INTERNATIONAL JOURNAL O F
SYSTEMATIC BACTERIOLOGY
Vol. 16, No. 3
J u l y 1966
pp. 313-340
METHODS FOR CHARACTERIZATION
OF STREPTOMYCES SPECIES'
E. B. Shirling and D. Gottlieb
Department of Botany and Bacteriology
Ohio Wesleyan University, Delaware, Ohio
and
Department of P l a n t Pathology
University of Illinois, Urbana, Illinois
ABSTRACT. T h e m e t h o d s u s e d b y c o l l a b o r a t o r s
i n t h e I n t e r n a t i o n a l S t r e p t o m y c e s P r o j e c t (ISP)
f o r . e m e n d a t i o n of d e s c r i p t i o n s of t y p e a n d
n e o t y p e s t r a i n s of t h e g e n u s S t r e p t o m y c e s
(Actinomycetales) a r e presented.
-------------
An international cooperative effort, now i n p r o g r e s s , is
d i r e c t e d toward collection of type cultures of t he Streptom y c e s s p e ci e s fo r deposition with the Centraalbureau voor
Schimmelcultures (CBS), Baarn. F r o m this cent er the refe r e n c e cultures will be supplied t o other culture collections
s o that they a r e available throughout the world.
An e s s e n t i a l adjunct t o this activity is the r edes cr i pt i on
of each type culture i n t e r m s of c u rrent l y acceptable c r i t e r i a and methods. Th e u rg e n t need f o r an authentic r e f e r ence collection, accompanied by standardized char act er i zations f o r each s p e c i e s , h a s been pointed out by spokesmen
f o r the s e v e r a l meetings and conferences which culminated
i n this project.
(See, f o r example, Gottlieb, 1959, 1961;
Mor e than 40 investiKiister, 1959; Krasil'nikov, I961 .)
g a t o r s t representing 17 countries a r e participating i n this
r e s e a r c h . E a c h culture is d e s c ri b e d independently by t h r e e
of t h e s e cooperating s p e c i a l i s t s i n different l abor at or i es
before i t is deposited i n the re fe re n c e collection.
This project is supported i n p a r t by a r e s e a r c h grant f r o m
the National Science Foundation, U. S. A. The Subcommittee
on Actinomycetes of the Committee on Taxonomy, A. S. M.
a n d the Subcommittee on,Taxonomy of Actinomycetes of the
International Committee on Bacteriological Nomenclature
a r e co-sponsoring advisors.
P a r t i c i p a n t s i n the 1964-1965 studies a r e l i s t ed on p. 338.
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INTERNATIONAL JOURNAL
This manual contains the c r i t e r i a and methods adopted
f o r the project. It re fl e c t s the re s u l t s of two extensive cooperative studies d i re c t e d toward selection of stable prope r t i e s and reproducible p ro c e d u re s f o r char act er i zat i on of
streptomycetes. One study conducted under t he direction of
the Subcommittee on Actinomycetes of the Committee on
Taxonomy, A m e ri c a n Society f o r Microbiology was r epor t ed
by the Chairman, Dr. D. Gottlieb (1961). A similar p r e l i m i n a r y study on an international basis was r epor t ed f o r
the Subcommittee on Taxonomy of Actinomycetes of the International Committee on Bacteriological Nomenclature by
the S e c r e t a r y , Dr. E. Kiister (1961,1964). The des cr i pt i ve
c r i t e r i a a r e e s s e n t i a l l y t h e s a m e a s those included i n t he
recommendations of this international subcommittee f o r
descriptions of Actinomycetales appearing i n patent applications (Gottlieb, 1963). The methods i n mimeographed
f o r m have been u s e d successfully f o r the description of type
and neotype s t r a i n s of m o r e than 200 named s peci es submitted t o ISP collaborators during 1964 and 1965, and a r e
now i n u s e f o r a continuation of the project. Only mi nor
e d i t o r i a l changes have been m a d e except that the t e s t f or
n i t r a t e reduction (including m e d i u m 8, Bacto-nitrate broth)
h a s been omitted. This c h a r a c t e r i s t i c proved unstable and
h a s been dropped f r o m the study.
It i s hoped that the c h a ra c t e ri z a t i o ns used i n t hi s manual
will be included in fu t u re descriptions of Streptomyces spec i e s s o that d i r e c t comparison can be made with des cr i ptions f o r type cultures i n the re fe re n c e collections.
MATERIALS AND GENERAL METHODS
CULTURE MEDIA
P r e p a r e Difco% 4 dehydrated culture media as i ns t r uct ed
on labels of containers. If t h e dehydrated media a r e not
used, u s e f o rm u l a s and i n s t ru c t i o n s in this manual as a
guide t o preparation of the media.
All culture media d e s c ri b e d i n this manual have been pr ep a r e d especially f o r the I.S. P. by Difco Labor at or i es as
pref ormulat ed dehydrated media. This important cont ribution by Dif co La b o ra t o ri e s is gratefully acknowledged. When
Difco dehydrated m e d i a a r e used, instructions on labels
s u p e r s e d e i n s t ru c t i o n s i n the manual.
Difco L a b o ra t o ri e s , Detroit, Michigan, U. S. A. 48201
SYSTEMATIC
BACTERIOLOGY
315.
S t e r i l i z e culture media i n t h e autoclave at 121 C. S t e r i l i z e loosely packed'tubes o r flasks containing less than 500
m l f o r 15 minutes; h e r i l i z e l a r g e r quantities f o r 20 minutes.
(Do not autoclave carbon compounds t o be u s e d i n the c a r b o n
utilization t e s t s . Special instructions f o r s t e r i l i z i n g t h e s e
compounds a r e given with medium 9.) Adjust pH of m e d i a
with NaOH o r HC1 before addition of a g a r and before sterilization.
O
T r a c e s a l t s solution ( U s e as d i r e c t e d i n media 3, 4, 5 and 7
i f p r e p a r e d f r o m formulas. Do not add t o t h e c o r r e s ponding Difco dehydrated media.)
. . . . . . . . . . . 0.1 g
. . . . . . . . . . . . 0.1 g
. . . . . . . . . . . . 0.1 g
. . . . . . . . . . . 100. 0 ml
F e S 0 4 . 7Hz0
MnClz*4H20
ZnS04.7Hz0
Distilled w a t e r
,.
,
Medium 1: Tryptone-yeast e x t r a c t broth ( P r i d h a m and
Gottlieb, 1948)
........
......
...........
Bacto-Tryptone (Difco)
Bacto-Yeast E x t r a c t (Difco)
Distilled w a t e r
pH 7.0 t o 7.2 before autoclaving
5.0 g
3.0 g
1.0 liter
Dispense 5 ml of broth into test tubes with a d i a m e t e r
of 20 mm o r m o r e . U s e t h e s e t e s t tubes f o r initiating
growth f r o m lyophile pellet. One tube will be needed f o r
e a c h culture studied.
Dispense 50 ml of t h e broth into each 250 ml E r l e n m e y e r f l a s k ( o r 25 ml into 125 ml flask). T h e s e f l a s k s
will be used f o r preparation of washed inoculum (p. 322).
One f l a s k will be needed f o r e a c h culture studied.
Medium 2: Yeast extract-malt
al., 1956-57)
-
e x t r a c t a g a r ( P r i d h a m eJ
......
......
.......
...........
-.............
4.0 g
Bacto-Yeast E x t r a c t (Difco)
Bacto-Malt E x t r a c t (Difco)
10.0 g
4.0 g
Bacto-Dextrose (Difco)
1. 0 liter
Distilled w a t e r
Adjust t o pH 7.,3, then add
Bacto a g a r
20.0 g
Liquefy a g a r by steaming at 100" C f o r 15-20 minutes.
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INTERNATIONAL JOURNAL
Dispense appropriate amount for a slanting into at l e a s t
Sterilize by autoclaving; cool
tubes a s slants. Use the a g a r slants for preparation of
stock cultures (page 321).
6 tubes f o r each culture.
Also sterilize medium 2 i n flasks f o r pouring the sterili z e d medium into P e t r i dishes. Seven standard 90 mrn
dishes containing 25 ml p e r plate will be needed f o r each
culture. (Pages 325 and 330).
Medium 3: Oatmeal a g a r (KUlster, 1959a).
..............
...............
Oatmeal.
20.0 g
Agar.
18.0 g
Cook o r s t e a m 20 g oatmeal i n 1000 ml distilled water
for 20 minutes.
F i l t e r through cheese cloth.
Add distilled water t o r e s t o r e volume of filtrate
to 1000 ml.
Add t r a c e salts solution (page 315).
..
1.0 ml
Adjust to pH 7 . 2 with NaOH.
Add 18 g a g a r ; liquefy by steaming at 100°C f o r
15-20 minutes.
SYSTEMATIC BACTERIOLOGY
317
S t e r l i z e i n f l a s k s f o r pouring into P e t r i d i s h e s . Seven
s t a n d a r d 90 mm d i s h e s containing 25 ml p e r d i s h w i l l be
needed f o r e a c h culture. ( P a g e s 325 a n d 330)
S w i r l m e d i u m before pouring t o a s s u r e even distribution
of the oatmeal.
Medium 4: Inorganic s a l t s - s t a r c h a g a r (KUster, 1959a. )
Solution I: Difco soluble s t a r c h 10. 0 g. Make a p a s t e of
the s t a r c h with a s m a l l amount of cold distilled w a t e r
and b r i n g to a volume of 500 ml.
Solution 11:
K2HP04 (anhydrous b a s i s )
1.0 g
MgS04.7HzO.
1.0 g
NaCl
1.0 g
(NH4)2S04.
i
2.0g
CaC03
2.0 g
Distilled water
500 ml
1 . 0 ml
T r a c e s a l t s solution (p. 315)
pH should be between 7.0 and 7.4. Do not a d j u s t
if i t is within t h i s range.
Mix s t a r c h s u s p e n s i o n and salts solution.
20.0 g
Add a g a r (Difco).
Liquefy a g a r by s t e a m i n g at 100°C f o r 15-20 m i n u t e s .
......
............
................
........... .
...............
...........
..........
S t e r i l i z e i n f l a s k s f o r pouring into P e t r i d i s h e s .
Seven s t a n d a r d 90 mm d i s h e s containing 25 ml p e r d i s h
will be needed f o r e a c h culture. ( P a g e s 325 and 330)
Medium 5: G l y c e r o l - a s p a r a g i n e a g a r ( P r i d h a m a n d Lyons,
1961)
....
...............
.......
............
.....
1.0 g
L - a s p a r a g i n e (anhydrous b a s i s ) .
Glycerol.
10.0 g
K2HP04 (anhydrous b a s i s )
1.0 g
Distilled w a t e r .
1. 0 l i t e r
1.0 ml
T r a c e s a l t s solution (page 315).
The pH of t h i s solution is about 7. 0-7. 4. Do not adjust
i f it is within t h i s range.
Agar.
20.0g
Liquefy a g a r by s t e a m i n g at 1 0 0 ° C f o r 15-20 m i n u t e s .
................
S t e r l i z e i n f l a s k s f o r pouring into P e t r i d i s h e s .
Seven s t a n d a r d 90 mm d i s h e s containing 25 ml p e r d i s h
318
INTERNATIONAL
JOURNAL
will be needed f o r e a c h culture. ( P a g e s 325 and 3 3 0 )
The f i n a l pH of the m e d i u m a f t e r s t e r i l i z a t i o n with
a g a r and solidification i s about 7 . 4 .
Medium 6 : Peptone-yeast e x t r a c t i r o n a g a r ( T r e s n e r and
Danga, 1958)
Bacto-Peptone I r o n Agar, dehydrated (Difco) 3 6 . 0 g
1. 0 g
Bacto-Yeast E x t r a c t (Difco)
Distilled w a t e r
1-0 liter
pH should b 7.0-7.2 before autoclaving; adjust i f
necessary.
Liquefy a g a r by steaming at 1 0 0 ° C f o r 15-20 minutes.
........
...............
Dispense a p p r o p r i a t e amount f o r slanting into 2 t u b e s
f o r e a c h culture. S t e r i l i z e and solidify as slants. ( P a g e
334). (Note that l e s s than 1 l i t e r of t h i s m e d i u m i s
e a s i l y p r e p a r e d by using a proportionately s m a l l e r
amount of the dehydrated peptone i r o n a g a r and adding
y e a s t e x t r a c t i n proportion of 0. 1% of w a t e r used.)
Bacto-Peptone I r o n A g a r , dehydrated, contains t h e
following ingredients when reconstituted as 36. 58 g r a m s
p e r l i t e r of water: Bacto-Peptone, 1 5 g; P r o t e o s e P e p tone, Difco, 5 g; F e r r i c Ammonium Citrate, 0. 5 g;
Dipotassium Phosphate, 1 g; Sodium Thiosulfate, 0.08 g:
Bacto-Agar, 15 g.
Medium 7: T y r o s i n e a g a r (Shinobu, 1958)
...............
............
...........
........
..............
..................
15.0 g
Glycerol.
0.5 g
L-tyrosine (Difco)
1.0 g
L- a s p a r agine (Dif co)
0.5 g
KtHP04 (anhydrous basis).
0.5 g
MgS04.7Hz0.
0.5 g
NaCl
0.01 g
FeS04. 7 H 2 0 .
1. 0 liter
Distilled w a t e r
1.0 ml
T r a c e s a l t s solution (page 3 1 5 ) .
Adjust t o pH 7.2-7.4
20.0 g
B a c t o - A-g a r .
Liquefy by steaming at 100°C f o r 15-20 minutes.
..............
..............
.....
..............
Dispense appropriate amount f o r slanting into 2 tubes
f o r e a c h culture; s t e r i l i z e and solidify as slants. ( P a g e
333).
SYSTEMATIC BACTERIOLOGY
319
Medium 8: Nitrate broth (Deleted because of unreliability
of the nitrate-reduction test).
Medium 9: Carbon utilization medium (Modified f r o m
' P r i d h a m and Gottlieb, 1948)
A. 'Sterile carbon s o u rc e s
Use chemically p u re carbon s o u r c e s cer t i f i ed t o be
f r e e of admixture with other carbohydrates o r contaminating ma t e ri a l s . Carbon s o u rc e s f o r this t e s t a r e :
No carbon s o u rc e (negative control)
D-glucose (positive control)
L-arabinose
Sucrose
D-fructose
Rhamnose
D xyl o s e
I-inos itol
Raffinose
D-mannit ol
Ce llulos e
-
S t e r i l i z e without heat by one of the following methods:
1. Filtration. F i l t e r s t e r i l i z e 10% solution through
bacteriological filter. (i-Inositol and cellulose a r e
not sufficiently soluble f o r s t e ri l i zat i on
by t h i s method-use one of the methods des cr i bed
below.)
2. E t h e r sterilization.
Weigh an appropriate amount of
the dry carbon s o u rc e and s p r e a d as a shallow l a y e r
i n a p re -s t e ri l i z e d E r l e n m e y e r flas k fitted with a
loose cotton plug. Add sufficient acetone-free ethyl
ether (C2H5)20 t o cover the carbohydrate. (OBSERVE
PRECAUTIONS AGAINST FIRE ! Allow e t h e r t o
evaporate at ro o m t e m p e r a t u r e under a ventilated
fume hood overnight o r longer. When all e t h e r h a s
evaporated add s t e r i l e distilled wat er aseptically t o
make a 10% w/v solution of the carbon source.
3. Ethylene oxide sterilization. (Judge and P e l c z a r , 1955).
Make a 10% w/v solution of the carbon source. Cool
the liquid i n an i c e bath t o 3-5'C and add 1 volume
p e r cent liquid (cold) ethylene oxide with a chilled
pipette o r syringe. Agitate the solution. Leave it i n
t h e cold i c e bath under a ventilated fume hood for
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INTERNATIONAL JOURNAL
1 hour. T r a n s f e r t o a w a r m w a t e r bath (about 4 5 ° C )
UNDER FUME HOOD t o p e r m i t complete volitilization
of the ethylene oxide. The vapors a r e toxic and
explosive.
Carbon s o u r c e s s t e r i l i z e d by one of t h e s e t h r e e methods
will be added t o the b a s a l m i n e r a l s a l t s a g a r t o give a
final concentration of 1%. F o r example, add 1 0 ml of
10% solution t o 100 ml b a s a l medium, o r 100 ml of a
10% solution t o 1000 ml b a s a l medium.
B. P r i d h a m and Gottlieb t r a c e salts (only 1 ml of t h i s
solution i s u s e d p e r l i t e r of final m e d i u m )
............
.............
.............
............
...........
CuS04~5Hz0.
FeS04* 7Hz0
MnClZ*4H20
ZnS04*7Hz0.
Distilled w a t e r
0.64
0.11
0.79
0.15
100.0
g
g
g
g
ml
S t o r e at 305°C until r e q u i r e d f o r use. Bring t o r o o m
t e m p e r a t u r e before using. P r e p a r e f r e s h solution e a c h
month. D i s r e g a r d any precipitate o r s c a l e (probably
i r o n salts) that f o r m s during storage. (Only 1 ml of t h i s
solution will be u s e d i n t h e medium.)
c.
B a s a l m i n e r a l salts a g a r (use analytical reagent g r a d e
chemicals)
2.64g
(NH4)$04.
KH2P04*anhydrous
2.38 g
K2HPO4'3HzO
5.65 g
MgS04*7H20
1.00 g
P r i d h a m and Cottlieb t r a c e salts (B)
1.00 ml
Distilled w a t e r
1.00 liter
Dissolve ingredients and check pH. Adjust, i f
n e c e s s a r y , t o 6.8-7.0 with 1 N NaOH o r 1 N HC1.
Add a g a r (Difco)
15.0 g
.............
.........
............
............
...........
..........
D. Complete m e d i u m
S t e r i l i z e b a s a l a g a r m e d i u m (C); cool it t o 60'C and
add s t e r i l e carbon s o u r c e (A) aseptically t o give a concentration of approximately 1%. Agitate the m i x t u r e and
pour 25 ml of m e d i u m p e r dish into 9 cm P e t r i dishes.
E a c h o r g a n i s m will r e q u i r e 2 P e t r i d i s h e s with no carbon
(as a negative control) plus duplicate plates f o r e a c h c a r bon s o u r c e tested. (Page 3 3 5 )
SYSTEMATIC BACTERIOLOGY
321
METHODS FOR INITIATING GROWTH AND PREPARING
STOCK CULTURES FROM LYOPHILE P E L L E T
1. Make a file s c r a t c h on ampoule at a location n e a r upper
p a r t of looped c o r d ( s e e Fig. 1).
2. I m m e r s e the unbroken ampoule into 7070 ethyl alcohol.
3. Enclose the alcohol-moistened ampoule with a piece of
s t e r i l e cotton o r cotton and gauze; then snap o r b r e a k i t
at the file s c r a t c h (Fig. 2).
4. Use s t e r i l e f o r c e p s o r a s t e r i l e stiff w i r e hook t o t r a n s -
f e r the looped s t r i n g and pellet t o the labeled tube containing 5 ml of s t e r i l e tryptone-yeast e x t r a c t broth
(Fig. 3). If a pellet f a i l s to come out attached t o t h e
s t r i n g , the c e l l s o r s p o r e s on t h e , s t r i n g will usually be
adequate t o start a good culture.
5. Shake the tube by hand until the pellet dissolves.
6 . Incubate the tubes i n a slanted position o r on a mechani-
c a l s h a k e r t o give good aeration. Use of a s h a k e r i s the
p r e f e r r e d method. Incubate at, 25-28°C f o r 24-28 h o u r s
( o r until t h e r e is evidence of s p o r e germination o r
growth).
7. Look f o r possible contaminants with the microscope.
Also s t r e a k one loopful of t h e broth culture onto the a g a r
s u r f a c e of a P e t r i dish containing medium 2 ( y e a s t
extract-malt
e x t r a c t a g a r ) . This plate can be examined
a f t e r a few days to confirm absence of contaminants.
8. Inoculate 6 o r m o r e t e s t tube s l a n t s of medium 2 ( y e a s t
extract-malt
e x t r a c t a g a r ) and of medium 3 ( o a t m e a l
a g a r ) with 0.1-0.2 ml of t h e 24-48 hour growth. S t r e a k
m a t e r i a l o v e r e n t i r e s u r f a c e of a g a r slant.
9. Incubate the s l a n t s at 25928°C f o r 14 days to get m a t u r e
stock c u l t u r e s f o r u s e i n p r e p a r a t i o n of inoculum ( s e e
section which follows). Then s t o r e stock s l a n t s i n r e f r i g e r a t o r (6-10°C) until r e a d y f o r use. Generally stock
c u l t u r e s f o r p r e p a r a t i o n of inoculum f o r c h a r a c t e r i z a t i o n
t e s t s should be used within one month.
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PREPARATION OF INOCULUM
Use stock culture s l a n t s (p
_ - re p a re d as des cr i bed i n t he
preceding section) fo r preparation of (A) gener al inoculum
f o r all inoculations except carbon utilization t e s t , and ( B ) a
s p e c i a l w.ashed inoculum fo r determining carbon utilization
patterns
~
.
A. P r e p a r a t i o n of g e n e ra l inoculum
1. P r e p a r e a supply of stoppered t e s t tubes containing 3-5
ml of s t e r i l e distilled water.
2. Use a w i re loop and standard a s e p t i c technique t o t r a n s f e r s p o r e s , o r m y c e l i a l growth, f r o m a stock culture
slant t o one of t h e tubes of s t e r i l e distilled water.
a. If sporulation on the stock slant is good, t r a n s f e r
sufficient s p o re m a t e r i a l to make a v e r y t ur bi d s us pension in the distilled water. Normally m o s t of the
s p o r e s u rfa c e f r o m a stock s l a n t will be required.
If n e c e s s a ry , use m o r e than one slant t o get a turbid
suspension.
b. If s p o r e s a r e not formed, u s e the wi r e loop t o t r a n s f e r
mycelial m a t e r i a l t o the tube of s t e r i l e distilled water.
T r i t u r a t e t h e mycelium i n the distilled wat er with a
s t e r i l e glass rod o r the t i p of a s t e r i l e pipette. P r o duce a v e r y t u rb i d suspension of mycelial fragments.
Do not u s e a mycelial suspension i f a good s p o r e suspension (a) c a n be obtained.
3. T h i s distilled w a t e r suspension of s p o r e s o r m ycel i al
f r a g m e n t s m a y be u s e d immediately a s gener al inoculum
o r m a y be held at room t e m p e ra t u re 3-4 hours. P r e p a r e
f r e s h inoculum suspensions f o r t e s t s per f or m ed on
different days.
B. P r e p a r a t i o n of washed inoculum
1. P r e p a r e 5 ml of turbid suspension of s p o r e s o r mycelium
i n s t e r i l e w a t e r as d e s c ri b e d f o r general inoculum.
2. T r a n s f e r 4-5 ml of this suspension t o 50 ml of medium 1
(tryptone-yeast e x t ra c t broth) i n a 250 ml E r l e n m e y e r
f l a s k ( o r 25 ml i n a 125 ml flask).
SYSTEMATIC
BACTERIOLOGY
make f i l e i c o n here
loop of cord
- - - - - - - - - - - p e l l e t dried on lower loop of c o d
Figure 1:
Scaled evacuated nmpoule containing lyophile p e l l e t and cord
f i l e scores break here
s t e r i l e cotton
Figure 2:
Ampoule scared and wrapped in s t e r l l e
cotton ready t o be broken
/
Figure 3:
Aseptic transfer of looped cord
and attached dry pellet t o 5 ml.
of tryptone-yeast extract broth
3 2.3
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3. Incubate the fl a s k f o r 48 hours at 25028°C;. If possible
t h e flask should be a e ra t e d on a mechanical s haker during
the incubation.
4. Use vigorous agitation with s t e r i l e glass beads, a
mechanical blender such a s the Waring Blendor, o r other
appropriate m e a n s t o b re a k up the 48-hour growth.
5. T r a n s f e r 5-10 ml of this fragmented broth culture into
s t e r i l e centrifuge tubes equipped with s t e r i l e caps.
6. Centrifuge the suspension.
7. Decant the supernatant broth. Add ste,rile distilled wat er
( o r s t e r i l e 0. 85% NaC1) t o r e s t o r e the original volume i n
the centrifuge tube. Mix and resuspend t he washed sediment with a s t e r i l e rod o r pipette.
8. Repeat steps 6 and 7.
(Compare amount of sediment
f r o m different cultures. When amount of sediment is
much less than n o rm a l amount, use proportionately l e s s
w a t e r f c a the final resuspension.)
9. Use the resuspended inoculum at once o r within 3 hour s
t o inoculate carbon utilization t e s t s .
PROCEDURES FOR CHARACTERIZATION OF CULTURES
I. MORPHOLOGICAL CHARACTERIZATIONS
Accurate morphological characterization of the Actinom y c e t e s producing catenulate s p o re s i s obviously dependent
upon u s e of a culture medium giving good sporulation. F o u r
media which gave good performance in this r es pect i n p r e vlous cooperative studies a r e l i s t e d below as "standard"
media. If t h e s e fo u r media all fail t o give good development
of t h e sporulating a e r i a l mycelium, then an additional mediu m promoting good s p o re formation should be used.
If it
is n e c e s s a r y t o u s e a n additional medium, al s o include a
r e c o r d of observations of t h e growth on the standard media.
Spore chain and sporophore morphology should be det er mined by observation of a fully m a t u re d culture with good
Cooperators i n the ISP will p l e a s e communicate the formula of any additional medium used t o Dr. Shirling, who will
then f o r w a r d the fo rm u l a t o the other two cooperating
investigators studying the culture.
5
SYSTEMATIC BACTERIOLOGY
32 5
s p o r e formation. Do not u s e a culture i n which degeneration
through autolysis, hygroscopic p r o p e r t i e s , o r e x t r e m e dehydration m a y have a l t e r e d the morphology.
A. Culture m e d i a f o r morphological studies
1 . The "standard" culture m e d i a f o r morphological
studies f o r all cultures will be medium 2 ( y e a s t
extract-malt e x t r a c t a g a r ) ; medium 3 ( o a t m e a l a g a r ) ;
medium 4 (inorganic salts-starch
a g a r ) ; and m e d i u m
5 (glycerol-asparagine agar).
2. P o u r 7 plates of e a c h medium using 25 ml of m e d i u m
i n each 9 c m d i a m e t e r P e t r i dish.
Media should be cooled t o about 50°C and dispensed
aseptically into s t e r i l e P e t r i dishes. P o u r e d m e d i a
should be held f o r a minimum of 24 hours at 25-28°C
to promote m o d e r a t e drying and t o check s t e r i l i t y
before inoculation.
B. Inoculation of plates f o r morphological studies
1. Use general inoculum p r e p a r e d according t o directions
on page 322.
2. P l a c e about 0.05 ml of the inoculum onto the a g a r
s u r f a c e n e a r one edge of the P e t r i dish (1 d r o p f r o m
a 1 ml serological pipette). This d r o p will s e r v e a s a
"pool" of inoculum.
3. Use a f l a m e - s t e r i l i z e d w i r e loop t o make 5 equallyspaced s t r e a k s a c r o s s the plate, dipping the loop into
the pool of inoculum p r i o r t o each s t r e a k ( s e e Fig. 4).
4. Make c r o s s h a t c h s t r e a k s a s shown in Fig. 5.
5. Inoculate a given culture onto at least 7 Petri d i s h e s
f o r e a c h medium.
6 . Incubate plates i n the d a r k at 25-28OC.
F o r each
culture observe two plates of each medium a f t e r
7 days, two at the end of 14 days, and two at the end
of 21 days. One e x t r a plate is inoculated in c a s e of
accident
.
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Figure Irr Five i n i t i a l s,treaks;
loop dipped i n t o inoculum f o r
each streak
Figure 5: Cross streaks made
without picking up additional
inoculum
C. Determination of micromorphological c h a r a c t e r i s t i c s of
the spore-bearing hyphae
1. Determine the c h a ra c t e ri s t i c s of the spore-bearing
hyphae and s p o re chains by d i re ct m i cr os copi c examination of the culture s u rfa c e on opened dishes of the
crosshatched cultures. Use a magnification adequate
t o establish the p re s e n c e ( o r absence) of chains of
s p o r e s . Th i s will usually be lOOx 700x. A minim u m of 10 m i c ro s c o p e fields at lOOx should be
examined.
-
2. Determine the number of s p o r e s at the end of m a t u r e
hyphae. State whether s p o r e s occur (a) singly, (b) in
p a i r s , ( c ) i n chains of 3-10, o r (d) i n chains of m o r e
than 10. If t h e r e i s variation state the range of variation and then choose the m o s t repr es ent at i ve s p o r e
number (a, b, c, o r d ) f o r the culture on a given medium. Make the observation on each medium which
shows good sporulation.
3. The f o r m of the s p o re chain and spore-bearing hyphae
should be d e s c ri b e d only with m a t u r e cultures on
which many well-defined s p o re chains can be seen.
D e s c ri b e sporulating cultures i n t e r m s of t he morphological groups of P ri d h a m et 51. (1958), modified
according to Baldacci ( s e e Figs. 6-13). In s ome c a s e s ,
p a r t i cu l a rl y with cultures producing open loops apd
SYSTEMATIC
327
BA C T E R I O L O G Y
primitive s p i r a l s , m o r e than one morphological type
m a y be observed. On the b a s i s of extensive observation of the culture s u rfa c e , r e c o r d &l m a t u r e sporophore-spore chain types s e e n with an es t i mat e of the
p e r c e nt of s p o re -b e a ri n g a e r i a l hyphae falling into
each category.
SIMPLE
-
VERTICILLATE
Monove rticillus (MV)
Monove r t i ci l l us
Spi r a (MV-S)
Biverticillus (BIV)
Bive rticillus-Spira
( BIV-S)
Undetermined (U)
Straight Rectus (R)
Flexible Flexibilis (F)
Open loops RetinaculumApertum (RA)
Sp i r a l s S p i ra (S)
-
-
-
4. Photograph typical morphology of the sporulating
hyphae, o r make s i m p l e drawings.
5. Retinaculum-Apertum: Record t he approximate
d i a m e t e r of c h a r a c t e r i s t i c open loops and primitive
s p i r a l s of RA cultures.
6 . Verticillate: (a ) Record a v e ra g e di amet er of the m ai n
hyphae f r o m which v e rt i c i l s originate. (b) Recor d
whether o r not v e rt i c i l s a r e evenly spaced along the.
m a i n hyphae.
D. Special morphological observations
It is a l s o important t o look f o r the following c h a r a c t e r i s t i c s (usually identified with other actinomycete groups):
1. P r e s e n c e of globular sporangia as i n Actinoplanaceae
2. P r e s e n c e of flagellated s p o r e s , as i n Actinoplanes
3. Fo r m at i o n of conidia-like s p o r e s on the s ubs t r at e
hyphae
4. Tendency of the s u b s t ra t e mycelium t o f r agment
5. Occurrence of s c l e ro t i a .
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Rectus (R)
o r straight
=gum 6.
(RA)
Retinacul&pertn
Open loops, hookn, o r extended
s p i r a l s af wide diamsfer
Figure 9. Spira (S)
Simple spirale (not on v e r t i c i l s ) ,
Spirals may be short and compact
or long, extended, o r open
VEKTICaLATE
True v e r t i c i l l a t e s have main
axis of wider diameter than
branching hyphae, and have
unif om spacing betpeen
verticile.
Figure 1 o : ' - ~ r t i c i l l u s (Hv)
Primary v e r t i c i l s or whorls
distributed on a long axLs or
branch; 110 s p i r a l s
Mgure 12.
Biverticillus (BIV)
Compound v w t i c i l s or whorls on,
a long axis; no ~ p i r e l s
F i g u r e s 6-13.
M a e ll.
GnoverticiXtusSpira (HVS)
Rimarg v a r t i e i l e or whorls
diatributed on a long axis;
elemente of verticils spiralled
Biverticillns-Spira (BIVS
Compound v e r t i c i l s ; elemants of
secondv e r t i c i l s spirallea
zl.
Reproduced f r o m P r i d h a m et
(1958) with
p e r m i s s i o n of authors and publisher.
SYSTEMATIC BACTERIOLOGY
32 9
E . Spore morphology and s u r f a c e
L a b o r a t o r i e s having a c c e s s to a n e l e c t r o n m i c r o s c o p e
should include e l e c t r o n m i c r o g r a p h s of t h e s p o r e s u r f a c e
as one of the descriptive c h a r a c t e r i z a t i o n s f o r each type
culture. E l e c t r o n m i c r o g r a p h s should a l s o show s p o r e
chains. Suggested method:
1. U s e the s a m e c r o s s h a t c h P e t r i dish c u l t u r e s p r e p a r e d
f o r observation under the light microscope.
2. E l e c t r o n m i c r o s c o p e specimen g r i d s coated with
F o r m v a r o r collodion a r e gently p r e s s e d t o the a e r i a l
s u r f a c e of a culture with m a t u r e s p o r e s .
3. Spore chains which a d h e r e t o the coated s u r f a c e of the
grids a r e observed and photographed i n t h e e l e c t r o n
m i c r o s c o p e without fixing o r shadowing at a magnification of 8, OOOx 10, OOOx.
-
4. P r e s e r v e the picture r e c o r d t o include with t h e r e p o r t .
In addition to t h e photographic r e c o r d , s p o r e silhoue t t e s should be c h a r a c t e r i z e d as Smooth, spiny, h a i r y ,
w a r t y ( s e e Fig. 14).
F i g u r e 14.
11. COLOR DETERMINATIONS
Color determinations should be made f o r ( 1 ) t h e m a s s
color of m a t u r e , sporulating a e r i a l s u r f a c e growth, ( 2 ) f o r
the color of the s u b s t r a t e mycelium a s viewed f r o m the r e v e r s e side, and ( 3 ) f o r diffusible soluble pigments other
than melanins. The T r e s n e r - B a c k u s color s e r i e s , adopted
by participants i n the 1962 Montreal Workshop (Kiister,
1964; T r e s n e r and Backus, 1963) i s specified f o r s p o r e m a s s
color determinations, In this s y s t e m , seven color s e r i e s
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a r e delineated by color wheels made with t a b s f r o m t h e 3 r d
o r 4th edition of the Color Harmony Manual (Jacobson c t =l.
1948; E c k e r s t r o m and FOSS,1958). 6 The T r e s n e r - B a c k u s
color guide i s not designed f o r determination of s u b s t r a t e
mycelium colors o r c o l o r s of soluble pigments. Although a
color guide i s not required f o r t h e s e colors, the s u b s t r a t e
mycelium color may be identified i n t e r m s of a special
guide7 p r e p a r e d f o r the I.S. P. by H. P r a u s e r (1964) o r i n
t e r m s of the Bondartsev (1 954) color scale.
A. Culture media f o r color determinations
Use the following media: Medium 2 (yeast extractm a l t e x t r a c t a g a r ) ; medium 3 (oatmeal a g a r ) ; medium 4
(inorganic s a l t s - s t a r c h a g a r ) ; and medium 5 (glycerolasparagine agar). The c r o s s h a t c h plates that w e r e p r e p a r e d f o r morphological studies may be used f o r t h i s
purpose.
B. 0bs e r vat ions
1. Observe plates at 7, 14 and 2 1 days as p r e s c r i b e d f o r
morphological studies. Color determinations should
be r e c o r d e d only f o r m a t u r e cultures.
2. Use north-window daylight on a bright day. If this i s
noh possible, u s e a balanced and reproducible s y s t e m
of a r t i f i c i a l light simulating north sky daylight.
C. Determination of a e r i a l m a s s color
1. Observations should be limited t o m a t u r e c u l t u r e s
with a heavy s p o r e m a s s surface.
6 A complete s e t of T r e s n e r - B a c k u s color guides h a s been
provided to each participant i n the I. S. P. P r i n t e d p a t t e r n s
f o r assembling the color wheels, together with r e p r i n t s of
the T r e s n e r - B a c k u s paper can be obtained without cost f r o m
P r o f . E. B. Shirling, I. S. P., Ohio Wesleyan University,
Delaware, Ohio 43015, U. S. A. Color Harmony Manual chips
used i n the guide may be purchased f r o m Color Standards
Department, Container Corporation of A m e r i c a , 38 South
Dearborn St. , Chicago, Illinois 60603, U. S. A.
7 P r e p a r e d f o r I. S. P. participants by H. P r a u s e r
(1964)
f r o m color tabs of Baumanns F a r b t o n k a r t e Atlas I.
S Y S T E M A T I C BAC T E RIOLO'GY
331
2. Determine the Tre s n e r-B a c k u s color s e r i e s (Red,
Yellow, Greeb, Blue, Violet, G r ay o r White) by comparing m a t u r e s p o re mass color on all appropriate
m e d i a with t h e Tre s n e r-B a c k u s color wheels. U s e
only the mat (dull) s u rfa c e of the color tabs. Most
cultures will fall within the range delineated by one
wheel. Th e re a r e a few instances i n which color t abs
a s s i g n e d to two different wheels a r e similar i n color.
If a culture is intermediate between t hes e two t abs
f r o m different wheels, it m a y be difficult t o choose
one s e r i e s as better than the other f o r describing t he
culture. I n t h e s e r a r e c a s e s c l a ss i f y the culture i n
both s e r i e s .
-
3. Identify by number-letter code (printed on the tab),
the one color t a b which m o s t n e a r l y c h a r a c t e r i z e s t he
s p o r e -m a s s color. Also r e c o r d the ISCC-NBS (U. S.
Dept. of C o m m e rc e , 1955) name l i s t ed i n t h e color
wheel folder f o r the t a b chosen t o char act er i ze t he
s p o r e -m a s s color. If the color is i nt er medi at e between two tabs, designate both.
D. Color of s u b s t ra t e mycelium ( r e v e r s e color)
The color of the s u b s t ra t e mycelium is det er mi ned by
observing the r e v e r s e (under) side of mass growth on the
various media. A method f o r removing m o s t of t he a g a r
medium is d e s c ri b e d below. A s i m pl e guide f o r u s e i n
cutting off the a g a r is provided.
1. P l a c e the cutting guide onto l a y e r s of f i l t e r paper o r
paper towel s a t u ra t e d with wet disinfectant solution
( s e e Fig. 16). (Also provide a beaker of disinfectant
f o r discarding work tools.)
2. Use a c o rk b o r e r ( o r similar sharpened cylinder).with
a d i a me t e r slightly l e s s than t h e hole in the template
t o cut an a g a r plug f r o m typical m a t u r e mycel i al
growth on a P e t r i .dish culture ( s e e Fig. 15). Determine color f o r (a) m a t u r e s u b s t rat e mycelium and
(b) t h e relatively young edges of the spreading growth.
3. Pu s h the plug f r o m the cylinder with a gl as s rod so
that it t u r n s ' o v e r and drops inverted onto the cutting
guide (Fig. 16). P u s h the inverted plug into the hole
i n the cutting guide.
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4. Slide a r a z o r blade a c r o s s the guide slicing off all
ag,ar medium which extends above the l e v e l of the
surface (Fig. 17).
5. It i s now possible t o observe the r e v e r s e side of the
culture plug remaining in the cutting guide. Very
little a g a r will r e m a i n to i n t e r f e r e with color d e t e r mination.
6. Assign the culture to one of the following color groups,
(Szab6 and Marton,
yellow- brown
yellow-brown
yellow-brown
yellow-brown
1964)
t r e d ( o r orange)
t blue o r violet
t green
7. In addition, if e i t h e r P r a u s e r o r the Bondartsev
color code o r the complete Color Harmony Manual i s
available, identify the color with the n e a r e s t matching panel on one of t h e s e color guides.
8. Determine the response of color of s u b s t r a t e myceliu m t o pH change by adding a drop o r m o r e of 0 . 0 5 N
NaOH and 0 . 0 5 N HC1 t o the specimen p r e p a r e d a s
d e s c r i b e d i n s t e p s '1-4 above. Make observations
immediately and a f t e r 10 to 15 minutes.
E. Soluble colors other than melanoid pigmentation
If soluble c o l o r s other than brown o r black a r e produced on any medium ( o r i f brown is distinctly modified
with r e d , yellow, green, blue, o r violet), 1 ) r e c o r d the
medium; 2 ) r e c o r d t h e color a s the s i m p l e unmodified
color name ( r e d , orange, yellow, green, blue, violetu s e no other color terminology); and 3 ) i n e v e r y c a s e of
soluble color other than melanoid pigment, d e t e r m i n e
response of color t o pH change by addition of a d r o p o r
m o r e of 0.05 N NaOH and 0.05 N HC1 to the colored a g a r .
Record whether o r not soluble color i s affected by pH
change. Make observations immediately and a f t e r 10 to
15 minutes.
SYSTEMATIC BACTERIOLOGY
Figure 15: Cut agar plug
frm a broad mass of mature
grovth.
Figure 16: Push plug s o that it drops
inverted into the cutting guide.
Figure 17: Use razor blade t o cut off and
discard a11 agar which extends above the
surface of the cutting guide. Observe
the exposed reverse side of the sycelfrm
which remains in the cutting guide.
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INTERNATIONAL JOURNAL
334
III. P
h
A. MELANIN PRODUCTION
1 . Culture m e d i a and inoculation for melanin production
Determine production of melanoid pigments on
a g a r slants of medium 6 (peptone i r o n a g a r , Difco,
supplemented with 0. 1% yeast e x tr act ) , and medium 7
(Shinobu's modification of Masumoto's t yr os i ne agar).
Also observe other organic media, especially medium
1 (tryptone-yeast e x t ra c t broth, used f o r obtaining
growth f r o m lyophile pellet).
Use a g a r slant c u l t u re s p re p a r ed a s des cr i bed
under MATERIALS AND GENERAL METHODS as
inoculum f o r media 6 and 7. Cultures used as inocul u m s o u rc e should be l e s s than 3 weeks old (except
f o r cultures unusually slow i n producing' m a t u r e a e r i a l
growth). Use a heavy inoculum of s p o r e s and a e r i a l
mycelium picked up on a standard wi r e loop. S t r e a k
this inoculum up the surface of the a g a r slant. Inoculate each experimental culture onto 2 slants of medium
6, and 2 s l a n t s of medium 7.
-
2. Observations and interpretations
a. Observe melanoid pigments on medium 6 and
medium 7 a ft e r 2 days and a ft er 4 days. Compare
inoculated tubes with uninoculated controls. Cult u r e s forming a g re e n i s h brown to brown t o black
diffusible pigment o r a distinct brown pigment
modified by other color s h a l l be r ecor ded as positive (t). Absence of brown t o black col or s , o r
total absence of diffusible pigment, s hal l be r e corded as negative (-) f o r melanoid pigment product i on.
b. Other organic m e d i a containing peptones o r tyrosine, including medium 1 (tryptone-yeast ext r act
broth) u s e d f o r initial growth f r o m the lyophile
pellet, should be observed f o r production of m el anoid pigments. The p re s e n c e of pigments i n t hes e
media should be included in the record.
S Y S T E M A T 1-C - B A C T E R I 0 L 0 G Y
335
B. CARBON UTILIZATION
1. Basal medium and carbon sources
Detailed instructions for medium 9 (Pridham and
Gottlieb carbon utilization medium) including carbon
source sterilization a r e given on pages 318-319.
After autoclaving the basal agar medium, cool i t to
60°C and add sterile carbon source aseptically to give
a concentration of approximately 1%. Agitate the
mixture and pour 25 ml of medium per dish into 9 cm
P e t r i dishes. P r e p a r e duplicate plates of each carbon compound for each culture to be tested. Store
medium in refrigerator.
Carbon sources and controls required for the test
a r e repeated below:
N o carbon source (negative control)
D-glucos e (positive cont r 01)
L-arabinose
Sucrose
D-f ructo se
Rhamnose
D xylo s e
I-inositol
Raffinose
D-mannitol
Cellulose
-
2. Procedures f o r carbon utilization t e s t s
a. P r e p a r e a washed inoculum a s described on
pages 323-324.
b. Dry the uninoculated plates by leaving them at
room temperature for 4 hours after they a r e
freshly poured o r after removing them f r o m refrigerator storage.
c. Place approximately 0.05 ml of washed inoculum
(1 drop from a sterile 1 ml serological pipette o r
dropping pipette) onto one edge of the agar surface.
Streak the drop straight a c r o s s the dish (see Fig.
18). Repeat with a second drop. Inoculate duplicate plates. Use only one culture per plate t o
avoid false positives due to c r o s s feeding.
d. Observe plates at 10-16 days. Always compare
growth on a given carbon source with the two cont r o l s ; growth on basal medium alone, and growth
on basal medium plus glucose.
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e. R e c o r d r e s u l t s a s follows:
Strongly positive utilization (tt), when growth on t es t ed
carbon i n b a s a l medium i s equal t o o r g r e a t e r than
growth on b a s a l medium plus glucose.
Positive utilization (t), when growth on t es t ed carbon is
s i m i f i c a n t l y better than on b a s a l medium without
carbon, but somewhat l e s s than on glucose.
Utilization doubtful (f), when growth on t es t ed carbon is
only slightly b e t t e r than on t h e b as al medium without
carbon and significantly less than with glucose.
Utilization negative (-), when growth i s similar t o o r
l e s s than growth on b a s a l medium without carbon.
(Always r e c o r d utilization as negative i f growth is
not b e t t e r than no-carbon control.)
F i g u r e 18.
REFERENCES
Bondartsev, A.S. 1954. Scale of colors. The Academy of
Science, USSR.
E c k e r s t r o m , R. and C.E. F o s s . 1958. Color harmony
manual, 4th ed. Container Corp. of Am er i ca, Chicago.
Gottlieb, D. 1959. Agenda f o r Round Table evaluation of
c r i t e r i a f o r taxonomy. Intern. Bull. Bact. Nomen. and
Taxon. 2:13-14.
1961. An evaluation of c r i t e r i a and pr ocedur es us ed
i n the description and c h a ra c t e ri z a t i on of the streptomycetes. Appl. Microbiol. 2:55-65.
-.
SYSTEMATIC BACTERIOLOGY
.
337
1963. Recommendations f o r description of s o m e
Actinomycetale s appearing i n patent applications.
Intern. Bull. Bact. Nomen. and Taxon. 13:159-160.
Jacobson, E . , W. C. Granville and C. E. Foss. 1948.
Color harmony manual, 3 r d ed. Container Corp. of
A m e r i c a , Chicago.
Judge, L.F. J r . , and M. J. P e l c z a r , Jr. 1955. The s t e r i lization of carbohydrates with liquid ethylene oxide f o r
microbiological f e rmentation t e st s Appl. Mi cr obiol.
3:2 92 -2 95.
Krasil'nikov, N. A. 1960. Resolution of the "Conference on
Taxonomy of Actinomycetes, I ' held i n Moscow, J u n e 810, 1960. Intern. Bull. Bact. Nomen. and Taxon. I l :
23-28.
Kiister, E. 1959a. Outline of a comparative study of c r i t e r i a used i n c h a r a c t e r i z a t i o n of the actinomycetes.
Intern. Bull. Bact. Nomen. and Taxon. _9:98-104.
1959b. Introductory r e m a r k s and welcome, "Round
Table Conference on Streptomycetes" on occasion of the
7th International Congress of Microbiology, Stockholm,
August 4-5, 1958. Intern. Bull. Bact. Nomen. and
Taxon. 2:57-61.
1961. Results of a comparative study of c r i t e r i a
used in classification of the actinomycetes. Intern. Bull.
Bact. Nomen. and Taxon. 2 : 9 1 - 9 8 .
1964. Report on the activity of t h e Subcommittee on
Taxonomy of the Actinomycetes, 1958-1 962. Intern.
Bull. Bact. Nomen. and Taxon. 14:1-3.
P r a u s e r , H. 1964. Aptness and application of colour codes
f o r exact description of colours of streptomycetes.
Z e i t s chrift f. Allg. Mikrobiologie 4:95- 98.
P r i d h a m , T . G . , P. Anderson, C. Foley, L.A. Lindenfels e r , C. W. Hesseltine and R. G. Benedict. 1956-57.
A selection of media f o r maintenance and taxonomic
study of streptomycetes. Antibiotics Ann. 1956/57,
pp. 947-953.
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, C. W. Hesseltine and R. G. Benedict. 1958. A guide
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s e ctions Appl. Mi c r obiol. 6:52 7 9.
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Shinobu, R. 1958. Physiological and cultural study f o r the
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LIST O F PARTICIPANTS
1. Shirling, Dr. E. B. (Coordinator) (Addressee f o r all
I. S. P. correspondence). Dept. Botany and Bact.,
Ohio Wesleyan Univ. Delaware , Ohio, U. S. A.
2. Gottlieb, Dr. David (Coordinator)
Dept. Plant Pathology, Univ. of Illinois, Urbana,
Illinois , U. S. A.
3. Anderson, Lucia (Sara Wold)
P a r k e , Davis and Co. , Detroit, Michigan, U. S. A.
4. Baldacci, Prof. Elio
1st. Patol. Vegetale, Univ. Milano, Via Celoria N. 2,
Milano , Italy
5. Brinkmann, Dr. Rolf
Organic Chem. Inst. , Univ. of Gottingen, Gottingen,
Germany
6. Crook, Dr. Kenneth E. , J r .
(Carol S. Cassidy)
Bristol Labs, , Inc. , Syracuse, New York, U. S. A.
7. C r o s s , Dr. T.
(A. Maciver)
Dept. Biol. Sciences, Bradford, Inst. Tech.,
Bradford 7 / Yorkshire, England
8. deVries, Dr. G. A.
Centraalbureau voor Schimmelcultures , Baarn,
Netherlands
9. Dietz, Alma
Upjohn Co. , Kalamazoo, Michigan, U.S.A.
SYSTEMATIC BACTERIOLOGY
339
10. Falczo de Morais, Dr. <.O.
Inst. de Quimica da Uoiv. do Recife, Recife,
P e rnambuco, Brazil.
1 1 . Gause, Prof. Dr. G. F. (Preobrashenskaja, Kudrina,
Maximova, Sveshnikova)
Acad. Med. Sciences of USSR, Inst. New Antibiotics,
MOSCOW,USSR
12. Higgens, Mr. C a l v i n E .
E l i Lilly and Co. , Indianapolis, Indiana, U. S. A.
13. Hirsch, Dr. P e t e r
Dept. Microbiol. , Yale Univ. , New Haven, ConnectiCut, U.S.A.
14. Hiitter, Dr. Ralf
Inst. fGr Spez. Bot. , Eidg. Tech. Hochschde,
Ziirich 6, Switzerland
(T. Kos)
15. Johanides, Prof. Dr. V e r a
Zabod z a mikrobiologiju, Zagreb, Pierottijeva, 6,
Jugos lavij a
16. Krasil'nikov, Prof. N. A.
Inst. Microbiol. , Acad. Sciences, Moscow B-133,
USSR
17. Kuznetsov, Dr. V.D.
USSR Res. Inst. f o r Antibiotics, MOSCOW,USSR
18. McClung, Dr. Norvel
Dept. Bact. , Univ. Georgia, Athens, Ga., U.S.A.
19. Mach, Prof. Dr. F.
Inst. f i i r Mikrobiologie, Greifswald, Ludwig-Jahn-Str.
15, Germany
20. Margalith, Dr. P.
Lab. of Microbiol., Israel Inst. of Tech. Haifa, Israel
21. Nakazawa, Dr. K.
Takeda Chem. Ind. , Ltd. , Juso-Higashiyodogawaku,
Osaka, J a p a n
(Mayama, T a w a r a )
22. Nishimura, Dr. Haruo
Shionogi Res. Lab. , Shionogi and Co., Ltd. ,
Fukushima-ku, Osaka, J a p a n
(Nonomura)
23. Ohara, Dr. Y.
Faculty of Engineering, Yamanashi Univ., Kofu, J a p a n
24. Okami, Dr. Yoshiro
Nat'l. Inst. of Health of Japan, Shinagawa, Tokyo,
Japan
25. Oliver, Mr. Thomas
Abbott Laboratories, N. Chicago, Illinois, U. S . A.
26. P r a u s e r , Dipl. Biol. H.
Inst. Mikrobiol. Exp. T herapie, Beuthenbergstr. L 1 ,
Jena, Germany
340
INTERNATIONAL JOURNAL
27. P r i d h a m , Dr. Thomas
U. S . Borax Corporation, 4 1 2 C r e s c e n t Way,
Anaheim, California, U. S. A.
28. Radziminska, Ina
Hoffman-LaRoche, Nutley, New J e r s e y , U. S. A.
29. EehSEek, Dr. Zdene'k (Alona 6iEicovg)
Czech. Acad. of Science, Inst. of Microbiol.,
P r a g u e 4, Budejovicka No. 1083, Czechoslovakia
30. Routien, Dr. John B.
(Corinne Clevenger)
C h a r l e s P f i z e r and Co., Inc., Groton, Conn., U.S.A.
31. Sanchez-Marroquin, D A.
Miami 40, Mexico 18 / D.F., Mexico
32. Shewan, Dr. J.M.
(T.G. Mltchell)
T o r r y R e s e a r c h Station, Aberdeen, Scotland
33. Spyvee, Mr. J.
(J. Elliott)
Boots P u r e Drug Co., Ltd., Antibiot. and F e r m .
Div., Nottingham, England
34. Srivistava, Dr. O.P.
C e n t r a l Drug Res. Inst., Lucknow (U. P.), India
35. Szab6, Dr. I.
Magyar Tudomanyos Akadgmia, Talajtani ks
Agrokgmiai Kutat6 Intkzete, Budapest, Hungary
36. Tanaka, Dr. Nobuo
Inst. of Appl. Microbiol., Univ. of Tokyo, Bunkyo,
ku, Tokyo, J a p a n
37. Thirurnalachar, Dr. M.J.
Hindustani Antibiotics Res. Center, P i m p r i , N e a r
Poona, India
38. T r e j o , Dr. W m . and Dr. Ralph Bennett
E. R. Squibb and Sons, New Brunswick, N. J . , U. S. A.
39. T r e s n e r , Dr. Homer
L e d e r l e Labs., P e a r l R i v e r , New York, U. S. A.
40. Tsyganov, Dr. V.A.
Res. Inst. of Antibiotics, Leningrad, L-20, USSR
41. Vernon, Dr. T.R.
Dept. Scientific and Ind. R e s . , P r i v a t e Bag,
Auckland C1, New Zealand
42. Wallhtiuser, Dr. K a r l H.
Mikrobiolog. Unters, F a r b w e r k e Hoechst AG,
HBchst, Germany
F r a n k f u r t a/M
43. W i l l i a m s , Dr. S. T.
Hartley, Botan. Labor., Univ. of Liverpool, Liverpool, England
44. Woznicka, Dr. Wanda
Pa6stwowy Zaklad Higieny, ul. Chocimska 24, Warszawa 12, Poland
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