m02tkb.

wpd 20 April 2000 Tobias Kieser

Chapter 2

Growth and preservation of Streptomyces

CONTENTS

Selective isolation of streptomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Making a Streptomyces spore suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Plating out a Streptomyces spore suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Streptomyces cultures on agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Growth of mycelium on cellophane overlays, nitrocellulose filters or agar . . . . . . . . . . . . . . . . 48
Growth of Streptomyces mycelium in liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Growth of Streptomyces for physiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Conditions for spore germination and reproducible exponential growth
and antibiotic production by Streptomyces coelicolor . . . . . . . . . . . . . . 50
Germination of Streptomyces spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Procedure 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Procedure 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Preparation of aerial mycelium and spores separately . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Aerial mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Sporulation of Streptomyces in submerged cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Preparation of protoplasts from Streptomyces lividans 66 and S. coelicolor A3(2) . . . . . . . . . . 56
Preservation of Streptomyces strains and phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Preparation of lyophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Alternative I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Alternative II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Growing a culture from the lyophil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Selective isolation of streptomycetes

Most genetic studies are made on strains that are available from stock culture collections or
colleagues, but here we give an indication of methods that have been developed to isolate streptomycetes
and other actinomycetes from nature. Streptomycetes can be isolated from a wide variety of habitats but,
because they are aerobic, they are less readily recovered from water-logged soils than facultative
anaerobes. Adding lime to the soil can enrich for streptomycetes (Wellington and Toth, 1994).
Most isolation techniques involve extraction from an environmental sample followed by biomass
dilution to allow culturing on solid media. Chemical disruption aids dispersal and subsequent
dissociation of spores and mycelium from soil particles with which they are normally intimately
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

associated. This can be achieved by gently shaking soil samples with the ion-exchange resin Chelex-100
(Bio-Rad) and an eluent, resulting in exchange of Na+ for divalent cations such as Mg2+ and Ca2+, which
bind soil aggregates together (Herron and Wellington, 1990). Spores can then be separated from the bulk
of mycelial material by differential centrifugation and filtration, which also serves to concentrate the
biomass. The final extract can be plated on selective media such as arginine glycerol salts agar (El-
Nakeeb and Lechevalier, 1963), HV agar (Hayakawa and Nonomura, 1987), colloidal chitin agar (Hsu
and Lockwood, 1975), or reduced arginine starch salts agar (Herron and Wellington, 1990).
Inhibitors may be incorporated into isolation media to aid selection of streptomycetes
(Wellington and Toth, 1994). Detergents like SDS, which kill vegetative cells, have been used to isolate
spores (Nonomura and Hayakawa, 1988). It is also useful to incorporate antifungal agents such as
cycloheximide and nystatin into solid isolation media to prevent overgrowth by fungi. Most
streptomycetes can then be isolated on the selective medium at 28o, whereas thermotolerant strains can
be isolated at 40o, and acidophiles on media adjusted to pH4.5 (Wellington and Toth, 1994). For
isolation of particular species groups, Wellington et al. (1987) chose selective media based on taxonomic
data, reflecting specific carbon or nitrogen source utilisation or resistance to various compounds.
Even on selective media, bacteria such as pseudomonads and bacilli may overgrow target
colonies. New methods are therefore being developed to allow specific recovery of desired propagules
using immunocapture. This facilitates initial separation of species from environmental samples, after
which enrichment on non-selective media and then on selective media provides sensitive recovery of a
target species. A monoclonal antibody to a major spore coat polypeptide exclusive to cluster 21
Streptomyces species, including S. lividans, was developed by Wipat et al. (1994). As a test, soil
samples were seeded with S. lividans spores, which were recovered by immunomagnetic capture using
magnetic beads coated with the antibody, and a magnet to concentrate the bead-spore complex.
Streptosporangium fragile was recovered from soil by indirect immunomagnetic capture using a rabbit
polyclonal primary antibody against the spores (Mullins et al., 1995); the antibody was added to soil
samples after treatment with blocking agents. Antibody-labelled spores were captured using magnetic
beads coated with sheep anti-rabbit second antibody, and concentrated on a magnet.

Making a Streptomyces spore suspension

Most streptomycetes produce copious, haploid, unigenomic spores under suitable culture
conditions. Although Streptomyces spores arise in chains in the aerial mycelium, individual spores can
usually be readily separated by suspending and vortexing in water; for some strains, however, a wetting
agent such as 0.1% Tween 80 or 0.001% Triton X100 is needed. The resulting suspensions are used for
many purposes, such as inoculating liquid medium to produce mycelium for isolating plasmid or
chromosomal DNA, RNA or enzymes, or for preparing protoplasts; for the isolation of mutants; and for
the analysis of recombination or plasmid transfer in crosses. Concentrated spore suspensions (c. 109
ml1) are crucial for purposes like starting reproducible cultures for physiological or fermentation studies
(p. 49), so it is worth experimenting with different media to find one that is good for a particular strain.
MS (mannitol soya flour; p 409) agar is particularly good for S. coelicolor and S. lividans.
Suspensions of spores in 20% glycerol, kept frozen at -20o, will usually remain viable for years,
even if they are repeatedly thawed and re-frozen for sampling purposes (but not if they start to
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 
germinate). Non-sporulating strains, such as whi and some bld strains, die rapidly on freezing in glycerol
(though bldA strains retain viability). Spore suspensions frozen in water usually lose about 50% of their
viability on freezing and thawing. Suspensions can also be kept frozen in DMSO at -20o (useful for
glycerol-sensitive mutants).

Fresh plate culture (or slant) of the strain

Sterile water
20% glycerol in water (sterilised by autoclaving)
Rigid inoculating loop; pipettes; filter tubes containing non-absorbent cotton wool
(see Fig. 2.1); centrifuge tubes; screw cap containers, e.g. 7.5 ml ("Bijou"
bottles) and 20 ml (wide-necked McCartney bottles or "Universal" containers)
Vortex mixer; bench centrifuge; compressed air supply

The following should be carried out in a laminar flow hood or other sterile environment.

1. Add c. 9 ml of sterile water to the plate (or slant). It is convenient to keep a supply of
20 ml screw cap bottles (wide-necked McCartney bottles or "Universals") containing 9 ml
amounts of sterile water ready for making spore suspensions. Plate cultures usually yield more
spores than slants and are routinely used, provided there is access to a laminar flow hood for
inoculating them. Slants are less prone to contamination, if this is a special problem.

2. Scrape the surface of the culture with an inoculating loop, first with gentle
pressure and then gradually more vigorously, so as to suspend the spores. Gentle
scraping is important for agarase-producing species such as S. coelicolor, otherwise lumps of
agar medium will clog the filter. Some use a small piece of cotton wool, held in sterilised
forceps, to rub the surface of the culture gently to suspend the spores. Others collect the spores
dry by rolling glass beads over the culture surface and then suspend the spores in water when
needed.

3. Pour the crude suspension back into the container that held the sterile water, or
use a sterile syringe or pipette, and agitate the liquid as violently as possible on
a vortex mixer for about a minute to break up spore chains.

4. Filter the suspension through non-absorbent cotton wool, using a filter tube (see
Fig. 2.1). If a piece of cotton wool was used to suspend the spores the suspension may be free
enough of mycelial fragments and pieces of agar medium that filtration is superfluous.

5. Pour the filtered suspension into a centrifuge tube and spin for 5-10 min at c.
1000 g to pellet the spores. The centrifugation step is to remove compounds dissolved
from the growth medium. These may include growth factors that could interfere with the
selective use of auxotrophic markers, or "staling" compounds that might reduce the longevity of
the spores, or inhibit germination.
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Compressed
air line

A
Glass
tube
Rubber
bung
F

B Cotton wool
C plug

A
15 x 125 mm test
tube with hole
blown in bottom B

D Cotton wool
collar D
C

Small cotton
wool pad

E
25 x 150 mm
E test tube

Fig. 2.1. Filter tube for spore suspensions. Pour crude spore suspension into inner
tube. Apply air pressure by holding rubber bung of compressed air supply gently
over cotton wool stopper. Filtered spore suspension will pass through non-
absorbent cotton wool filter pad into the receiving outer tube. Remove inner tube
and cotton wool collar to recover spore suspension.

6. As soon as the centrifuge stops, pour off the supernatant. If the spore pellet is left in
the tube, even for a few minutes, after the centrifuge has stopped, it will often become detached
from the wall of the tube.
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 

7. Agitate the tube on the vortex mixer for a few seconds to disperse the pellet in the
drop of water remaining in the tube. It is easier to disperse the spores in a minimum
volume of liquid than in the final volume.

8. Add sterile 20% glycerol (usually 1-2 ml for the spores from a well-sporulating
slant or plate) and briefly agitate again. Transfer the suspension to a screw cap
bottle for freezing at -20E; 7.5 ml "Bijou" bottles are convenient for this. If the
spores are just for immediate use, they can be suspended in water; if you then decide to keep
them, add a roughly equal volume of 40% glycerol and freeze.

Plating out a Streptomyces spore suspension

Streptomyces spores (and even mycelial fragments) are quite resistant to osmotic damage and so
dilutions can be made directly into distilled water, even from 20% glycerol or concentrated sucrose
solutions. When using frozen spore suspensions, take care to keep them on ice on the bench if you plan
to re-freeze them, because they rapidly lose viability on re-freezing if spore germination has begun.
We usually transfer 0.5 ml or 1 ml of suspension using conventional glass pipettes to 4.5 ml or
9 ml of water, respectively, for each successive tenfold dilution step, but smaller volumes can of course
be handled accurately using automatic micro-pipettes (such as the Pipetman manufactured by Gilson).
A fresh pipette or tip must be used for each successive dilution step.
For plating, we usually spread 0.1 ml of spore suspension on each standard Petri plate using a
glass spreader. The same spreader can be used for a series of plates at the same dilution or when moving
from more dilute to less dilute suspensions, because any carry-over of spores is then negligible. However,
use a different spreader for selective plates containing different growth factors since the carry-over of
traces of these compounds can cause background growth. Note that, in plating undiluted spore stocks in
20% glycerol, significant amounts of this potential carbon source are added to the plate; the spores may
need to be spun down and resuspended in water if this may be a problem.
When preparing dense lawns, for example when looking for pocks (pp. 153, 255) or for making
"plate-crosses" (p. 152), we normally dry the plates in a laminar flow cabinet after spreading; drying them
before-hand, so that the suspension rapidly soaks in, may lead to patchy growth. When plating for isolated
colonies, for example of recombinants from a cross, there is usually no need to dry the plates; just incubate
them with the agar surface facing upwards for the first day or so.

Streptomyces cultures on agar

To obtain confluent cultures on agar, the organisms have to be inoculated over the entire surface
of the medium. This is because Streptomyces colonies, in contrast to those of most moulds, will spread
only over a very limited distance within a reasonable time and so point inoculation of Streptomyces will
  &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

not yield a confluent culture. It is best to use a suspension of inoculum in liquid as starting material. This
need not be a carefully prepared spore suspension, although this is very suitable.
It is undesirable to propagate cultures by successive rounds of mass culture. Instead they should
be plated, or streaked out, and a single colony taken to start the next culture. This precaution reduces the
accumulation of revertants and the gradual loss of unselected plasmids, which is otherwise an ever-present
possibility when the variant forms grow or sporulate better than the original genotype; as a visual
manifestation of this, mass subculture very often yields obvious morphological heterogeneity.
It is not uncommon to observe heterogeneity even in confluently grown lawns. Two features in
particular have been noted. One is the appearance of tiny plaque-like holes in the aerial mycelial mat,
often reminiscent of plasmid-induced pocks (p. 153). It has usually proved difficult to establish the cause
of such spontaneous "self-pocking", although there are reports of material resembling defective phages
being associated with some such pocks (Ogata et al., 1992). The other feature probably results from in
situ germination of spores produced on a culture that has sporulated, so that white tufts may appear, or
the whole culture surface may even become white again (Dowding, 1973). Such germination is perhaps
stimulated by the gradual diffusion of nutrients up the aerial mycelium, causing a second developmental
cycle.

Growth of mycelium on cellophane overlays, nitrocellulose filters or agar

Streptomycetes, because they grow naturally on solid surfaces, or at water/air interphases in the
soil, are usually ill-adapted to undergo their full cycle of physiological and morphological development
in liquid culture, and so they often show particular aspects of their phenotype, such as antibiotic
production or sporulation, best on a solid surface. (Much of the early development work done on a
promising new antibiotic-producer in an industrial laboratory is often aimed at finding a medium, and
appropriate culture conditions, that allow the organism to make its antibiotic in submerged liquid culture.)
This explains why small-scale, academic work is sometimes done with mycelium scraped from cellophane
laid on the surface of agar plates. For antibiotic production, even agar-grown cultures can be used. The
agar can be chopped up and extracted with an organic solvent, as in the series of studies on "hybrid"
polyketides described by McDaniel et al. (1995 and references therein). Water-soluble metabolites can
easily be recovered from agar plates by freezing them whole at -20o and then allowing them to thaw,
whereupon the gel collapses and up to 60% of the total volume of the agar can be poured off as a crude
aqueous solution.
Cellophane discs need to be made of normal wettable cellulose acetate film, such as dialysis
membrane, not certain kinds of non-wettable plastic. The discs can be bought as pre-cut "jampot covers",
or can be cut from larger sheets (much cheaper than dialysis tubing). They are conveniently sterilised by
autoclaving in glass Petri dishes, interleaved with wet filter paper. (They can also be autoclaved dry but
must then be wetted before use.) After the mycelium has been grown it can be scraped easily from the
cellophane and harvested in water or a suitable buffer. Streptomyces hyphae can grow through
nitrocellulose filters of 0.45 m pore size into the agar (Wolf and Schoppmann, 1989); this was turned
to advantage by Kelemen et al. (1995) who peeled off the aerial mycelium on the filters and examined
ectopic sporulation in the remaining substrate mycelium.
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 

It should be noted that, at least in our hands, and on some media, growth on cellophane discs
differs somewhat from that observed when cultures are grown directly on the same medium. The amount
of biomass, particularly substrate mycelium, appears to be reduced, and the onset of sporulation is
accelerated.
When aiming to obtain a series of plates developing synchronously - for example so as to harvest
groups of plates at intervals to make a RNA time curve for developmental S1 mapping - it is very important
to dry the plates carrying the cellophane discs thoroughly in the laminar flow cabinet. Otherwise damp
patches develop on the surface and hinder the development of aerial mycelium and spores.

Growth of Streptomyces mycelium in liquid

For many purposes, such as for preparing protoplasts for fusion, transformation or transfection,
or for isolating plasmid or chromosomal ("total") DNA or RNA or proteins, as well as for physiological,
biochemical or fermentation studies, streptomycetes are grown as mycelium in liquid cultures started from
an inoculum of spores. Since streptomycetes are highly aerobic, the cultures need to be shaken during the
incubation, usually on an orbital incubator shaker, in vessels with sufficient space for good aeration; for
example 25-50 ml cultures are grown in 250 ml Erlenmeyer flasks, or 500 ml cultures in 2 l flasks. Many
strains, including S. coelicolor A3(2) and S. lividans 66, tend to grow as rather compact masses or pellets
of mycelium unless steps are taken to encourage dispersed growth. Dispersed growth is favoured on
particular media, for example those containing polyethylene glycol, or Junlon (Hobbs et al., 1989), or a
high concentration of sucrose and/or by the use of flasks containing baffles. A simple baffle is obtained
by inserting a stainless steel spring into an ordinary Erlenmeyer flask. For each 250 ml flask we use a 30
cm length of spring, 1.3 cm diameter coil, 19 sw gauge, marine grade stainless steel wire (p. 419). The
new springs need to be washed very thoroughly with detergent before use to remove traces of inhibitory
material.
The growth of mycelium for the preparation of DNA and RNA is described in Chapters 8 and 16
as parts of the protocols for extracting nucleic acids. Here we deal with the growth of organisms for
physiological purposes and for preparing protoplasts for genetic work.

Growth of Streptomyces for physiological studies

Because bacteria are small the only way in which physiological and biochemical events can be
measured is to work with populations. For the events that are measured in the population to be a true
reflection of what is happening in the individual cell, all or most of the cells must be in the same
physiological condition. For studies of primary metabolism, this is usually considered to be achieved
when culture growth is exponential. A problem with streptomycetes is that one of the aspects that makes
them such interesting bacteria to study physiologically, their multicellular lifestyle, makes that study very
difficult. Streptomycetes grow by elongation at the tips of the mycelium and by branching. This permits
the achievement of exponential growth by young mycelium. However, physiological homogeneity is not
readily sustained over many generations because older, central parts of the mycelium may become
nutrient-limited. This problem is exacerbated by the tendency of initially separate microcolonies to
  &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

aggregate into larger clumps. Some species, such as S. rimosus, fragment in liquid medium to give fairly
homogeneous suspensions, but many, including the genetically most studied S. coelicolor and S. lividans,
form large, dense mycelial clumps unless special media or growth conditions are used.
One way to carry out biochemical and physiological tests on a homogeneous cell population is
to work with spore germlings, but the disadvantage is that very large numbers of spores are needed to
obtain enough biomass for most assays. This technique was used by Riesenberg and Bergter (1984) with
S. hygroscopicus. They were concerned to obtain balanced growth (i.e. growth in which each cellular
component increased by the same factor) and they found that growth remained balanced for up to five
doublings. Liquid medium was used, but spores can also be germinated on cellophane discs (p. 48)
overlaid on a solid medium and the germlings scraped from the cellophane.
To obtain large amounts of biomass that can be easily manipulated the ideal solution is to grow
the organisms in a liquid medium. Several techniques have been developed to allow dispersed growth of
S. lividans and S. coelicolor A3(2). There is a rule of thumb that the more complex the medium, and so
the faster the growth rate, the less likely is "clumping" of the cells. Addition of 34% sucrose, which S.
lividans and S. coelicolor A3(2) cannot catabolise, to complex media such as YEME, and the use of
stainless steel springs as baffles, allows the generation of cultures that are relatively dispersed. The
problem comes when a fully defined salts medium is required, as in physiological studies where the inputs
and outputs in a culture need to be precisely measured. Addition of Casaminoacids enhances growth rate
and hence aids dispersal, but amino acids can act as nitrogen, carbon and energy sources, as in NMMP
medium (p. 413).
Several materials have been used as dispersants in liquid minimal media. Table 2.1 lists their
advantages and disadvantages. Some kind of baffle is always needed, in addition to the dispersant, or the
culture has to be vigorously stirred. Stainless steel springs are convenient as baffles, but the disadvantage
is that antifoam (p. 413) must be used to avoid frothing (Hodgson, 1982). An alternative is to use 0.3 cm
diameter glass beads (c. 100 per 250 ml flask) (Doull and Vining, 1989).
Good dispersed growth depends on the state of the inoculum. The single most important point
is to use a large inoculum and to pre-treat spores by a procedure that allows germination but not
necessarily outgrowth. It is also important that the germinated spores are disaggregated before being used
as inoculum. They can either be vortexed vigorously or subjected to a short burst of ultrasound. Several
methods of pregermination have been used (see below). The recommended inoculum is that which gives
an initial OD450 of at least 0.03 ($107 spores ml1). This inoculum size should allow at least six doublings
before stationary phase. When inoculum size was investigated in detail it was found that there was a clear
co-operative effect: the larger the inoculum the shorter the lag phase. It was speculated that carbon
dioxide might have been responsible for this cooperativity (Hodgson, 1980).

Conditions for spore germination and reproducible exponential growth and

antibiotic production by Streptomyces coelicolor

These conditions were developed (by Strauch et al., 1991) for S. coelicolor M145 (prototrophic,
SCP1, SCP2), and may require modification for other strains. In SMM, which contains 0.2% (w/v)
Casaminoacids, M145 grows exponentially with a doubling time of about 2.2 h; in the absence of the
Casaminoacids, the doubling time is about 4 h. The cultures enter stationary phase approximately 14 h
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 

Table 2.1 Dispersants used in liquid minimal medium

Dispersant Advantages Disadvantages

Sucrose Fully soluble to give a clear solution Very high concentrations

(350 g 1 w/v, 1.1M) for OD measurements. Not
Hopwood et al. (1985) metabolised by most streptomycetes.
Can be removed by simple washing.
Unlikely to affect most enzymes.
needed; high osmotic potential.
Cannot be autoclaved at higher
concentration and autoclaving
in the presence of other media
components releases glucose
and fructose.

Polyethylene glycol Fully soluble to give a clear solution
8000 for OD measurements. Can be
(50 g 1 w/v, 6 mM) autoclaved or filter-sterilised as a
Hodgson (1982) double strength solution. Removed
by extensive washing of cells. Not
metabolised by streptomycetes.
Causes foaming and so
antifoam is necessary. Low
concentrations inhibit some
enzymes and Folin-Ciocalteus
reagent. Autoclaved 10%
solution acquires growth
inhibitor on standing.

Junlon Low concentrations are effective. Insoluble colloidal gel makes

(2 g 1 w/v; p.419) Not metabolised by streptomycetes. OD measurement difficult, but
Hobbs et al. (1989) not impossible. Cannot be
removed. Inhibits many
enzymes and interferes with
RNA isolation.

Starch Unlikely to affect most enzymes. Relatively high concentrations

1
(50 g 1 w/v) Can be removed by amylase. needed. Must be autoclaved
Doull and Vining (1989) with medium. Colloid, making
OD measurement difficult.
Can be metabolised by most
streptomycetes.

Agar Low concentrations are effective. Catabolised by S. coelicolor

(1 g 1 w/v) Unlikely to affect most enzymes. A A3(2) as carbon, nitrogen and
Hobbs et al. (1989); colloid, but low concentrations energy source.
Magnolo et al. (1991) unlikely to affect OD measurements.
Could be removed by agarase.

Carboxy-methyl- Gives a clear solution for OD Catabolised as carbon and

cellulose measurements. Can be removed by energy source by most
cellulase. Unlikely to affect most streptomycetes.
(10 g 1 w/v)
enzymes.
C. Potter (personal
communication)
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

after inoculation at an OD450 of 1.5-2.0, yielding about 1 mg ml1 dry weight after 7-8 cell doublings. The
high spore inoculum, coiled springs and PEG provide relatively dispersed growth. A higher
concentration of Casaminoacids will give a higher final OD450, but the increase in size of the mycelial
pellets may lead to greater physiological heterogeneity. Note that the cultures are nitrogen-, not carbon-
limited.

A dense spore suspension (c. 1010 spores ml-1) of S. coelicolor

20 ml volumes of 2 YT medium (p. 414)
SMM (Supplemented Minimal Medium) (p. 413)
250 ml siliconised flasks containing coiled springs. (We use 250 ml siliconised
PYREX flasks with stainless steel coiled springs (p. 419). To minimise differences in
aeration and clumping between flasks, use the same length of spring (about 26 cm, with
4.5-5 turns per 2 cm length) in each, shake at 300 rpm at 30o and check frequently;
universal containers; pipettes
Bench centrifuge, vortex mixer; water bath; incubator shaker; sonicator.

1. Inoculate 20 ml of 2 YT in a 250 ml flask containing a coiled spring with 20

l of the dense spore suspension. We use 20 ml because this is convenient for spinning
down in a Universal, but 50 ml can also be used. If a smaller volume is required, use a Universal
with c. 10 ml of medium, containing a short coiled spring, instead of a flask.

2. Incubate at 30o on the shaker at 300 rpm for 6-8 h, by which time emerging germ
tubes should be visible.

3. Spin germinated spores down in a Universal at 1000 g for 5 min, preferably in

a swing-out rotor.

4. Decant the supernatant, add 5 ml of SMM and vortex mix. Sonicate on ice for
5 sec only to disperse aggregated germlings. We use a MSE Soniprep 150 with fine
sonicator tip at full power and tuned to 50%. To sterilise the tip, sonicate in absolute ethanol for
a few sec and store under ethanol; air dry immediately before use.

5. Add a portion of the sonicated cells to the desired final volume of SMM to give
an OD450 of c. 0.1 measured against water. It is important to mix the samples well
immediately before measuring the OD. SMM has an OD450 of about 0.06 (mostly from the
antifoam).

6. Mix well. Dispense 50 ml samples into fresh 250 ml flasks and incubate as
above. This should be equivalent to a spore inoculum of 4 106 ml1, but remember that some
of the germlings will still be clumped, and that sonication may cause reduced viability.
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 

Germination of Streptomyces spores

Apart from the procedure just described, several others have been used for the germination of
spores. Here are two.

Procedure 1 (This method was included in Hopwood et al. (1985) and is probably still useful.)
A dense spore suspension (c. 1010 spores ml-1), either fresh or frozen
Sterile TES buffer (0.05M, pH8)
Double strength Germination Medium (Difco yeast extract, 1%; Difco
Casaminoacids, 1%; CaCl2, 0.01M (prepared and autoclaved separately as 5M solution;
add 2 l per ml of medium)).
Screw cap bottles; centrifuge tubes; pipettes
Bench centrifuge; vortex mixer; 50o water bath; 37o orbital shaker;
spectrophotometer set at 450 m; cuvettes; microscope.

1. Pellet the spores on the bench centrifuge and re-suspend in e.g. 5 ml of TES
buffer. It is important to remove glycerol from the spore suspension, otherwise it may impair
spore viability during heat-shocking.

2. Heat shock the spores (e.g. 50o for 10 min) and cool under cold tap. Various
combinations of time and temperature are suitable: e.g. 45o for 15 min or 50o for 10 min for S.
coelicolor; other strains may need different conditions.

3. Add an equal volume of double strength Germination Medium. Incubate at 37o

with shaking (300 rpm) for 2-3 h. Malt extract is not present; it hampers synchronous
germination. About 50% of spores should have microscopically visible germ tubes at the end
of this stage.

4. Pellet the germinated spores in the bench centrifuge and re-suspend in water or
TES buffer. Agitate on vortex mixer to disperse the clumps. Quite vigorous
agitation (or even a short burst of ultra-sound) is needed to disperse the clumps caused by
adhesion of the germ tubes.

5. Inoculate growth medium to OD450 of 0.03-0.05. A dense inoculum is needed: the

spores from one or more slants (c. 2 107 spores) will be required for each 100 ml culture.
Spores treated in this way should establish exponential growth in c. 5 h or less in a chemically
defined medium, depending on the carbon source.
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Procedure 2 (From Mikulik et al., 1984)

Streptomyces spores
Chemically defined germination medium: 2.5mM of each of 20 amino acids; 0.01%
each of adenosine, guanosine, thymidine, uridine and cytidine; 0.02M potassium
phosphate, pH7.0. Complex germination medium: Casaminoacids, Difco, 0.2%; Yeast
extract, Difco, 1.0%; KH2PO4, 0.68%; (NH4)2SO4, 0.2%; MgSO4, 0.02%; CaCl2,
0.001%; pH7.0
Ultra-Turrax Turbomixer (p. 420) or High Speed Homogeniser System (p. 419) with
speed regulation from 0-20,000 rpm; reciprocating shaker

1. Homogenise 1 g of spores in 2.5 ml of cold water at 20,000 rpm in the Ultra-

Turrax Turbomixer at 0o (5 times 45 sec run followed by 30 sec cooling).
Homogenised spores prepared as above can be mixed with an equal volume of 40% (v/v)
glycerol and stored at -70o. The spores remain viable for several months of storage, and
germinate synchronously.

2. Inoculate 0.5 ml of the homogenised spore suspension into 50 ml of germination

medium in a 500 ml flask and incubate on a shaker at 28o. After 3 h of cultivation,
outgrowth of germ tubes is microscopically visible. Synchronous germination takes place
through a sequence of time-ordered events. RNA and protein synthesis start during the first 5
min and net DNA synthesis at 60-70 min of germination.

Preparation of aerial mycelium and spores separately

This procedure was supplied by K. Mikulik. It was developed for physiological studies of
sporulation and spore germination in S. granaticolor (Stastn, 1977; Mikulik et al. 1977, 1984; Stastn
and Janda, 1983).

Streptomyces mycelium
Sporulation agar (Yeast extract, 0.4%; malt extract, 1.0%; glucose 0.4% Oxoid No 4
Agar, 2.0%; pH7.0)
Cellophane discs (see p. 48)
Ultra-Turrax Turbomixer (p. 420) or High Speed Homogeniser System (p. 419) with
speed regulation 0-20,000 rpm; laminar flow hood; preparative centrifuge; vortex
mixer

1. Pour a 7 mm layer of sporulation agar (45 ml in a 9 cm dish) in each Petri dish

and dry in a laminar flow hood for 30-40 min.
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 

2. Lay a cellophane disc on the agar and remove air bubbles with a sterile spreader.

3. Inoculate 3 ml of a culture of vegetative mycelium, spread the cells over the

cellophane, and incubate at 28o. Then proceed to either 4a or 4b.

Aerial mycelium

4a. After 48 h growth, strip the white layer of aerial mycelium and residues of
vegetative cells from the cellophane with a spatula.

5a. Homogenise stripped cells in ice-cold water at 15,000 rpm for 30 sec in the
Ultra-Turrax Turbomixer.

6a. Spin the mixture at 7,000 g for 10 min at 4o. Vegetative hyphae sediment at
the bottom of the tube while hydrophobic aerial mycelium floats in the
supernatant.

7a. Transfer the supernatant fraction to a centrifuge tube and remove water from the
pellet by suction using a Pasteur pipette with its tip wrapped in glass wool.

8a. Repeat steps 5a-7a.

9a. Freeze the white aerial mycelium in liquid nitrogen and store at -70o.

Spores

4b. After 14 days growth, scrape the grey hydrophobic spores from the surface of the
cellophane.

5b. Transfer the harvested material to cold sterile distilled water and wash by
vortexing for 1 min.

6b. Spin the mixture at 7,000 g for 10 min. Hydrophobic spores may float in the water
phase.

7b. Collect the suspension of spores and remove water by suction as above.
Spores can be stored either frozen at -20o or mixed with an equal volume of 40% glycerol
and kept at -70o.
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Sporulation of Streptomyces in submerged cultures

Although the typical reproductive cycle of Streptomyces takes place on solid surfaces, some
strains can sporulate in submerged liquid culture. This feature was first observed with S. griseus
(Kendrick and Ensign, 1983) and has led to the use of certain strains of this species for studying
physiological events associated with sporulation (Ochi, 1987; McCue et al., 1992). Subsequently, other
species have been found to sporulate in liquid medium under suitable conditions: S. viridochromogenes
(Koepsel and Ensign, 1984); S. roseosporus (Huber et al., 1987); S. acrimycini and S. albus (Daza et al.,
1989); S. venezuelae (Glazebrook et al., 1990); S. albidoflavus (Lee and Rho, 1993). In most or all
respects, the spores from such cultures seem to resemble typical aerial spores of the same strain. In
contrast, only "spore-shaped bodies" lacking the normal resistance of spores to moist heat were produced
by S. coelicolor A3(2) and S. lividans 66 (Daza et al., 1989). When the S. coelicolor whiG gene,
encoding a sporulation-specific sigma factor, was cloned at high copy, sporulation occurred in submerged
liquid culture, but not to an extent usable for large-scale study (Chater et al., 1989). Recently, apparently
normal heat-resistant spores were found in submerged growth of S. coelicolor cultures provided they
were not shaken (E. Takano, personal communication).

Preparation of protoplasts from Streptomyces lividans 66 and S. coelicolor A3(2)

Protoplasts are used both for fusion and for transformation and transfection. This protocol
describes their preparation up to the first wash (see the appropriate technique for their further handling:
pp. 157, 232, 301 respectively). It was originally based on the work of Okanishi et al. (1974) and was
adapted and optimised for high transformation frequency by Bibb et al. (1978) and Thompson et al.
(1982). Many different Streptomyces species can be protoplasted and regenerated using this procedure.
Sometimes the age and/or physiological state of the mycelium is important. For example, the
best transformation or transfection frequencies in S. lividans are obtained with protoplasts prepared from
mycelium grown to "late exponential" phase (36-40 h), by which time the culture develops a pink/orange
tinge, whereas for transfection of S. parvulus protoplasts needed to come from much younger mycelium
(c. 18 h). The details may also depend on the particular genotype: many morphological mutants are
particularly idiosyncratic.
It is worth emphasising that a poor spore inoculum leads to a bad batch of mycelium, and this
in turn results in poor protoplasts and a low transformation frequency.

Streptomyces spores, fresh suspension (c. 109 spores ml1) or frozen stock in 20%
glycerol (pp. 44-47)
YEME medium (containing 34% sucrose, 5 mM MgCl2, 0.5% glycine) (p. 412)
P buffer (p. 415)
10.3% sucrose
Lysozyme solution (1 mg ml-1 in P buffer, filter sterilised)
250 ml baffled Erlenmeyer flasks (see p. 419 for springs); 20 ml screw cap bottles;
pipettes (25 ml, 10 ml, 5 ml, 1 ml); filter tubes plugged with cotton wool (p. 46);
plastic tubes (disposable sterile tubes, e.g. 16 100 mm, round bottom, polycarbonate
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 
test tubes, Cat. No. 142AS made by Sterilin, Teddington, Middlesex. These are much
preferable to tubes which are washed and re-used because traces of detergent are thereby
avoided.); pro-pipettes; 30o orbital incubator; microscope; bench centrifuge;
30o water bath; refrigerator; -20E or -70E freezer.

1. Add 25 ml YEME medium to a baffled flask. Add c. 0.1 ml spore suspension

and required growth factors. Incubate 36-40 h at 30o in an orbital incubator
shaker. Cultures of S. lividans and S. coelicolor are ready for harvesting when they start to
produce red pigment

2. Pour culture broth into a 20 ml screw cap bottle and spin in the bench centrifuge
(e.g. 1000 g, 10 min). Before centrifugation, examine the culture for contamination by
unicellular bacteria, usually indicated by turbidity: the Streptomyces mycelium will fall to the
bottom in a few moments, while unicellular contaminants remain suspended. In case of doubt,
check in the microscope.

3. Discard the supernatant.

4. Re-suspend pellet in 15 ml 10.3% sucrose and spin in bench centrifuge as above.

Discard supernatant. Occasionally, mycelium will not pellet from the growth medium
containing 34% sucrose on centrifugation; in this case add 10.3% sucrose or water to reduce the
density of the medium and so facilitate pelleting. Take care in pouring off the supernatant from
the loose pellet.

5. Repeat step 4. The mycelial pellet, without added liquid, can be frozen at -20o before
resuspension.

6. Resuspend mycelium in 4 ml lysozyme solution; incubate at 30o, 15-60 min. For

most purposes, lysozyme is dissolved in P buffer; use of L buffer (p. 416) gives more efficient
transformation of S. lividans by plasmid DNA. Lysozyme treatment times vary from strain to
strain, e.g. 15 min is usually long enough for S. lividans whereas S. coelicolor requires 60 min;
other strains may take longer. Protoplast formation can be monitored using the phase-contrast
microscope. For S. lividans the time (15-60 min) is not critical but protoplasts of other strains
may lyse (especially in L buffer) if left too long. If this happens, P buffer + lysozyme can be
substituted for L buffer and lysozyme treatments may be done at a lower temperature (e.g. room
temperature) or for a shorter time.

7. Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.

8. Add 5 ml P buffer. Repeat step 7. This helps to free protoplasts from the mycelium so
that they will pass through the cotton wool filter used in step 9. At least with S. lividans, it is
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

possible to obtain transformants with unfiltered material, but the washing (steps 9-10) is still
needed to remove lysozyme.

9. Filter protoplasts through cotton wool (using a filter tube: p. 46) and transfer to
a plastic tube.

10. Sediment protoplasts gently by spinning in a bench centrifuge (e.g. 1000g,

7 min).

11. Discard supernatant and suspend protoplasts in 1 ml P buffer. At this and any other
steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of
liquid by tapping the side of the tube repeatedly with a finger until the protoplasts are dispersed
to form a creamy suspension, then add the suspending P buffer (otherwise the protoplast pellet
is difficult to disperse). Avoid vortexing, which induces foaming and consequent lysis.

12. See one of the three techniques (protoplast fusion p. 157; transformation of
protoplasts (p. 232) or transfection of protoplasts, p. 301) for further
manipulations. To freeze the protoplasts for storage, place samples of the
protoplast suspension in small plastic tubes, close them and place them in ice in
a plastic beaker. Place the beaker at -70o overnight. Free the frozen protoplasts
in their tubes from the ice and store at -70o. To thaw, shake the frozen tube
under running warm water (i.e. freeze slowly, thaw quickly). To assess the
proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in P
buffer and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies
arising after dilution in detergent are likely to have arisen from non-protoplasted units.

Preservation of Streptomyces strains and phages

Strains that are needed only for short-term use can be kept as cultures on agar slants or in sealed
Petri dishes at 4o, where they will usually remain viable for months, provided they sporulate before
refrigeration. Non-sporulating cultures need to be sub-cultured frequently.
A very convenient method for keeping strains for longer periods is to prepare spore suspension
in 20% glycerol (see "making a spore suspension", p. 44) and freeze them at -20o. Even with repeated
thawing and re-freezing for sampling purposes, viability remains high for years, provided the spores are
not allowed to remain at room temperature for extended periods before re-freezing: when you work with
them on the bench, keep them in an ice bucket. Another procedure, using mycelium instead of spores
and employed routinely at the Eli Lilly Company (E. T. Seno, personal communication), is to grow the
strains in a complex medium such as Tryptic Soy Broth (Difco) for two days, add an equal volume of
20% glycerol/10% lactose, and store in the vapour phase of liquid nitrogen. Again the cultures remain
viable for years.
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV 
For long-term storage, and as a back-up to frozen spore suspensions, lyophils (on filter paper
wicks) are prepared as described below.
Phages are usually kept in suspension in Difco Nutrient Broth (DNB; p. 412) at 4o in the dark.
Lyophils of phages may be prepared by dipping filter paper wicks into broth suspensions and proceeding
in the same way as for spores.

Preparation of lyophils (From Hopwood and Ferguson, 1969)

Fresh slant cultures or spore suspensions in 20% glycerol

Skim milk (10% solution of Difco Bacto Skim Milk powder sterilised by autoclaving
at 121o, 5 min; this shortened time is to prevent caramelisation)
Glass ampoules or bottles previously sterilised by autoclaving; filter paper wicks
(strips of Whatman 3MM paper, cut to fit the container); inoculating loop; long-jawed
forceps
Freeze dryer, either with manifold to accept ampoules or chamber to contain
bottles; gas flame, ampoule tester

Alternative I (using a freeze dryer with manifold)

1. Add 1 ml skim milk to a slant culture and scrape the surface to make a crude
spore suspension. If working from an existing spore suspension in glycerol, add
0.05 ml of dense spore suspension to 0.5 ml skim milk or, if the suspension is not
very dense, centrifuge it and re-suspend the spores in 0.5 ml skim milk.

2. Using forceps, place several sterile filter paper wicks in the spore suspension and
allow them to become saturated.

3. Using long-jawed forceps (e.g. stainless steel angled aural forceps) place one
wick in each ampoule.

4. Constrict the neck of the ampoule to about a quarter of its original diameter by
drawing it out in a fine flame, at a point c. 30 mm from the open end, and place
each ampoule, after constriction, on the manifold of the freeze dryer.

5. When the manifold is full, switch on the freeze dryer and leave it pumping until
a low reading (<10 mm mercury) is obtained on the vacuum gauge (this takes
several hours, so overnight pumping is convenient).

6. Seal off each ampoule in turn and remove it from the freeze dryer.
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

7. Each ampoule (or a sample of them) should be tested with a hand-held vacuum
tester to ensure that they contain a good vacuum.

Alternative II (using a freeze dryer with chamber and press for closing bottles: e.g. from Edwards High
Vacuum Ltd. p. 419)

1. Previously sterilise the bottles, each containing (say) 4 filter paper wicks, with
the rubber stopper loosely in place.

2. Prepare a spore suspension in skimmed milk as in (I, step 1).

3. Pipette 0.2 ml of spore suspension into each container so that the wicks soak it
up.

4. Replace the stopper loosely and place the container in the chamber of the freeze
dryer.

5. Pump for several hours as in (I, step 5).

6. Close the containers by screwing down the press, remove them from the chamber
and crimp on a metal closure.

7. Test the ampoules as in (I, step 7).

Growing a culture from the lyophil

To grow a culture from a lyophil, open the container, remove the wick (or one of them if several
were included in the same container, then re-evacuate and re-seal it) and place it on a plate of agar
medium (CM is better than MM and may be better than R2YE). When it has soaked up some moisture,
move it about over the agar surface to distribute the inoculum. Incubate.

Bibb MJ, Ward JM, Hopwood DA (1978) Transformation
of plasmid DNA into Streptomyces at high frequency. Nature,
274, 398-400.
Chater KF, Bruton CJ, Plaskitt KA, Buttner MJ, Mndez
C, Helmann J (1989) The developmental fate of S. coelicolor
hyphae depends crucially on a gene product homologous with
the motility sigma factor of B. subtilis. Cell, 59, 133-143.
Daza A, Martin JF, Dominguez A, Gil JA (1989)
Sporulation of several species of Streptomyces in submerged
culture after nutritional downshift. J.Gen.Microbiol., 135,
2483-2491.
Doull JL, Vining LC (1989) Culture conditions promoting
dispersed growth and biphasic production of actinorhodin in
shaken cultures of Streptomyces coelicolor A3(2). FEMS
Microbiol.Lett., 65, 265-268.
Dowding JE (1973) Characterization of a bacteriophage
virulent for Streptomyces coelicolor A3(2). J.Gen.Microbiol.,
76, 163-176.
El-Nakeeb MA, Lechevalier HA (1963) Selective isolation
of aerobic actinomycetes. Appl. Microbiol., 11, 75-77.
Glazebrook MA, Doull JL, Stuttard C, Vining LC (1990)
Sporulation of Streptomyces venezuelae in submerged
cultures. J.Gen.Microbiol., 136, 581-588.
Hayakawa M, Nonomura H (1987) Humic acid-vitamin
agar, a new method for the selective isolation of soil
actinomycetes. J.Ferment.Technol., 65, 501-509.
Herron PR, Wellington EMH (1990) New method for the
extraction of streptomycete spores from soil and application
 &KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV
to the study of lysogeny in sterile amended and nonsterile
soil. Appl.Env.Microbiol., 56, 1406-1412.
Hobbs G, Frazer CM, Gardner DCJ, Cullum JA, Oliver
SG (1989) Dispersed growth of Streptomyces in liquid
culture. Appl.Microbiol.Biotechnol., 31, 272-277.
Hodgson DA (1980) Carbohydrate utilization in
Streptomyces coelicolor A3(2). Ph.D.Thesis, University of
East Anglia, Norwich.
Hodgson DA (1982) Glucose repression of carbon source
uptake in Streptomyces coelicolor A3(2) and its perturbation
in mutants resistant to 2-deoxyglucose. J.Gen.Microbiol.,
128, 2417-2430.
Hopwood DA, Ferguson HM (1969) A rapid method for
lyophilizing streptomyces cultures. J.Appl.Bact., 32, 434-436.
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ,
Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf
H (1985) Genetic Manipulation of Streptomyces. A
Laboratory Manual. John Innes Foundation, Norwich.
Hsu SC, Lockwood JI (1975) Powdered chitin agar as a
selective medium for enumeration of actinomycetes in water
and soil. Appl. Microbiol., 29, 422-426.
Huber FM, Piper RL, Mertz FP (1987) Sporulation of
Streptomyces roseosporus in submerged culture.
J.Ind.Microbiol., 2, 235-241.
Kelemen GH, Plaskitt KA, Lewis CG, Findlay KC,
Buttner MJ (1995) Deletion of DNA lying close to the glkA
locus induces ectopic sporulation in Streptomyces coelicolor
A3(2). Mol.Microbiol., 17, 221-230.
Kendrick KE, Ensign JC (1983) Sporulation of
Streptomyces griseus in submerged culture. J.Bacteriol., 155,
357-366.
Koepsel R, Ensign JC (1984) Microcycle sporulation of
Streptomyces viridochromogenes. Arch.Microbiol., 140, 9-14.
Lee KJ, Rho YT (1993) Characteristics of spores formed by
surface and submerged cultures of Streptomyces albidoflavus
SMF301. J.Gen.Microbiol., 139, 3131-3137.
Magnolo SK, Leenutaphong DL, DeModena JA, Curtis
JE, Bailey JE, Galazzo JL, Hughes DE (1991)
Actinorhodin production by Streptomyces coelicolor and
growth of Streptomyces lividans are improved by the
expression of a bacterial hemoglobin. Bio/Technology, 9,
473-476.
McCue LA, Kwak J, Babcock MJ, Kendrick KE (1992)
Molecular analysis of sporulation in Streptomyces griseus.
Gene, 115, 173-179.
McDaniel R, Ebert-Khosla S, Hopwood DA, Khosla C
(1995) Rational design of aromatic polyketide natural
products by recombinant assembly of enzymatic subunits.
Nature, 375, 549-554.
Mikulk K, Janda I, Maskov H, St'astn J, Jiranov A Microbiol., 140, 2067-2076.
(1977) Macromolecular synthesis accompanying the
transition from spores to vegetative forms of Streptomyces
granaticolor. Folia Microbiol., 22, 252-261.

Mikulik K, Janda I, Weiser J, St'astn J, Jirnova A
(1984) RNA and ribosomal protein patterns during aerial
spore germination in Streptomyces granaticolor.
Eur.J.Biochem., 145, 381-388.
Mullins PH, Gurtler H, Wellington EMH (1995) Selective
recovery of Streptosporangium fragile from soil by indirect
immunomagnetic capture. Microbiol., 141, 2149-2156.
Nonomura H, Hayakawa M (1988) New methods for the
selective isolation of soil actinomycetes. In Okami, Y.,
Beppu, T. and Ogawara, H. (eds.), Biology of Actinomycetes
88. Japan Scientific Societies Press, Tokyo, pp. 288-293.
Ochi K (1987) Changes in nucleotide pools during
sporulation of Streptomyces griseus in submerged culture.
J.Gen.Microbiol., 133, 2787-2795.
Ogata S, Matsubara H, Harada Y, Umeda A (1992)
Formation of spontaneously developing pocks and production
of phage taillike particles in thiostrepton-producing
Streptomyces laurentii ATCC 31255. Actinomycetol., 6, 29-
32.
Okanishi M, Suzuki K, Umezawa H (1974) Formation and
reversion of streptomycete protoplasts: cultural conditions
and morphological study. J.Gen.Microbiol., 80, 389-400.
Riesenberg D, Bergter F (1984) Establishment of a system
for analysis of balanced and unbalanced growth of
Streptomyces hygroscopicus. J.Gen.Microbiol., 130, 2543-
2548.
St'astn J (1977) A method of rapid wetting and synchronous
germination of streptomycete spores. Folia Microbiol., 22,
137-138.
St'astn J, Janda I (1983) Production, long term
preservation and synchronous germination of aerial spores of
Streptomyces. J.Microbiol.Meth., 1, 267-273.
Strauch E, Takano E, Baylis H, Bibb MJ (1991) The
stringent response in Streptomyces coelicolor A3(2).
Mol.Microbiol., 5, 289-298.
Thompson CJ, Kieser T, Ward JM, Hopwood DA (1982)
Physical analysis of antibiotic-resistant genes from
Streptomyces and their use in vector construction. Gene, 20,
51-62.
Wellington EMH, Toth IK (1994) Actinomycetes. Methods
of Soil Analysis, Part 2. Microbiological and Biochemical
Properties-SSSA Book Series, no. 5. Soil Science Society of
America, Madison, pp. 269-290.
Wellington EMH, Al-Jawadi M, Bandoni R (1987)
Selective isolation of Streptomyces species-groups from soil.
Dev. Ind. Microbiol., 28, 99-104.
Wipat A, Wellington EMH, Saunders VA (1994)
Monoclonal antibodies for Streptomcyes lividans and their
use for immunomagnetic capture of spores from soil.
Wolf H, Schoppmann H (1989) Streptomycetes can grow
through small filter capillaries. FEMS Microbiol.Lett., 57,
259-264.
 &KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV