HYPHAL ANASTOMOSIS AND CYTOLOGICAL ASPECTS

OF STREPTOMYCES SCABIES1
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Abstract
Two strains of Strepto?izyces scabies and a Strepto~i~yces sp., strain T 1 2 , h a w been
sti~diedfor hpphal anastomosis ancl its cytological consequences. The presence
of multiple short hyphal bridgcs between neighboring hpphae and the absence
of dcrnonstrable cell walls or ~ncmbranesa t the points of contact are presented
a s cvidence for anastomosis. T h e formation of "initial cells", a s a result of
hyplial fusion, \\.as not obser\led. Two types of s\\rollen boclies, decply stained by
the acid-Giemsa method, were noted. Onc type, identified as the residue of the
parent spore, passcd t h r o ~ ~ ga hstage during,w!lich it did not stain by this lncthod
but s ~ ~ b s e q u e n t lbecame
y highly c h r o m a t ~ ~ l ~ The
c . s c c o ~ ~type
d was aln.ays
terminal and developed concurrently with or subsequent t o "secondary
mycelium". Aerial hpphae \\.ere observed t o arise directly from "prilnary
mycelium" in S . scabies. Hyphac wit11 cytological characteristics intermediate
between primary a n d secondary types mere observed in Strepto~izyccs sp. T12.

Introduction
Genetic recombillation in Streptomyces coebicolor Reiner-i\/Iiiller (15) and
the production of heterolcaryons in several other species of Strepto,nzyces
For personal use only.

(1, 16) have been reported without comment as to the mechanism b y \vhich
nuclei fro111 two parental strains may beconle associated within a single
hypha. Similar phenomena alllong the filamelltoils fungi have been ascribed
to hyphal anastomosis (13). Cytological demonstration of anastomosis in
the streptomycetes, however, is equivocal. Carvajal (2) reported observing
hyphal fusions during electron microscope s t ~ ~ d i of e s Streptomyces griseus b u t
did not p ~ ~ b l i ssupporting
h photographs. I<lienebergcr-Kobe1 (8) assumecl
t h a t fusion occurred \vithin "nests" of hyphae or agglomerations of hyphae
described as slceins, netwol-I;, and scrolls. She observed t h e development of
new cellular eleme~ltsa t the points ~vlleretwo filaments were in close contact.
These new bodies were designated "initial cells" since they elongatecl and
divided to form a "secondary m y c e l i ~ ~ m " .This secolidary myceli~impossessed
cleeply staining chromatinic cylinders, easily stainable transverse septa, and
fewer sicle branches than the "primary mycelii~n~".T h e seconclary m y c e l i ~ ~ m
~ ~ l t i m a t e lgave
y rise to conidia. Erilcson (4) challengecl these interpretations
on the grounds t h a t the "nests" described resulted fro111 branches o l h-)~phae
sticking together when the slicles were fixecl and clried. Furthermore, she
stated t h a t a general absence of anastomosis is one of the characteristic
f e a t ~ ~ r eofs the streptomycetes, \vherein they differ nlost marliedly from
fungi. P h a u et nl. (12) coulcl find 110clear cases of anastonlosis in S. gl.iseus.
S o ~ n e\vorlcers have observed snrellings in the llyphae of streptomycetes,
not originating from "nests" of tangled filaments, which they considered to
be initial cells (3, 9, 17). Some of these structures were found to be intercalary
' M a ~ ~ l i s c r i received
pt J l ~ l y4, 1956.
Cor~triblitio~z frotrz the Departinent of Bacteriology, Olttario Agricult~~ralCollege, G l ~ e l p l ~ ,
Owtnrio. Tlze iitoestigatio~t was part of tAe progranz of tlze Outario Potato Scab IZesearclz
 650 C . \ V A D I A S JOURS.412 O F MICROBIOLOGY. VOI,. 2, 1956

while others were attached to the hyphae b y short sta1l;s. Diclie~lso~l and
MacDonald (3) published electro~l micrographs which were intended to
show hyphal a~lastonlosisand the formation of initial cells via anastornosis.
These illustratio~lsare not entirely convincillg since the hyphal strancls are
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nlerely shown to lie in contact with one another, and proof of actual fusion is
lacliing. T h e work reportecl here was undertalten to explore further t h e
presence or absence of hyphal anastomosis in streptomycetes and its proposed
role in initial cell formation.

Materials and Methods

Streptomyces scabies (Thaxter) IATalisnlan & Henrici, O..A.C. strains A26
and A30, originally isolated from potato scab lesions and purified by sin-
gle spore isolations with the de F o ~ l b r u n e1nicrol11a11ipulator, were examined.
.A Sfreptonzyces sp., strain T12, in which initial cells have been described
(9), was 1;inclly supplied b y Dr. H. B. Newco~nbe,ancl was also studied.
T h e inocula used were prepared b y suspencling the sporulated surface
growth of cultures, five t o seven days old, in sterile 1 : 5000 Tween 202 and
filtering these conidial suspensions through sterile No. 1 U'hatinan filter
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papers t o remove mycelial fragments. T h e cultures were gro\\-11 on the

surface of sterile cover slips either b y the invertecl agar-blocli technique of
I<lieneberger-Nobel (8) or in liquid ~nediilmspread over the surface of the
cover slip. All cultures were incubated a t 28' C. over clistilled water in
a sealed chamber. T h e high humidity in this chamber prevented evaporation
of the meclia. I n some cases, the inverted agar-block cultures were removed
from the hu~nitlitychamber after 48 hr. incubation so t h a t slow drying would
favor sporulation. A \,east-beef brothQanc1 t u o aspnragine-gli~cose media
(6, 9) were employed.
Slicles were removed from the incl~batorrat hourly intervals for the first eight
hours, a t four hour intervals for the next 28 hr., ancl a t eight hour intervals
s u b s e q u e ~ ~ tfor
l \ ~ a total of five clays. T h e growth on each cover slip was
allo\vecl ~ ~ a r t i a l to
l y d r y in air and was hsecl for two minutes in the vapor of
274 osmic acicl. T h e agar bloclis were then removed from the invertecl
agar-block cultures ancl the cover slips iln~nersecl in ~nercuric chloride
(satill-atecl) for four minutes.
Three methocls of staining were usecl with all ages oi cultures. 'The
Robinow (14) acid-Giemsa methocl of nuclear staining nlas iollo~vecl. T h e
cover slips were in~merseclin N FICI a t 60' C. for eight minutes, rinsed wit11
clistilled water, ancl stained with G i e ~ n s astain (20 clrops o i stock solutioll
per 10 1111. phosphate buffer, pH 7 . 0 ) for 20 min. Permanent mounts were
~naclein a mixture of ~nineraloil ancl a c - b r o l ~ ~ o ~ ~ a p h t l ~according
: ~ l e ~ l e to the
proceclure of hginsavage (10). T h e tannic acicl - crystal violet stain for
rnembrnnes, as applied b y Klienebergel--Nobel (8), \\.;as used t o reveal
?./I w e t l i ~ ~c~gelzl
g obtained from l l ~ eAllas Powder Co., H f i l ~ i z i ~ ~ gDelawc~re,
to~~, U.S.A.
Yeasl exlracl, 3 . 0 gnt.; beef exlracl, I . 5 gnr..; peplone, 6 . 0 gnl.; gl.ucose, 1 . 0 g1rz.;
disfilled aualer, 1000 ~ 1 . ;PI1 6 . 5 .
 septations. These preparations were also mounted by the Millsavage
procedure. One minute in a m m o n i ~ ~ oxalate
m - crystal violet (7) provided

a clear picture of gross morphology.

Photomicrographs were made 1vit11 a Leitz Ortholux microscope sing a
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X 90 apochromatic objective, X 12 periplanatic eyepiece, 50 watt lour voltage

tu~lgstenfilament for illumination, I<odak Wratten Filter #58 (green), Leitz
extensible mirror reflex camera for plates 9 X 12 cm., and I<oclalc Ortho-X
film or, for low contrast material, Iiodalith Ortho-Type 2 film.

Results
De7norzstratio?z of A?~nstomosis
-An examination of young (less than 24 hr.) mycelia of the strains of
S . scabies and, to a lesscr extent, the mj-celium of Streptomyces sp. T12,
stained by the simple crystal violet stain, revealed Inany instances wherein
ttvo h).pl~ae,origin,\ting from different spores, appeared to be united b]. a
short hyphal strand. Such a n appearance could occur by the chance
juxtaposition of hjphae, ho~rever,cvithout the occurrence of fusion. T l ~ e
~lnionof separate hyphae by two or rnore strands in close proxinit>- uras
also observecl and considel-ed less lilcely to represent a chance occurrence.
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Figs. 1, 2, and 4 each sholv two hyphae, originating from different spores,
joined by two or morc hyphal strancls, while Fig. 5 shows two hyphae,
originating from the same spore, bridged by ut least three strands.
The tailnic acid - crystal violet stain for me~nbranesprovided a further
test as to whether fusion had actually occurred bet\veen hyphae. This
procedure stained the hyphae only lightly and dicl not render them opaque.
Hence, \\here one hyplia lay on top ol another, the stain appeared to be much
darker than ~vhcretwo 11g.phae merely touched olle another. Furthermore,
where t\vo hypllae came in contact without fusing, some of the ta~lllicacid
morcla~ntprecipitated and revealed the cell ~vallsseparating the two hyphae.
Anastomosis, as revealed by this stain, is shown in Figs. 3, 6, and 7.
Gsing the criteria of ~nultiple~ ~ n i obetween
n hyphae and the absence of a
demonstrable cell xvall or membrane between these h y p l ~ a eas evidence for
f ~ ~ s i o nit, was apparent that the three strepto~nycetesexanlilled possessed
the ability to ~l~itlergo anastomosis. Thc two strains of S. scabies appeared
to be subject to agreater f r e q ~ ~ e n of
c y hyphal fusions than dicl the unidentified
species T12. I t was apparent that a~lasto~nosis occurred both in the early
growth of the myceli,i (less than 16 hr.) and in later growth. T h e incidence
of ailastornosis wc\s not demonstrably influenced by the co~npositio~l of the
culture mecli~un.

Occurrence of Swollen Chromatinic S t r u c t ~ ~ r e s

The acid-Giemsa stain did not reveal ally unusual features in the chromati~lic
bodies (presumably nuclei) present a t the sites of allastornoses which would
indicate the fornlation of "initial cells1'. S\vellings in the hyphae, both
terminal and intercalary, were observed a t various stages of gro\vth, however,
 652 CASADI.AN JOURNAL O F MICROBIOLOGY. VOL. 2. 1956

which resemblecl those described by some worlrers as initial cells (3, 9). These
bodies stained deeply with the Giemsa stain follo\ving acid hydrolysis or
contained much chromatinic material. These structures appeared to be of
a t least two types.
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One type of structure has beell identified as the residues of spores froin
which the hyphae originated. When streptomycete conidia were placed
in a culture medium, they swelled, to give a three- to four-fold increase in
volume, prior to germination. Some spores germinated \vith one or two
hyphae (all cultures studied) or \vith three and rarely four hyphae (S. scabies
A26) (Fig. 8, A-D). For the first few hours of growth, the parent spore
was readily recognized. After incubatioil for about 12 hours, however, the
parent spore became unrecognizable when the young mycelium was viewed
subsequent to the acid-Giemsa stain (Fig. 9). Parallel series of slides stained
by alternate methods (e.g. crystal violet) still showed the originating-spore
very clearly (Fig. 10). As the mycelium developed furthcr (about 20 hr.
incubation), the parent spore once again was clearly revealed by the acid-
Gienlsa stain (Figs. 11 and 12). The spores now stained uniformly dark
with this stain or appeared to contain several tightly packed chron~atinic
bodies. T h e parallel series of slides, stained by the alternate procedures,
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made the identification of these structures as spore-residues quite clear.

Xfter about 44 hr. incubation, additiollal swollen bodies were observed.
Some of these were no larger than the spore residues clescribed above but
were Inore nearly spherical (Fig. 13). Others were several-fold larger and
irregularly shaped (Figs. 14 and 15). All of thcse structures observed were
terminal and stained deeply, \vithout diilcrentiation of interilal detail, wit11
the acid-Giemsa stain. T h e hyphae bearing these s\vollen bodies lilielvise
stained deeply without differentiation ol cytological detail. These bodies
appeared a few hours earlier in Sfreptomyces sp. T I 2 than in S. scabiesA26
but were essentially the same in both cultures.

Occurrence of "Secondary ilIycelizcm"

Mycelium similar to Iclieneberger-Nobel's (8) "secondary mycelium" was
observed in Strepfomyces sp. T12 as previously reported by i\/IcGregor (9).
T h e hyphae in this nlycelium possessed chrolnatinic cylinclers, illore or less
regularly spaced transverse septa, and fewer branches than those in the
"primary" myceliun~. T h e designations "priillary" and "secondary" a s
used here follow thc termiilology of I<lieneberger-Nobel (8). T h e identifi-
catioil of the mycelia is based solely on their cytological appearance. I t
proved to be extremely difficult to trace these secoi~daryhyphae to their
points of origin. In many cases, however, they could be traced t o regions
where the internal structure, as revealed by the acid-Giemsa stain, appeared
to be intermediate between the primary and secondary types (Fig. 16).
T h e mycelium of S. scabies could not be differentiated clearly into primary
and secoildary types. Hyphae with few branches and clearly de~noilstrable
- transverse septa were observed in old cultures (Fig. 17) b u t these hyphae
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FIGS. 1 a ~ ~2. c l r\~iastollioscsbct\\:ec~lhyphac origilintillg fro111selxlratc sporcs. S. scc~bics

.\?6, 16 hr., cryski1 \-iolct stain. X 1600.
FIG. 3. S. scclbics :\26, six c l a y , 11lclnl)ra11c sinill. X 2.i00.
Frc;. 4. ; \ ~ ~ ; i s t o ~ ~ ~ I,ct\\.ecu
o.;cs l~!.pl~;~c c]rigi~lat.i~~g frool s c p a ~ ~ tspcrrcl-;.
c .Ylrc~plo~lrycc,s
sp. 'f12,-16 Iir., cr~.slalviolet s l a i ~ ~ X. ?.iOO.
I . . . \ ~ ~ a s i o ~ ~ i o11ct\\.cc11
scs I I salrlc sl~orc. .C, .sc.c~/~ic.s
I~ypllaco r i ~ i l i a i i ~I ~~g ~ Illic :\2G,
cr!.slal violct stain. X 1600.
FIG. 6. S.src~bics.\30, 20 lir., mcml~rancs t a i ~ X ~ . 2.100.
FIG. 7. S. srtrbics 2\26, 20 l ~ r . ~ , i ~ c r i i b r as ~t a~ie~ ~X. 2300. Notc al~scllccoi ~ n c ~ ~ ~ l ~ r a ~ l c s
a t poi~lisot' col~tacl..
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ITrc;. 8. ( ; c r ~ ~ ~ i l ~ a t of of .Y. sctihicss ;\2h, 8 lir. X 2300. (;\) O11c liyph;~. ( R )

i c , c:o~~itli;l
i~
'l'\\.o Ii!.l)l~ac. (1) 'i'lircc liyphac. (I)) Four h).ph;~c.
I ; 0 .S. .scobic.s .\2h, 12 hr., ac.itl-(;icnlsa s t ; i i ~ ~ .1';1rc11(.spore ~ ~ rccognizaljlc.
o t X 2.300.
Frc.. 10. .S. scc~bics;\I(,, 12 l ~ r. . c:rystal , . \.iolct s t a i ~ ~ I'arcllt
. s l ~ o r cclearly r c c o ~ ~ i i z a l ) l e .
X 2300.
I . I I . S. sc-trliies .\26, 24 hr., acitl-(;ic~~ls;~ s t a i ~ ~ I'arc~ll.
. spores tlccply s l ; ~ i ~ ~ eXt l 2.100.
.
Frc;. 12. Slrr~/)lu~~iycc.s sp. TI', 20 hr., acitl-Gicms:~ S ( ; I ~ I I . I';lrc~lt spores tlecpl). stai~lctl.
X 2.300.
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I:rc.;. 1.;. S . st:nhic.s .\20, 72 I I ~ .;~c.itl-(;iclnsa

, stail]. l ~ t r i t l c ~ ~ ~ i ls\\-r~llc~t
ietl I)otlj-. X 2300.
I . 1 I.;lrgc i r r e ~ c ~ l a r lsll;~l)ctl
! 3 \ v o l l e ~I~otlics.
~ .C. .vt:c~hic,.s .\?(I, 48 11r., acitl-Gicn~sa
S ~ ; I ~XI I2~<00.
.
Frc;. 15. l.;crge s \ v o l l c ~I)otlj,.
~ .Yrc/)lomiyrc.s s11. '1'12, 48 hr., ucitl-Gicnlsa s r a i ~ i .X 2300.
1 , 0 I ; i ~ ~ t e ~ ~1 c tl i 1t " r i ~ r I" ' e : o t ~ c l r "I . .CIr(~pIo~~~yct~s
511. '1'12, 60 l ~ r . a, c i d - G i c ~ ~ ~s tsial i ~ ~X. 2.500.
1;;. 1 Sel,t;llic)~isit1 six-tl:~y.C. .sr-r~l~ir~.s 2\26, mcnll)rntle s t i l i ~ ~X. 1-300.
I . 1 . S c p t a t i o ~ l si l l 16-lir. .S. scclDii~.s.\26, ~ n c t ~ ~ l ~ stain. l - a ~ l cX 2300.
I:rc;. 10. Spusc iol.mation i l l S. sc.clbic,.s :\2h, six (la)..;, nlcml)sanc stail). X 2.iOO.
FIG. 20. ;\c:ri;~l hyllhnc origi~~at-iilg from " p r i n ~ a r ) ~ m " > . c c l i ~ ~ m.S.'. sc:tabie.s :\26, i 2 hs.,
:tc.itl-C;ic~~~s~s t n i ~ l .X 2.300.
 GREGORY: STREPTOMYCES SCABIES 653

clearly originated directly from inore profusely branched mycelium. Very

young hyphae also often shoxved similarly spaced, readily demonstrable
transverse septa (Fig. 18). Conidia were generally formed on short branches
derived from an abulldantly brailched hypha (Fig. 19). The acid-Giemsa
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stain did show deeply stained, little-branched hyphae which contrasted with
the lightly stained, branched inyceliurn containing discrete nuclear bodies.
i\/Iany of these hyphae coulcl readily be traced to their points of origin from
hyphae resenlbliilg those described as primary (Fig. 20). No special structure
or initial cell could be identified a t this point of origin.

Discussion
The abundant occurrence of multiple unions, by short hyphal strands,
of neighboring hyphae, together with the absence of demonstrable cell walls
or membranes a t the points of contact, indicate that l~ypllalanaston~osisis
of common occurreilce in the three strains of streptomycetes esarnined.
Although septa were clearly deinonstrable by the tannic acid - cr)-stal violet
stain, the cells xvei-e multinucleate. Hyphal fusion in these organisms coulcl,
therefore, readily produce a heterolcaryotic conditio~l.
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Erikson (4) has suggested that the dark-staining discoid structures revealed
by the tannic acid mordanting technique Inay not be true septa but may
result froin the action of tannic acid as a protein precipitant. T h e coherence
of the streptomycete mycelium, as compared to the reacly rupturing oi the
inyceliunl of the nocardias, where septa inay be observed tvitho~it prior
mordanting, was offered in further support of this view. T h e regular
occurrence of these structures, in manv hyphae of the strains examined,
and their similarity to the ~~lldisputed septa formed during sporulation, make
this interpretation less lilcely. I t must be rernelllberecl that although septation
may be follo\\recl by fragmentation of the mycelium anloilg some of the related
members of the Actinomycetales, many of the septate nlolds maintain a firmly
coherent myceli~im.
The failure to note any nuclear changes a t the sites of ailastomoses does
not, in itself, mean that such might not have occurred if further clevelopn~ent
hacl been permitted. The formation of large s\vollen initial cells as the result
of anastomosis appears to be improbable in these strains, however, since
none of the observed structures iulfilled the necessary criteria. T h e parent
spores are persistent and have the peculiar property of not containing
chromatinic nlaterial during the early development of the hyphae but of
staining deeply with the acid-Giemsa stain a t a later stage. If a spore had
germinated wit11 one hypha only, it would subsequently appear to be on the
end of a stalk, whereas if it had germinated a t t ~ v opoints, it would appear
to be in the middle of a hypha. If one were to esamine various ages of
streptomycete mycelium by the acid-Giemsa stain alone, these spore-residues
might seem to appear de novo. Hence, care should be talcen not to interpret
these structures as initial cells.
 654 C.\S.\DI.\N JOURKAL O F AIICIIOBIOLOGS. VOL. 2, 1956

T h e other swollen chromatinic structures observed were invariably terminal.

These could not, therefore, have arisen a t the sites of anastomoses. If their
formation were related to hyphal fusion, then a co~lsiderableamount of
growth must have occurred followi~lg the anastomoses and prior to the
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formation of tlie swollen bodies. In all cases, these structures developed

concurrently with, or subsequent to, the development of hyphae cytologic all^^
characteristic of the secondary mycelium. I t appears to be unliliely, therefore,
that the secondary myceliu~narose from them. T h e hyphae 011 which these
bodies were borne were u~ldifferentiated and not typical of either primary
or secondary t ~ p e s . The function ol these s\vollen bodies, i f anj., was not
apparent. IZnlargecl stl-uctures have also been clescribed in S. grisez~s (12).
E r i l i s o ~(~5 ) observed many large s\vollen cells in S. scubies and S. coelicolov
grolvn on ~iiediaco~ltaini~lg a detergent or pectin. She con side^-s that the
blodted shapes frequentlj- ellcounte~-edin acti~lomj-cetesresult fro111overfeeding
and othel- unlavorable conditions.
Iilieneberger-Sobel (8) considered that the structure of tlie secondary
rn>.celium favored tlie assuml~tionthat a fusion cell with a fusion nucleus
initiated its formation. Since h>-phaeapparentl~.representative of secondar)-
mycelium in S. scabces A126 may originate directl5- fro111 typical primary
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ml-celium (Fig. 20), initial cell formation is clearly not a necessary prerequisite
for the clcvelopme~itof secondary or aerial hyphae. Evidence for the direct
development of seconclary ml-celium from primarj- myceli~~ln in S f ~ e p t o r n y c e s
sp. T I 2 is less conclusive. 'The existence of hyphae ~ v i t h an appearallce
intermediate between these t\vo types, however, suggests t h a t , here too, no
fusion cell or iusio~lI ~ L I C ~ ~ Lneed
IS precede the formation of ~ n y c e l i u ~cyto-
n
logicallj. identifiable as secondar!l mycelium. These observations suggest,
therefore, that the development of "secondary" mj-celium in the strains of
streptomycetes studied results from physiological changes rather than a n
alternatio~lof haploid ancl diploid phases as suggested by Iilieneberger-Sobel
(8). These changes could be associated \vitIi exhaustion of nutrients, maturity
of the m y c e l i ~ ~ m or, some other factor. Sewcombe (11) noted one i~lstance
in 11-hich the environme~ltaffected tlie cj-tological appearance of the nuclei
in a streptom)-cete. Intermittent incubation (alternating with chilling)
resulted in nuclei being clearly visible in a lightl). stainecl cytoplas~n,whereas
~vitlicontinuous irlcubatioll the stainable material iiearlj. filled the cell.
T h e rarity of genetic reconlbinatio~iin conidia formed by heteroliaryo~is
of streptomycetes (1, 16) agrees with the concl~~sions presented here. If
colliclia were formed by meiosis from diploid iluclei of seco~ldarymycelium,
one \vould anticipate a much greater i~lcidellceof reco~iibiiiedcharacters in
the haploid conidia than has been observed.

Acknowledgment
This was assisted by a grant from the Xational Research Council
of Canada whose support is gratefull). acknowledged.
 GRBGOIIY: STIIEPTOMYCES SC.-\BIBS

References
1. BRADLEY,S. G. '111d LEDERBERG,J. Ilcterolcaryosis i n Streptomyccs. Bacteriol
I'roc. 1956 : 48.
2. C:\RV.\T.\L,F. The prod~rctionof spores in submerged cultirres by sorne Streptomyces.
Liycologia, 39 : 426-440. 1947.
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3. D ~ c ~ c ~ : s s oPs , B. and ~I.\cDoN.\LD,Ti. D . i\n elcctro~lrllicroscope examination of

the initial cell stage in Streptoinyces spp. J. Gen. hIicrobiol. 13 : 84-90. 1955.
4 ERII<SON,D. 'The nlorphology, cytology, and taxorlomy of the actinornycetes. .\nn.
Rev. hIicrobiol. 3 : 23-54. 1949.
5. ERIICSOS,D. L o ~ of s aerial myceli~rn~ ancl other cha~lgcsin strcptomycctc tlcvelopincnt
due to physical variations ol cultural conditions. J . G c r ~ .1Iicl.obiol. 13 : 136-148.
1955.
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