarity# the cyclic events will tm
described under the following headings« A* The Primary
Mycelium? B M The Secondary Mycelium# and C. Sporulation.
&*, Urn teeUm
XtadUUKft rnA 2»tofelak--a0 mature apora mea-
sured (0.9-l.lu) in diameter was usually of spherical form
and germinated by one or two germinal tubes (PI. 1.# fig.a#),
when nutrients w a wltMra^n from the medium containing
mature spares and only Nobel agar mm present# 1 mm than on®
per oent of the spores germinated, trader nomml nutritive
conditions# this percentage was increased to at least 95 per-
cent. Swelling of spores prior to# or during# germination
was observed to be very slight or not at all.
PLATE IS
v» • » ' * CJ
Hyphal attraction between two similar spores
SE2ES J&g, Development
fill the Primary Mycelium.—Germination and general growth rate
of the organism were affected by such obvious factor® ass
1) temperature, 2) pH, 3} nutrients, 4) amount of oxygen avail-
able, and 5) hydration. Vant Bo£f*s rule was roughly aprox-
imated between 10°-37° C. Optimum growth, and the shortest
germination period (6 hrs.) occurred at 32°-35°C. ;\bove
this point inhibition was observed, and thermal death oc-
curred at 42°C. The organism remained viable in a h range of
2,0-12.0# however, best growths were observed frcxn a h of
6.5-7.0. i/hile increasing the richnesss of the medium usually
produced an increase in the time and level of total primary
production, germination times were little affected by vary-
ing nutrient strengths. Inhibition of growth was noted when
media solids exceeded 60 grams per liter. The organism un-
questionably favored an aerobic pathway, for anaerobic con-
ditions greatly reduced the rate of growth. These data were
reinforced when malonate poisoning revealed incomplete in-
hibition. It will also be noted that flasks and slants under
anaerobic tensions demonstrated no pigments or ordors, even
after periods of two months.
The primary mycelium required high levels of hydration.
Desiccation was found to be a very successful method of con-
trolling the amount of primary growth in a chamber. When
IS
i t me ^iotdroblo to liiait tiho aim© of a or to p®s«
iR4ti«i® the M©«tei?y jfiagta, «i© coUa tieso rlacsix*
in a aeaica&toc isw on© op two howttu flits tSMtnonfe
naiaUy c&$ rsot ®to\* <gpsm$ihi frorawogv toteaapa gWRfHt was at;
a mOk Btmm M® «fi«ast mmli n a m * s**£>aetlttia.
ofi the Aai^w mm sot auoaeseftil in iwwslng the iftiiMtiosi*
28MSlitiKle — aporo gotnination ana bsira?©
brandling, m la f tnml of sceUrofttioa or log «$g*ma (PI. 2
££$* ."WJ)* Isuftfe of this period wsw greatly affected
tiy *£m &HfeM9 «%JJis©ci in tit» i*oooodinrj m f t e « ttie ts«sr-
ntnotlon of this period iaitiafeM an «*$loalv9 graft!* period
in tftlcftt mm mmm&m swtei»t@€ the um&m wim mAmI e»»
tetwtafis aaS iotewow* hyjfcal eSanfliite* a««i«dbm originate*
m #gt»r:l©aa. Mt t i age at mm si Cm o£ hyriww. tmmm
mmlU&m ma» W f coobiittwHk of teimiaal scorer)
®cfemrl 2 laterally irtdl* e t i l l saiateiMag their bulboid
a ^ s i a w s <PL. 2,, f ig. d • P h m « « M s&cnocoegy
eoooc&oaAtty swoolod livegular acptetioci at the z-rtiwary
egreeMly «fe tihl* early atag© of growth. St me
ftirthear aoted that: 00 tin® cwlesjy iswmwssl ia siise ttie
-liAwsfccr of t!"> «» ly f«sms<2 ttygtsae OtadaUlhea*
"Siila the gaminel taboo oad ini t ia l ly fiornx?a israndbos
wop© 0.* u to 1.0 a. to at«wfew, t t» I r i t w at the txrnBm
of a l a w «ol»y w w fcwy times maxima to &m 0.4 u
to 0»7 u» t» stuping & nature 00^007 i t wme ionuS t in t
* *
If W-
© large rost&cm tho jsseija&ry asiigtel boswath thfc
surfsoe o£ the i»dim» fi l ls "rtiiaoi&al• off stteiirSee®
laiycMiIISRi# vit£h it© twisted sa l otKLlodl ocxifiguration, node
i t irnxMatcfty d&MNNnalb&B Skis tin© nee© regularly damA
ftORfeise g m t o g ea t t » swrfsei® of £h» mdtttRu Vha ifsJ,8©id&3,
t i j j aw « a e fiMBwd us staple t»nctais of the pelmvy
a^wMmt (91. 2# £1q. ©I # ^ t d h paastaraMl the tasMUUm in
en oppGXtK& smfexitxqi;MU» >Ms>otMM»* Shis growth
wmi icg&UM&od to tiha i*o«auctian o£ the MBs ®f t t e go&*
<-Mr» faaftfema fiafiliilsfcift nigarstsafiffi HBBB SO t»W}»W^ in
maa-l «ygy» sail
unaaCTacn grores. —. ttheoagb tho <AoMHnwtion o£ inoiiiteS
eAToco pelxs s»S t r iplet#, i t wis &aa! tftet tftse«i <arsist®S>
pcnfoBoofeial « f f » ® t m end birax&ing betMeon the hggSiMi
«ssi*t end uriex*nraon pexesital Agoree* 1M.S p » f e r «
«%iai #rwth ofibaa rswtltoa in -..esr.ianottt f!ts3fxi»,I
aosxtacta" (PI. X, ficj,. JVwTr Pi, 3, Fig- A-8r Pl„ 4? SLo*
iwij» AttvaefeioR wae wtsea. i s 75-as j e r Mssfe of a t o t a l
of ss tiboottvet&one* An orcecrt pesoeategc eould not be
ototod fcr ssa© data hod to be dlee&vded <3ae t o Isiesed
i«tootles® e£ s^eotoaae* M&rocfeiea tms dboaervad t o %s
raoct dnsetatio i a the oarly ptmess ©£ gsocrth, and tAm
oixare© ©f dl£8nraot ftaeetxfeal ©rigfe w s ttoed tel. 3* •
f ig , /**3) . Attraction and contact of hyphae was not
20
PLATE
S • 4, - ? ^ V ••
y/ ?-V .
Production of secondary hyphae from one spore
21
UnteaS to tfc© fiUtaeMMto found e* the aurfiee© the latdiUBu
1% nne cdso observed ia gfetsotdsd •tenanto# lisiefe «afc titaoe
booacia infe&aato within «f» aolid itee itoelg • Tt ar®aoxx*&
1Ms the ceaponM ootid fee etiUMfeive In xogavA to ttmcfeion
a# mil as plane* fls®§ aoofeaetMi IAmhmImmi nam £*adnBa&
«t rt*fchor rwtei w«eiafet«iii3 in ytiiati cell mils cad pwq-
branea %ioto ari oroRfcly niaixita£tte&« hile to odUito of
ecmtoofe wcmm uetftcdly Mftintoinodr m m or both wufeara
«£ tho udoa oewKKwily oontsiiised to gratr CpI. 3# a<j,,. .3) •
3fteee sussocltifciotts tisaallf c&ialto? ia a tiMmwwo in the
lag jliaaa md a otoe^er GsqpesMHt&aA JIKII® «c £&$*&*
ftepewlsietos® fboie of the Pftswy HyoaUua
She sctiaosy nStoMsw p»p^pte3 iaee&f m$0&mA.votj
% £ro*y**itation. &Bg?odiaisl«Hi ©f this n a m e
®towfwt! ia slid® ealtuver T*»«v«r, in «mit*to& fiasls
otUsiCQst &®?p«tsfeafetaa m m «£<paretttly SI <KSdt£KeBie3»y
©ftuett'W: &oprddb(&iv« cSumnel. 3!w togfthoe did not be*
mm. »ptoriy fifvJUtei ia x*oceas. Batter# verieue
«aA%© of fcgnlaae noiiM taosfe kote ta o parent c©I«^ ead
then tea the molouo of s »*r oeloegf»
Mtesr tS» rayoeUUsa tad ceptatod a»a lioa
toegim to £rognent Into aporoc, tfhe mgmMxm,
would aStsMt sml undergo Mtolfst©* At tlhia tto© it « w
teeqaenfety netod tSwt eAdto-Wae etoaatocoo fiwaea.
tb© walls o£ the i-rlraery tQ&tiee (Pi. e, £ig. B). A poenible
22
PLATE 3
T
I n H
Hyphal attraction between two dissimilar spores
P L A T E 4 23
I S
urn
1 /
f
j
l ir
*p*
*
i r^~
vmwWr-^
- ' . : .-» ••• •'• • • *
A J R w 9
Vlfl
HaKgg I -#'HP * ""• .w w'
•PPOH
Hyphal attraction between three similar spores
^0
ftotie&httx fior those ^ s s s s t e w oould safe 1*
feoft# £or th«o© wtio£go®miib£>& pswoii fc©
* « 1 2 a vitaan %© £reusfh ra&Sia.
flfe; I) ife VO0 X # 21 it; %
A i a
titat jgdtoasy i s
sWKSUid a tieg&
3| if
.d&&$*4ggp3
,i® by «spfea# oad ©©©fe xwa&Uy
lire Ci© fenioti#
ioraoata #f 2J3i® ac unllfco | f i w s f t^t iae
to t$M» I«g^&fe# @«©ia o£ |,*»AWfe^» (!?1. 3, fig.,, C) a®3
lotad 0roco (?1, 21* S a i n i t i a l ouel.tingo gorsiRC&ort
fltfoy fipota tho twSlvM* Ite wswwiiatiwiag stwfeiott aetsM bo
eewtgpaod to t&ooe ©jiwci-ajjsa etmetemje, £or it wM €£j»
poor ttefe tho ooBditlenis i?®®niRK§ £or their wn
laMfetaai to tews i#MMOb et tbeir 9agtAiw%ti»*
fte vum a£ apoBiQstiovo cmilg»Rife4i» Mid vAaBenfc
pCDdaatitti «s (ssaoaomlo tool® tor© jswwwl lay #» &3,l0viag
fitnOinasa Slsttt* it mui ifae* &ak m tmtmM, ootony
:*«&KX>a 2r«3 aao ox«jr© ooalu slmltanocusly boar m
to fit© iSiifewiitte a&mmsfaam OM&gnga&lmu
eis te keanc&cx3, -gghmttm s|4«al hooka, and
me cftNMrwoA thofc fip»
c oia ia qpoco titaagpoa«te»* {ipMed aid iMqj£gaMMI
csetaitel oauia flii»aimMMM0iy wwtlfc tuitfe cttter
affacfeoviatiGs *«ti$&y oanofcaat) • flstjs tos
ot pigmmt m® usually only aafciooa itwt spove OUftfetcnui
ware M#i to ±iS9vonk oo&mlae Stam «»toefeii #*fcf£k£?k **M&* «TO*?li*£IIKr *
afl aytf a gMWtott IsSX IJilS* ****" ®6®
s BMMm 3h9&D0fcoftl jcdufe o£ ¥i<» it ay/«a*ou tfeafc tho
maeatay upoi&taa wm atf^tttonally tfefMontaBfe «t tfto
Sdnuey jtwygii for it m# ganeffc&ly ieol steS fa® t$® txMum
<1*g to c©a»3ctliig lamidhiao it me 1st diseeb eoateasfc wit2»
2C>
t j J l© i f tossy PWGhlJM&BMtS* ##?/#• j&Vfe$KHP fescHU*
I S w S t t l fSdlMft S l i p t t l i l S l i y ^ f p & C i l m M O t w ^ l S ^ W ^ w w JSTOSfl
n s t t v i t j w M s o t omtimia t o ^ w # b a t w o u l d i r n v t
l a y # : to t t » p p t a w y i * fease . , t i b i l f t t h l o w i ^ w w a 2 £
fluggNfe tihft d ^ a n A t t M » o f t h o M ^ t e f j - t e f t © o n t f h »
; 4 T i » a i r y t f n u x b A s x t t a r I s i A U t t M l t h e f c
4 S | S ® S l O S S & 3 M & 9 & t K t i ^ *jj3vp g r t f w
o t r r r t e 0 0 w e l l * I t w w o b n e r v o t i t h a t t w o © p v t a n g y
o p t e t o e C f i p o e o £ a * » « e l a s ? f t q $ h G « # w n M } . « o d a o e a
flgaa.ll » © t l f l f e O f dOCKHVlaXY Cf lMf l f t ih S 9 * l t & f M f t t l i O S B ' f e t i . W t o WW .JST *^P -W?r Sgpfjefr "Sr ^np1 " ^^^wlPfSr-leapy ^B^psW',wpKr *9^5^^ flSPfp1 wjepwssfx?
« n t t 3 & t ^ m l i . t c i B T m & b o o n M t w m S t s f i « i 4 f s g w i t h a
i j e X a n o a d s a l t a o & i s t t i e a * ' S b i a o c c u r r e d I n a t g g m d j a a f e e t y
t n w f t y p a r a * a & o f tih* d t t l t w c o B l a v e e t i g e t a g * 9 tacsw3,
l e v a l * © f » § e « s i i i i ? y h j f t t v i g r o w t h i » e w m O w A b y
tslfc® 8 & t . t W S t t S l ^ l #
a a ^ i a t t i «
s p s a l i w i i w m tttfualXf* £ € o O u a o & i n t h o e a r l y ^ e t t e a a r f j i i s a #
© £ t b a ^ w n H i W f « » s « » e < i i i t t l c f t t o i i o f t 3 »
a r « s o g o m » i i j f t i e f l o « n s M 1 » r r a M t t u s o l y & i s i s K w l t ? f iw to**
jCX3l#iUSx! <^t . | .<a « a y i f c « i . t i 4 » i ^ p y ^ - p y s y y g y s @ 3 4 W S <SO £§1©®%. g S C l o d l S
o i z a & l d c k w d o e e f c i o n , V a r i a t i o n o f p i t ( w l t i i i a fcbo
j ^ W B t o & o g U n l b w & s a t te i « o v 4 o u » p Q K a g N 0 » ) t i e d
l i t t e & t o r a © e & B e e t s ® t h e * W i s © o f r e d u c t i o n o £ t b t
i ^ w s a s c y e ^ o e t S i M B i f ^ » p a » f e i » , o n t i » o t t o * 1 » ® 2 # c l i c l
4S» t
definitely affect rfete of secondary growth.. Of the
factors involved in initiation of secondary mycelium, it
would appear that the oxygen available to the organism
would be most important. The organisms were subjected to
anaerobic conditions at various stages prior to and after
secondary formation. She following data resulted from
this study:
1) Organisms growing under anaerobic conditions could
experience spore germination and full development of the
primary mycelium, but could not produce secondary mycelium,
odors, or pigments.
2) Colonies which had almost achlm*^ fell development
of the primary mycelium under aerobic conditions and which,
at taiis time, were transferred to anaerobic conditions
would occasionally produce secondary mycelium.
3) Colonies, which were sterile of secondary mycelium
at the onset of anaerobic conditions war© more apt to bear
secondary hyffciae when their positions allowed them to be in
close proximity to or in direct contact with sporogenous
colonies.
4) Colonies which bore secondary hyphae at the onset
of anaerobic conditions continued their growth after a short
period of acclimation.
Malonate at 300 ppu (H 6.0) inhibited secondary hyphae
pemanently.
The Pasteur effect was not observed when pure oxygen
was passed over the organism? rather, stimulation was
observed.
C. Sporulation
13i© time required for sporulation varied greatly,
but it usually began four to five days after innoculation.
At this time of maximal extension of the secondary
mycelium, the septa would begin to form. The septa formed
approximately at the same time along the filament, usually
starting from the distal end. Desiccation appeared to be
the dhief stimulus for fragmentation of the spores and
usually occurred simultaneously with the autolysis of
the primary mycelium.
CHAPTER IV
DISCUSSION
The primary mycelium was found to toe a basically
aquatic and highly adaptive somatic foundation which
propagated itself by elongation, branching# and
irregular fragmentation. In addition, the primary my-
celium appeared to be capable of mutalistic associations
with similar organisms. These associations with similar
organisms being similar to those described by Dickenson
and Macdonald (2), and Davis (1), were observed to be
random permanent contacts produced by preferential
directed growth of the primary hyphae and did not result
in the formation of "initial cells" or anastamoses as
described by Klienberger-Nobel (8) or Gregory (5)*
respectively. When spores of different parental origin
were studied, hyphal bridges (PI. 3, fig# a.) very simi-
lar to Gregory * s report of anastamosal structures (his
Jigs.1-7} formedi however, in opposition to his obser-
vations, the bridges were not permanent and passed over
29
30
or under opposing hyphae (PI. 3, fig. b.)• While
Gregory's photograph® seemed to be quite convincing# it
was unfortunate that he found it necessary in recording
his observations to use stains# which terminated future
development.
On the basis of the following observations# the
association of primary hyphae was thought to be a pos-
sible syntrophic or heterokaryotic mechanism*
1) Association was noted to reduce the lag phase
of primary growth. This became increasingly important as
physiological and nutritional conditions varied from the
optimum.
2) The tendency of the organism to produce nonpig-
mented colonies was suppressed through association,
3) The ability of a sporogenous colony (formed under
aerobic conditions) to Initiate sporogeny in a nearby
sterile colony (on the sudden removal of oxygen from the
environment) was found to be enhanced by association of
the primary hyphae.
31
Vhe m&omSmsf mymlin® was clmracrfcerissea fey being larger
than the primary hypJiae, usually pigmented, and apparently
hydrophobic* Hopwood1e observations (7) ware Qorx&rtaod in
t3wt the eewsdarf' was dhnm to result ffcaa the
simple tewiAiaf of th© primary ntyeeUiMn without prior
hypfial fesioas at the points of secondary origin- Mao, it
w m shown that eaoondary nqpseUa could b@ bom© froa an
iaolatacl colmy produced &©» a single spore.
She secondary aorodXlun appeared to be e ppp&iGfe of th©
primary mycelium during its stationary phase of growth,
since thor production of mmMary liyghaa « « M b© xapomatsure*-
ly triggered in the primary colony fey inducing unfavorable
conditions mdh as derteaa&leo/ or Mfmovml of isttri**
ante from the sssdiwis* In addition to tfce «®rgett3@ of the
©wc^asy mycelium, tMa period of d®ro!oiw»fe- w i also w -
IfMtcd lay t3s® colony's production of soltible pipssafes and
©dor oon^ounda.
%ha role of the pffinecy tnycwllun in tfte #@v@lop«fc of
wwoadary sywlium s©«si»3 to to vital, Because ovon tlhm
fmmg maomSaxf tad* (hyphal elements) wore transplanted to
fresfc media they reverted to primary raye»lium. A parasitic
rslatim^Mp taMeen the two morphologic phases was tether
suggested in that the soeorvdary fsyceliuia urns usually spacially
separated from the nutritive substrate, m& that the primary
ageellwa after ®sfc6asive msMng would produce small mounts
of peoondary myc&Hum without additional nutrients*
<> ,«1
The genus Streptamvces has classically "been defined as
a basically aerobic group (9). nonetheless, recent; workers
"have shovm that members of this group can carry out fermenta-
tion under anaerobic situations (4),(6),(10). The major
criticism of such work was that little or no attention was
devoted to the morphological selection occurring during
anaerobiosis. The present study indicated that the primary
mycelium was a facultative aerobe probably utilizing the
tricarboxylic cycle as the major metabolic pathway while
the secondary mycelium appeared to be an obligate aerobe
when grown under continual anaerobic conditions or in the
presence of malonate. However/ when anaerobic conditions
were introduced late in the development of the primary my-
celium, the secondary mycelium occasionally reflected
facultative characteristics. The facultative nature of the
secondary phase was most evident when secondary hyphae were
already present at the time of oxygen replacement. Erikson,
(3), using crude techniques, implicated the role of oxygen
ill the-.initiation of secondary mycelium, but her studies did
not reveal the facultative nature of the sporogenous phase.
Colonies producing secondary mycelia under anaerobic
conditions were thought to utilize metabolic pools accumu-
lated in the primary mycelium under aerobiosis. These pools
were apparently also the source of accumulated nutrients
33
v t l l i o r t by prtaKUQT eo&cnloe a f t e r boing wsdbofl wi th b a l a m M
a o l t sataftioas t o 2«odhie» o©s©sia«,sif tnsooliun* AASitiOQaX
Mis | . 4« r i^ i« is £or tho o t t h i s pbononaoon iwsaM
bo the peesIM© spst»ifil»G error duo t o i w i t i m of ougnpan
during aavoMc IJO IMs by t l ® agar tae&lVHft*
CoRtwasy t o M f t o y ' e Cl2) ©tacty o£ a thocnoHhilio cust*
ittoayoefcG j:mm cctygac Old not i n h i b i t tho oooaa&ary' k$oq&1is»*
Tno btfipogralo&ive 11 fo 'History zacepoaod % f t e d i ana
s i lvcy (IX) t o sc i .«ate tho aqpa&le £bob me*~
b w c£ t t a fcfailv coul& not bo iiict*ti£i©d
t i i t h i s stet$7* I t ^ould es&eor tfcsfc tho a c e t i c m g m l m
&MCtcl£m& Imm w m a truo mnsl btihuvosL aira i lcr ly
i n i t s p w s l t o HQiftjooa'a ooeli**
aoletag (7)„
tSho groat pKo^axtiofi o£ tb& (wgaaiflno ua&nr
9ta$f oliewoa tujoir & jaat ic esdfftoanee l a MI«©s §aS »«*
MNrvoiaro o£ tJ» Soi^l 'woteta United states t o be «p i t# sec-
cosaful. During the tdxntotr taontho tba oporo atcge noal4 be
anviaicood t o i n tho bottom suds* Stsa epeg©
wctt.ia gen&ORte i n tho tspriit^ {rt&^latod by lncrooso taa«
pomrtuios, end i w M bufta t o speisitefe® sKaaottauo isutr i t ivo
a# sfiKwmlateS « ^ i a i e mot#* <Stoc«®ii*ej vogafeo-
fetai* s a l oh&al by^«oduot0 %fhldfc tMmid bo avoiiisfblo « t th©
ixsriciotar ©r bottoa ©- tho l&'.jo or roaosvoir* I t tho onanism
darolfigse# a t tho psoritoefcor; i t s g r w t h would only be l lcdtod
VI/t
by the degradation of Mghor aquatic plants and alga© that
m m M be i«oduc5ed teasing the suraagtr in this region* afco-
trorJiic vQgeat«fcloa in this section would oenmly enter its
death jtfioa© in lute sua®® and %«mM allcs# i s ^ m l seoo&daxy
nsyoelial dovoloproant lay early fall. On tho oth&r tiead# organ-
imm that tiwald develop in tli® of mdb bodiee of
wetar would "have to grot? under saaarobie emditicsss during
mmt of tlio sisanarr hcuerwar, sourcoe ox aitricafcs in tho foam
of «fee«ap©s@a orfmsic ockso as "well ae variews fesss of vege-
tation would bo ex&Cttaefty riclh. By list® &mm% tho ©rga^es
would readh its stationary lihasa of grewth aad would peodooo
s@e©»l«y sapsoliun as contact with oxygon. 3he tooot common
contact© would be th© tanijorary disa|^«Mms» of tli© ai»-
robic regions (caramon to lake® and reservoirs of this region)
©y the full tiajmovor* Tim m^mkm would probaibly tho
ms£m& by sssoeiati<« with algal bloaa» and/or by tbe bydropho-
"b i e isateore of the secondary rapoelSunu Havo acttei wtUd
usually wash the alg®l«stirefJteK ©et© ootaplox t© slier© %<*harQ
tho MMseNcuSbuey iap»litia would e^erimce full dwrelopcaoot
e M on teslescatioa would &agnsat into spocttft. It wuld be
during this period of aosxaadary production that odor acta-
pm?«2s 90 a«SB«t to SaxtkMOBfeaKsi lapses and reservoirs would
bo irapcrtcd to the water.
35
On the basts of the following observations, the asso-
ciation of primary hyphae was thouc/ht to bo a possible
syntrophic or heterdkaryotic mechanisms
1) Association was noted to reduce the lag phase of
primary growth. This became increasingly important as phys-
iological and nutritional conditions varied from the optimum.
2) The tendency of the organism to produce nonpig-
mented colonies was suppressed through association.
3) The ability of a sporogenous colony (formed under
aerobic conditions) to initiate sporogeny in a nearby
sterile colony (on the sudden removal of oxygen from the
environment) was found to be enhanced by association of the
primary hyphae.
CHAPTER BIBLIOGRAPHY
1* Davis, G. H. G., "Interpretation of Certain ilorphological Appearances in Streptomvces BPP.," Journal of General i&crobiology, XXXI 11960) ,740-743.
2. Dickenson, P. B., and MacDonald, K. D., aAn Electron Micro-scope Examination of the Initial Cell Stage in Strepto-mvcea epp.,l- Journal of General Microbiology, XIII (1955), 84-90.
3. Erikson, D., "Differentiation of the Vegetative and Spor-ogenous Phases of the Actinomycetes 2. Factor Affecting Development of the Aerial Mycelium,Journal of General Microbiology, I (1947), 45-52.
4. Ganguly, S., and Roy, S. C., "Oxidation of Substrates by Streptomvces oriseus," Archives of Biochemistry and Bio-
' ' M X (1955) , 45-51.
5. Gregory, K. F., "Hyphal Anastamosis and Cytological As-pects of Streptonryces scabies, " Canadian Journal of Micro-biology, II (1956), 649-655.
6. Hockenhull, D. J. D., Hockenhull, F. K. H., Herbert, M., and Whitehead, B., "Glucose Utilization by Streptomvces ariseus," Journal of General Microbiology, X (1954), 353-370.
7. Hopwood, D. A., "'Phase-contrast Observation of Streptomvces coellcolor," Journal of General Microbiology, XXII (1960), 295-302.
8. Klienberger-llobel, F., 'The Life Cycle of Sporing Actinomy-ces as Revealed by a Study of their Structure and Septa-tion," Journal of General Microbiology, I (1347), 11-32.
9. Perlman, D., "Physiological Studies on the Actinccrvcetes," Botanical Review, XIX (1953), 46—97.
10. Prave, P., "Fermentation of Streptomvoes spp. Under Nitro-gen, " Archives of Microbiology# XXXII (1958), 286-295.
TH f
3?
11. Roach, A, w., agd silvey, J. K. G., -'The Morpholoigy and Life Cycle o£ Fresh Water Actlnomycetes," Trans-
^f§§g^ a|^^> awflaMi msmma&sai, §ssms,* u « m
12* weibley, D. w., "The Effect of (Oxygen on tho Grxwth and Msteboliera of the Jjeroblc Tharrao Silllc
J'immai of General Mare*. 122. XI C19
CHAPTER V
SUMMARY AND CONCLUSIONS
1. A slide culture technique was developed which allowed
the continual observation of the complete morphologic devel-
opment of an aquatic Streotomvcete and a means of investigat-
ing basic physiological and nutritional factors surrounding
the developmental morphology of the organism*
2. Spores in contact with a nutritional substrate germ-
inated with little or no swelling by one or two germinal tubes,
3. Growth of primary mycelium resulting from germinated
spores was found to occur in a wide range of physiological
conditions and was found to be easily terminated by mild
4. Fran a study of spore pairs and triplets it was shown
that hyphal attraction existed between like and unlike hyphae
which resulted in extensive association. These associations
appeared to be beneficial to the organism and were found to
be greatly intensified when spores of different parental
origin were studies.
5. The primary mycelium was found to be facultatively
aerobic.
6. The secondary mycelium required oxygen for its initia-
tion but assumed facultative characteristics after initiation.
38
7. socoodary hypha© wr® produced frora slagAe branches
of the prliaary myealiuta during ita stationary phase of growth#
3. Th« secondary pfoass of growth not only character-*
iwd by tho production of aocajadary hystows but wm also ths
period of odor and soluble pigment proaustion*
9* 3®ccmdary hypha© could result fro® the primary my-
celium produced fran mm ypora.
10* thm mycelium %mm found to be nutritionally
dapsndent on the prisary rayceliura and could not stereleq? »«-
parately.
11. avrar&l sporqphoro configurations could result from
a colony produced from one spore •
12. The primary stimulation for aporulaticn appeared to
be desiccation*
13. Wns aquatic iyafcinomreiitii under study ir*£l«etad true
3tr®pteiflvc.®ta> characteristics sad could not l» identified as
having a hisporulative life cycle.
8msoamsr?
B©©!» Orrikov, J. , Xnveflfe&gafetaB& ix&o the J4«sl»I©gf o£ fcba Kay
ftaagl, ••• 0^«is&g«s Lowta nod tA»0cavMMMl*
MiMiQUisssi, .3* 2-/ aid BoeidQVO* «. H,, "Amotialiiatloa la . .yaagwal of aaaagal
pSiaTrw*23. a*aKtaS>, s. Q.t ana t&kuto*BQt *T. ( t&toveSairsosts in
" gflasttft a£ QrafftsgMffinr* tissui, ar«gc*Ilo, D« R., and Sag balsfcij Oonafcic 2BtaKi€Jt4,m 001003 Strcr/4Mx.T oet©3; Is uoccfoar oaiD oad oynkatyoeis, of gafeteml JtesadflBiy o£ .igieaieo, I£CXZ
vJ», !?Ee «»l« Pfcie Caapafc-Geoefcle E«»Maa«
ar«f^ie» P« Ii., ma SsrybaiBltiL ihility, Mgfe »14«' Ctoop«tfv&Uar and tioa la aiuBBaatiBBPiattc#" aanalg. of Mmt Itoffg.
,HfSnfsf7 ftfc
carvajaX* P., on the -Sfewetewe of fi&Mft* '-cc: IIM7K 421
dqvIg, a# n. G.» "ftaeftio&og&d&S. iiy&wK&aaoa la
M«to«»b i?» 3«# aw5 SMtanald* K« D.« "M 21actroa :a.caro-soop© eg Ms® Initial Ooii Stag© in m
TTO/Two. Btilia®* d.» 'Pbe EiocsSio;
J«otsJLsiO0®s® ®, (m», 23-54
the
40
41 Erikson, D., "DifgeBoat&afclfla o£ the asa StxwoQps*" on© Manns of the 2. Factor Mfeefelag Dsvelcgxaeafe ©f tho Aerial 2$edl±im#" Jaaraal of tenml al*a<g, 1 U947), 45-52. Ganguly, S., and Roy, S. C«# Oscldation ©f Stbstratos by
P^fPtl) st ag »itgiK. «& iteliilgi* Gregory, SC. F. > "Hyphal Anaatcraosis a»3 cyfeotefieEa. Aspects ' fianguriian Jcttruai Oa llicro—
- WHn -M» MB85S»r fr
{fcKftariball, D, J. D. * HocCieraShttll, f« k. H., Heebart* H« end ilhlta£h@od# S.« :'Q2»ooee OtAMsaticsi by Jo*® * 0 •
HOpHOQdL 0» A ., •'Genofcic ftecnrclbiaatlea in sM. gea®g«ii !-
Jeiarnaa 11. KOpWOOd, D. A.
W 2 3 Observations m. SSa
HopsfD©<!$» D. A., simwefsi, ai*l Spaa-Sejpaoofci, Isabella, {i-lsfte»iifpm5is C36BM in ol iSMg4. i®9**20O ® Jons®/ sc. L., "Sho ter@l©pwit o£ tfaa Young vegetative tfpcal* te in Btrmtoau .,' tii ^^pstoat tf M®« Attfc ana
xii«n3x*rgar-B0k©l, 8., "*i» Life cycle of Sp«i»g as EmrooXod toy a 3t«ay of tfceir Straotaa© sod'Sieptatiao ' •TomrngLl. ot Amoral Mcacribiolocer* I Ci945), 23-32* iicClung, IT. II.* "VfctlabiUty ia oawtawgjeetaw," fiHffltirfft S& mm Tftaflt iteteg* zaaSQt llfS®), 379*«886. PtfVSlMMI, D< Physiological stadioff on tto* " ' * > SEES (1953), 46-071. Wave. P.. 'f*F®s»wftati« ©£ Tinder liltsogoa*
, 286-295.
2
&•* K* o„, " she MocgiM&ogjr ®k3 tASa c o x tscMth tj&teer AetAJMOKOGfea?, a*ajnaaafeigri &g &** tomtom. maMs^afn^p^ %&£»%>
3§®astifcl* Q«. aal $pod*>«&€ttn*Hifci, L.» "tSeastdte llsottialfssfeitea SttMBBBsaa/" aaam, ct&s&z l imm* m .
tjy IC* a., Bliss®!!# J. C., EuSdifi* £>, R.} ® l JSoOCMOsa* ion# >#» c,, 'Kttaafootea cadi Oatatfon faste®© and odoro# «».«-** , • — -'2^ U95C)
stuorfc, s>. d.# £*., m » staMcftnge o£ tls© ;*x&ao£d oafi In-ternal fleeiirfiii© 8&8teet& o£ S ^ ^ h w
w e w m C&OO), m*aai, #
#
vfaSj Bso# s„ A. , cin& HosixMolr A. » "St® iia ol tea's cjj silteaitefi of tho l^iws^^es#" Ja»««ii. of noota-
(1943), 337-341. M* « « r
ablay, ». w., '5$i» Sffixtft of OBafgnt on tho a w S t aas Mote* ©&t» of tfha Aeftotoio wciopa.lic Megg _ — - « — « - —
ytt%tib%A*fhaA Material
^ S M S m w i w m , •'wM&simm m m ^ ^ m s p ^ y partraenfc of Biology, Novth teas States tfnlvoreltv, Bmttbn, Trnwm,' i»62.
