ISOLATION AND

SCREENING FOR
SECONDARY
METAB0LITES

T HE OPENING SECTION OF THE 2 N D EDITION OF THE MANUAL

Industrial Microbiology and Biotechnology (MIMB2) described the
OF

initial stages of establishing an industrial microbiological process:

culture isolation and preservation, screening, small-scale fermentation, and
process optimization. Much of what was recommended in those discussions
remains valid and relevant for developing modern industrial processes;
what the contributors to the present Third Edition (MIMB3) provide is
new vision and options with which to approach such objectives. The cur-
rent rapid pace of scientific discovery and technological innovation is such
that completely novel options have become available to the industrial mi-
crobiologist in the quest for new products and processes since the last edi-
tion was prepared. Arguably the most dramatic event changing the face of
search and discovery has been the accelerating move from a culture-based
to a genomics-based strategy. This is set, not least, against the background
of a failure of combinatorial chemistry to deliver exploitable drug leads. The
challenge, therefore, rests on the evolutionary power of natural biological
processes to fulfill the requirement for new drugs and other products of this
increasingly biotechnology-dependent age.
The contributions in this section focus broadly on two themes, namely,
some central issues pertaining globally to industrial microbiology (isolation
and characterization of organisms), and the routes to biodiscovery. Al-
though each chapter addresses specific questions and provides appropriate
intellectual and practical support for their resolution, a number of recur-
ring themes can be found in the advice offered by authors having different
professional backgrounds and interests. Examples include the increasing
dependence on integrated skills in order to develop successful screening
protocols or uncover and exploit silent biosynthetic pathways (SBPs), and
the strong desirability of using efficient dereplication procedures at the
chemical, organism, and genomic levels so as to avoid unnecessary and
cost-ineffective redundancy during the early stages of process or product
development.
A recurrent source of embarrassment for microbiologists is the fact that
only a minute proportion of the estimated numbers of, in particular, bacteria
have been isolated and maintained in the laboratory. Slava Epstein and his
colleagues in chapter 1 tackle this dilemma head-on and argue that because
such a major part of microbial diversity remains “missing” it hampers prog-
ress in basic and applied microbiology: cultures enable the comprehensive
study of whole organisms and the elucidation of complete genomes. These
authors have developed techniques with which to target microorganisms
in their natural habitats such that they are grown in situ and subsequently
isolated and domesticated under laboratory conditions. All-importantly, the
approach recognizes the impact of naturally occurring growth conditions
2 ISOLATION AND SCREENING FOR SECONDARY METABOLITES
and microbial interactions in enabling cultivation in ways
that attempts at in vitro cultivation rarely achieve. In the
following chapter, Michael Goodfellow presents methods
for selectively isolating members of one particular group of
bacteria-the actinobacteria. Although these organisms are
viewed traditionally as typical members of soil and fresh-
water ecosystems, their distribution in the biosphere now
is known to be among the widest of all bacteria, and they
remain a prime choice in the search for novel industrially
important natural products and biocatalysts. Goodfellow
shows that intelligent manipulation of standard media com-
ponents and incubation conditions, often guided by ecologi-
cal and taxonomic database information and allied to the
probing of poorly studied and neglected habitats, continues
to yield isolates of new species and genera having biotech-
nological value. In this context, the isolation of members of
deep lineage actinobacteria remains as a timely challenge.
Two essential tasks need to be undertaken following
the isolation of an organism(s) that possesses the required
properties: culture curation (described in MIMBZ) and
taxonomic characterization. All too often, the latter exer-
cise is ignored or treated superficially, but as Giovanna Felis
and her coauthors opine in chapter 3, such characterization
is essential for organism identification, rigorous scientific
communication, securing intellectual property rights, and
establishing taxonomic road maps to genes and products.
The importance of a polyphasic taxonomic approach is
reiterated in this chapter, and “how-to” details are provided
with regard to prokaryotes (similar strategies being entirely
apt to eukaryotic microorganisms). Felis et al. also consider
the likely impact of whole-genome sequencing on taxon-
omy, which, given the speed and affordable cost of genome
sequencing, might be seen as the “ultimate source” for taxo-
nomic characterization. However, they rightly caution that
phenotypic analysis remains valid in order to complete the
organism’s characterization, to provide a more precise idea
of how it interacts with its environment, and to give clues
to its industrially exploitable biological activities.
Turning to the question of biodiscovery, the sine qua
non is to design search methods that avoid, or at least
minimize, rediscovery of known entities. To this end it is
desirable to isolate new organisms, explore new ecological
niches, design new screens, and apply new methods for
detecting natural products. These elements of the dis-
covery process are clearly revealed in chapter 4 by Don
Cowan and colleagues, who focus on obtaining biocatalysts
from extreme environments, the so-called extremozymes.
A n important part of this contribution is devoted to the
sampling of extreme environments and extracting DNA
from such extreme samples. There follow detailed pro-
tocols for producing metagenomic libraries, host-vector
systems for use with extremophiles and the expression of
extremozymes, and activity screens. In chapter 5, Stefan0
Donadio and Margherita Sosio describe cell-based assays in
screening for anti-infective compounds and argue the case
for these screens v i s - h i s cell-free systems. They guide the
reader along the critical path from target identification and
validation to assay development and assay implementation,
and introduce a range of options that include reporter, anti-
sense, and reversion assays. Important recommendations are
made with regard to screening algorithms and the evalua-
tion of target “hits.”
The final chapters in section I examine two rapidly
evolving components of the search and discovery toolbox,
namely, detecting the metabolic products of expressed genes
(metabolomics) and probing genomes for the potential to
synthesize new products (genome mining of SBPs). Me-
tabolomics is discussed by Jens Frisvad, who at the outset re-
minds us that the metabolome is a snapshot of an organism’s
metabolic capacity under a specific set of growth condi-
tions, and that its definition is crucially dependent on the
analytical techniques used for metabolite detection. Thus,
maximizing expression and detecting compounds again em-
phasize the necessity of an integrated skills platform. Frisvad
demonstrates the steps in metabolome analysis with refer-
ence to microfungi, but the protocols are pertinent to all
microorganisms. Finally, strategies for accessing microbial
secondary metabolites from SBPs are presented in detail in
chapter 7, by Robert Cichewicz and his coworkers. While
acknowledging the value of culture condition manipulation
as a means of triggering SBPs, these authors show that a
new suite of genomic-driven, mechanism-based, and hybrid
approaches is providing an unprecedented opportunity for
identifying SBPs. Such approaches include genome scan-
ning, heterologous and in situ expression of SBP genes, and
chemical epigenetic and genoisotopic methods. Yet again,
the integration of microbiological and chemical techniques
is a key element in the successful application of these tech-
niques. Especially helpful features of this chapter include an
analysis of the major strengths and weaknesses of the vari-
ous methodological approaches, and case studies that guide
the reader on such points.
Isolation
New Approaches to Microbial Isolation
SLAVA S. EPSTEIN, KIM LEWIS, DOMINICA NICHOLS, AND
EKATERINA GAVRISH
1.l. INTRODUCTION
It has been known for over a century that most microorgan-
isms do not grow under standard laboratory conditions (2,7,
20, 32,34-36,39). At first, this appeared to be a cell num-
ber phenomenon: cells in the sample grossly outnumbered
the number of colonies they produced on artificial media.
The introduction of the rRNA approach to the study of
microbial diversity (25) revealed that it was also a species
diversity phenomenon: the number of microbial species in
nature proved much larger than the richness of established
culture collections (10, 12, 14, 16, 18, 22, 29,33). In 1985,
a term was coined to describe these observations: the Great
Plate Count Anomaly (30).
Metagenomics approaches promised to provide access
to the uncultivated species while bypassing the problem of
uncultivability (11, 17). Many of these promises have been
fulfilled, resulting in remarkable findings (4). The applica-
tion of these and high-throughput sequencing approaches
also showed that the microbial diversity in nature is so stag-
gering that even the largest metagenomic efforts still sample
only a small fraction of the metagenomes of microbial
communities ( 18). Metagenomics libraries are dominated
by gene sequences with unknown functions, and this limits
the knowledge gained from the community genome. In
practical application, metagenomic approaches have not
yet produced a robust pipeline of bioactive compounds from
uncultivated species. Microbial culture thus remains a basic
necessity for the study of microbes in nature, and a prerequi-
site for drug discovery from natural sources. However, over
a hundred years after its discovery, the anomaly remains un-
resolved, and more than half of microbial divisions are still
without a single cultivable representative (27, 28). The fact
that the major part of microbial diversity remains “missing”
hampers progress in both basic and applied microbiology,
and is widely recognized as one of the most important chal-
lenges facing microbiology today (19,37).
This state of affairs set the stage for a recent resurgence
of cultivation efforts (3, 5, 6, 8, 9, 13, 15, 21, 24, 26, 31,
38; D. Nichols, N. Cahoon, E. M. Trakhtenberg, L. Pham,
A. Mehta, A. Belanger, T. Kanigan, K. Lewis, and S. S.
Epstein, submitted for publication). Informing these efforts
was the principal lesson from the past: traditional manipu-
lation of the standard media components likely brings only
incremental success. Significant departures from conven-
tional techniques were clearly in order, and indeed, the
3
new technologies substantially diverged from the cultiva-
tion tradition by adopting single-cell and high-throughput
strategies (8, 27, 39; Nichols et al., submitted), better
mimicking the natural milieu (3, 6, 13,31) and increasing
the length of incubation and lowering the concentration of
nutrients (9). Our research group contributed to the effort
by developing three cultivation methodologies (15, 21;
Nichols et al., submitted). Their gist is to replace the in vi-
tro cultivation by growing microorganisms in situ, and thus
provide target microorganisms with their natural growth
conditions, followed by domestication of the isolates for
growth under laboratory conditions. This chapter describes
the principle of these methods, details their application
and the initial results of this application, and summarizes
the lessons learned.
1.2. METHOD 1: DIFFUSION CHAMBER
1.2.1. Principle of the Method
Designing this method to cultivate environmental micro-
organisms, we considered that natural environments by
definition provide the necessary growth conditions to the
microorganisms inhabiting these environments. No matter
how transient such conditions may be for any given spe-
cies, they do occur, and the species grows. It follows that
by placing a sample of environmental cells into a diffu-
sion chamber, and incubating this chamber in the natural
environment of these cells, we could ensure that at least
some of them would be supplied by the required growth
factors via diffusion. If so, such cells should grow inside
the diffusion chamber just as their kin cells would outside
the diffusion chamber. The key element of this strategy is
incubation of the diffusion chamber in situ, which allows
diffusion to occur while preventing cells from migrating in
and out of the chamber. Reducing this idea to practice, we
tested a number of different membranes and different ways
to construct the chamber, and designed a simple device (21)
described below.
1.2.2. Diffusion Chamber Design
We construct the diffusion chambers (Fig. 1A) using
stainless steel washers (70 mm outside diameter, 33 mm
interior diameter, 3 mm thickness; Bruce Watkins Supply,
Inc., Wilmington, NC) and 0.03-pm-pore-size membranes
4 ISOLATION A N D SCREENING FOR SECONDARY METABOLITES
A. Diffusion Chamber
-__c ----
Agar matrix ’ 0 03 Pm membrane , Agar matrix,
Air
I 0.03pm
membrane
- c-
prevents
entry of
airborne
contaminants
I 0.03 pm --
Agar matrix
3coooc3c‘
bacteria
FIGURE 1 Design and application of diffusion chamber (A) and microbial trap (B). Explanations
in the text.
(Osmonics, Inc., Westborough, MA). After a membrane is
glued to one side of the washer using Silicone Glue I1 (Gen-
eral Electric, Waterford, NY), the half-assembled chamber
is kept in a laminar flow hood until bonding is complete.
Meanwhile, we prepare the inoculum by mixing environ-
mental cells with warm (and still liquid) agar (0.7 to 1.5%
final concentration). Note that the agar does not have to
be supplemented with nutrients, as they are supposed to dif-
fuse into the chamber during in situ incubation. However,
when targeting specific microorganisms, their selection may
be enhanced by relevant supplements, for example, with
cellulose when species capable of cellulose degradation are
of interest.
The diffusion chamber accommodates approximately
3 ml of the cell/agar mix; in a typical experiment this
inoculum contains between lo4 and lo5 cells. Our ex-
perience with soil and aquatic environments shows that
such inocula usually produce a number of colonies that is
optimal for observations and subculturing. Once the 3-mI
inoculum is administered into the chamber by pipetting,
it fills the space formed by the bottom membrane and
the opening in the washer. We then seal the chamber by
B. Microbial Trap
- 0
/---- -_
0 03 pm membrane ,
0.03 pm
membrane
contaminants
gluing the second membrane on the upper surface of the
washer and wait for the glue to bond in a laminar flow
hood. It is critical to provide extra moisture to the cham-
ber during this drying period and we accomplish this by
placing the drying chambers on moist paper. This second
membrane sandwiches the microbe samplelagar mixture
within the chamber between two membranes, and pre-
pares the chamber for incubation.
The chamber can be incubated in two different ways.
The first is to simply return the chamber to the place of
the cells’ origin. This is convenient for environments that
are both close to the home laboratory and experience
little recreational use. Often this approach is impractical
because one or both of these conditions are not met. In
these cases, the chamber can be incubated in a simulated
natural environment, such as a block of freshly collected
aquatic sediment or soil kept in an aquarium at ambient
temperature. In our experience, this mimics the natural
environment well for 2 to 4 weeks after the sample col-
lection. As the sample ages, it exhibits signs of a shift to
a new community represented by few abundant species.
This results in an impoverished collection of species
 1. New Approaches to Microbial Isolation 5

grown inside the diffusion chamber, dominated by strains
of uncertain environmental relevance. We note that this
observation is strictly qualitative, and we have not exam-
ined the phenomenon in detail.
The length of incubation largely depends on the nature
of the target environment, and could be best determined
empirically. Applying the method to boreal aquatic envi-
ronments and soils, we typically incubate the chambers for
3 to 4 weeks if the ambient temperature is between 10 and
20°C, and for 2 to 3 weeks at higher temperatures. When
the method is applied to samples from human microbiome
or sewage treatment facilities, incubations under 1 week
appear sufficient to obtain microbial growth adequate for
observation and subculturing.
After incubation, the diffusion chambers are retrieved
and opened by cutting off the upper membrane with a ster-
ile razor blade run along the interior diameter of the washer.
Direct examination of the grown material, e.g., under a
dissecting microscope, is not an efficient way to quantify
growth, because most of the colonies formed during incuba-
tion are of microscopic size (Fig. 2A). In addition, the trans-
lucent nature of the bottom membrane interferes with such
observation. Instead, we subsample the agar material by
taking randomly distributed, measured cores using Pasteur
pipettes as coring devices. As a rule, cores 1 to 30 pl in vol-
ume, depending on the density of growth, are sufficient for
further examination and colony counting. Placed between
a glass slide and coverslip, the flattened cores are examined

B
50% -

40%

2
e,
3> 30%
f2
75
i3 20%

10%

February March June July August September

FIGURE 2 (A) Microcolonies of marine microorganisms grown inside a diffusion chamber incu-
bated in simulated natural environment. Bars, 5 pm. (B) Growth recovery of environmental cells
in diffusion chambers plotted as percentage of inoculated cells forming colonies.
6 ISOLATION AND SCREENING FOR SECONDARY METABOLITES
in their entirety at 400 to 1,OOOX magnification under a
compound microscope equipped for differential interference
contrast (DIC), the latter being crucial for colony visualiza-
tion. In a typical application, we record between 1 to 5 and
several hundred microcolonies per replicate. The counts are
then used to estimate the total number of colonies grown
in the chamber, and these estimates are compared with the
total number of cells inoculated to calculate percentage of
recovery.
Subsampling grown colonies for subculturing represents
one of the more time- and effort-consuming aspects of the
overall protocol. If the colonies are rare in the diffusion
chamber agar, they may be difficult to locate. However,
increasing their number by inoculating more cells leads
to a new difficulty: frequent failure to sample cells from
one-and only one-colony. The next round of growth
thus results in a mixed culture, necessitating additional
purification efforts. One recommended way to pick up colo-
nies from the diffusion chamber-grown material is to place
a sample of this material under a compound microscope,
verify the target colony under high magnification (e.g.,
using a dry 40X objective with a long working distance),
and then sample this colony using tungsten wires (75-pm
shaft, <1 pm at tip; FHC, Inc., Bowdoinham, ME). This
material is then syringe mixed in a small volume of sterile
water (seawater in marine application) and inoculated into
a new diffusion chamber. If this produces a mixed culture,
a series of chamber-to-chamber transfers may be necessary
to achieve purity. Purity is confirmed by a combination of
light microscopy at high magnification, scanning electron
microscopy, and analyses of PCR-amplified 16s rRNA gene
fragments.
1.2.3. Diffusion Chamber Applications
We have applied the method across a variety of different
environments to quantify microbial growth inside the
chambers, and assessed what part of grown species could be
domesticated (subcultured under conventional laboratory
conditions).
1.2.3.1. Marine Sediments Application
In this application, we grew microorganisms inhabiting a
sandy tidal flat in Massachusetts Bay, close to one of our
home laboratories at the Marine Science Station of North-
eastern University, Nahant, MA (21). Microorganisms
from the uppermost layer of these sediments were detached
from sediment grains by vortexing 1-g sediment samples
in 5 ml of autoclaved seawater for 5 min. The heaviest
sediment particles in the supernatant were allowed to settle,
and subsamples of the supernatant were mixed with 2.5 ml
of warm (40°C) agar supplemented with 0.01% technical
grade casein (Sigma-Aldrich, St. Louis, MO). The cells/
agar mix was inoculated into the diffusion chambers as de-
scribed above. Some chambers received agar but not cells;
these served as a contamination control. Inoculated growth
chambers were incubated in an aquarium containing freshly
collected intertidal sediments and recirculating natural sea-
water. After l to 3 weeks of incubation, the chambers were
opened and microbial colonies were examined, counted,
and subcultured as described above. This experiment was
repeated 13 times over a period of 7 months; its main
results are given in Fig. 2B. The number of cells forming
colonies in diffusion chambers averaged approximately
22%, exceeding hundredfold what could be achieved us-
ing standard petri dishes. This strongly indicated that the
diffusion chamber-based approach to microbial cultivation
likely provides access to some of the previously uncultivated
species. Indeed, in this and follow-up research (5, 24) we
isolated numerous species that did not grow in petri dishes
inoculated with environmental samples, but could be grown
and maintained in the diffusion chambers.
Cultivation of novel species inside diffusion chambers
is a welcome development, but their properties and abili-
ties could be fully studied and utilized only if they could
be adapted for growth in vitro. We therefore examined
whether repetitive cultivation in a series of generations of
diffusion chambers facilitated domestication of the grown
strains, and discovered a positive correlation between the
number of cultivation rounds in situ and the probability
of obtaining a variant capable of growth in vitro (24). Of
the 23 strains tested, approximately a quarter acquired the
ability to grow on standard media after just one round, and
this proportion steadily grew to 70% after four cultivation
rounds (24). The nature of the domestication process re-
mains unclear, but the empirical observations suggest that
rounds of in situ cultivation adapt a significant number of
otherwise “uncultivable” strains to growth under conven-
tional conditions of standard petri dishes. This important
observation was confirmed in follow-up studies conducted
in different environments (see below).
1.2.3.2. Freshwater Sediment Application
We applied the diffusion chamber-based method to fresh-
water sediments to cultivate microbial species from the
Turtle Pond, a small freshwater pond in Boston, MA (5).
Pond sediment material was diluted in agar supplemented
with Casamino Acids (0.01%), yeast extract (0.01%), and
hot water extract from the sediment (0.1%), in various
combinations, and inoculated into diffusion chambers.
These were incubated on top of a freshly collected block
of sediment kept under a layer of aerated extant water in
an aquarium. After 4 weeks of incubation, the chambers
were opened, the material was homogenized by passaging
the material through a syringe equipped with a gauge 25
needle and diluted with sterile, filtered pond water, and
part of the material was incubated in the next generation
of diffusion chambers. The remaining material was used
to prepare standard pour plates supplemented with diluted
( 10% of manufacturer recommended) Luria-Bertani broth
(LB; BD, Franklin Lakes, NJ). Reincubation in new dif-
fusion chambers was performed once again, to a total of
three rounds of in situ cultivation, each time with parallel
subculturing in standard petri dishes. In this way, we could
assess if new species would appear by repetitive incubations
of the chambers, and what species from thus grown diversity
could be grown in vitro. Indeed, 70% of the 438 strains iso-
lated came from the diffusion chamber-reared material, and
several strains unique to this approach only appeared after
two or more rounds of cultivation in situ; notably, these
represented rarely cultivated phyla Vemomicrobia and
Acidobacteria. Overlap between species composition grown
in standard petri dishes inoculated with pond material di-
rectly versus petri dishes inoculated with cells first grown
in diffusion chambers was minimal (7%). This confirmed
a similar observation made in a different environment (24)
that one or more rounds of growth inside diffusion cham-
bers leads to domestication of novel microbial species that
are difficult to grow otherwise.
1.2.3.3. Application to Contaminated Environments
We also used this method in contaminated soils and ground-
waters in several areas around the Field Research Center,

Oak Ridge National Laboratory, TN. These data are being
prepared for publication, but the principal results mirror
those from the above-mentioned studies of marine and fresh-
water environments: the diffusion chamber-based method
allows for growth and domestication of novel species from
contaminated soils and groundwater. It also brings into cul-
ture organisms previously known only from the rRNA gene
and metagenomics surveys of the sampled areas (data not
shown). This indicates that some microbial species, includ-
ing the likely bioremediation players, do not grow under
standard conditions unless first preincubated in the diffusion
chambers deployed in situ.
1.3. METHOD 2: ICHIP
1.3.1. Principles of the Method
In a typical application, the diffusion chamber described
above is inoculated with a mix of environmental cells, and
so the first growth event leads to a mixed culture. This ne-
cessitates additional purification and isolation efforts, which
are often substantial and limit the throughput. To resolve
this bottleneck, we developed a variant of the diffusion
chamber method that transforms it into a high-throughput
platform for massively parallel microbial isolation. While
based on the same principle (placing microorganisms into
diffusion chambers for incubation in vivo), it differs signifi-
cantly from the original chamber design described above.
The key element of this new method is the Isolation Chip,
or ichip for short (Fig. 3; Nichols et al., submitted), which is
a combination of hundreds of miniature diffusion chambers
loaded with an average of one cell per chamber. The ichip
thus represents a tool helping to achieve microbial growth
and isolation in a single step.
1.3.2. lchip Design
The ichip consists of several elements: the central plate,
the main function of which is to house growing microor-
ganisms; semipermeable membranes on each side of the
plate, which separate the plate from the environment; and
two side panels, playing mainly a structural role (Fig. 3).
The central plate and side panels have multiple match-
ing through holes. When the central plate is dipped into
a suspension of cells in molten agar (Fig. 3A), its through
holes capture miniature volumes of this suspension that
A B
r’
i
i
\
FIGURE 3 Ichip design. Explanations in the text.
1. New Approaches to Microbial Isolation 7
solidify in the form of small agar plugs. These plugs are
sealed from both each other and the outside environment
by membranes pressed against the central plate by the side
panels, fastened with screws (Fig. 3C). The seal transforms
the assembly into an array of small diffusion chambers, each
filled with agar and cells. The number of cells inside each
through hole is the function of the initial dilution rate in
molten agar, and thus can be controlled. When an average
agar plug contains a single cell, the majority of through
holes will contain individual cells (Fig. 3B). The assembled
ichip is incubated in nature (or simulated natural environ-
ment) similarly to the diffusion chambers of the original de-
sign (Fig. 1A).The growth of individual cells automatically
leads to pure cultures inside the through holes, minimizing
isolation efforts.
The ichips currently in use in our laboratories were
manufactured by HI-TECH Manufacturing (Schiller Park,
IL). The plastic elements are machined from blocks of hy-
drophobic plastic polyoxymethylene, known as DelrinB. All
the through holes are uniform in their diameter (1 mm), and
are arranged in two arrays with 192 through holes per array.
The size of the array is such that it can be completely cov-
ered by standard 25- or 47-mm-diameter membranes. It is
also possible to use commercially available sheet membranes
that could be cut to the appropriate size in advance.
Prior to assembly, we sterilize the plastic components
in ethanol, dry them in a laminar flow hood, and rinse
the parts in particle-free DNA grade water (Fisher Sci-
entific, Hampton, NH). After incubation, ichips are
washed vigorously in particle-free DNA grade water, and
disassembled. The central plate is then examined under
a compound or high-power dissecting microscope for
colony count. The colonies are removed from the ichip’s
central plate by touching agar plugs with unwound and
sterile #1 gauge paper clips for further analyses and/or
subculturing (e.g., in microtiter plates).
In a series of preliminary tests, we checked whether the
pressure applied by tightening the screws was sufficient to
prevent cells from migrating in and out of the agar plugs.
Ichips were loaded with sterile 1% agar (BD, Franklin
Lakes, NJ), assembled, submerged into Escherichia coli
K-12 culture growing in LB, and incubated for 24 h.
After incubation, the content of ichips was examined for
growth under a compound microscope. In parallel, ichips
loaded with E. coli K-12 cells mixed with 1% warm LB
8 ISOLATION AND SCREENING FOR SECONDARY METABOLITES
agar were incubated for 24 h while bathed in sterile LB.
The outside medium was then examined for growth. In
neither case were microbial cells observed crossing the
barrier established by the membranes (Nichols et al.,
submitted).
1.3.3. lchip Applications
In a proof-of-concept study, we used the ichip to grow
marine water column and soil microorganisms, recorded
the colony count, and compared the rRNA gene diversity
of ichip-grown microorganisms and their colony count
with microorganisms from parallel incubations in stan-
dard petri dishes (Nichols et al., submitted). Seawater
and waterlogged soil samples were obtained from the
respective environments in the vicinity of the Marine
Science Center of Northeastern University, Nahant, MA.
Microbial cells were dislodged from soil particles by two
10-s-long sonication pulses (Sonics Vibra-Cell VC130; 3-
mm stepped microtip; Sonics & Materials, Inc., Newtown,
CT) and collected after larger particles settled for 60 s.
Seawater samples were used without sonication. All cells
were enumerated with 4’,6-diamidino-2-phenylindole novelty; it does so in a time- and effort-efficient manner,
(DAPI) (Sigma-Aldrich, St. Louis, MO) and diluted in
warm diluted (0.1% wtlvol) LB agar to a concentration
of lo3 cellslml, with or without supplementation with 4%
sea salts (Sigma-Aldrich, St. Louis, MO). In the present
ichip configuration (Fig. 3), each through hole captures
approximately 1.25 p1 of agar, or 500 p1 of cell/agar mix
per assembly. Given the dilution, each loaded through
hole contained 500/384 = 1.25 cells (on average). To ap-
proximate the species diversity inoculated into ichips, for of through holes with wells of microtiter plates paired with
conventional cultivation we established miniature petri
dishes with 500 p1 of the cell/agar mixes in 24-well culture match the wells will allow massively parallel growth and iso-
plates (Corning Costar, Corning, NY). The ichips were
returned to the respective environments of cells within,
70
60
1
50
3
I-
?0
40
5
a 30
z
5
20
10
0
97-1 00 94-97
Known New
species species
FIGURE 4 Novelty of seawater and soil microbial strains grown in ichip and petri dish. Equation
of sequence novelty, in percentage of diversion from the known species, and taxonomic rank of
novelty (genus, family level, etc.) is approximate. O W , operational taxonomic unit.
incubated for 2 weeks, and disassembled. Approximately
50 agar plugs from each ichip were removed for a detailed
microscopic examination and colony counting, whereas
the rest served as a source of DNA for 16s rRNA gene
sequence-based identification of grown species. Agar ma-
terial from the conventional petri dishes was processed in
an identical manner.
This study led to three important observations. First, 40
to 50% of cells incubated in ichips formed colonies, which
exceeded the petri dish recovery fivefold. Second, the ichip-
and petri dish-grown collections of microorganisms were
dramatically different and shared only one species (Vibriosp.).
This lack of overlap between the two species lists was not due
to undersampling, because petri dishes inoculated with sea-
water and soil samples did share several species, as did ichips
inoculated with microorganisms from these two sources.
Third, the novelty of isolates grown in ichips significantly
exceeded that of petri dish-reared species (Fig. 4). Our
general conclusion is that the ichip-based method enables
growth of a significant fraction of microbial diversity, and
produces unique microbial collections of high phylogenetic
compatible with contemporary tools for high-throughput
analyses, such as microtiter plates.
We note that the flexibility of the ichip design makes it
useful for a variety of applications beyond those described
here. For example, a single incubation of the device cur-
rently in use (Fig. 3) affords tens of microbial cultures,
and cultivation efforts can be easily scaled up via minimal
changes in configuration of the ichip. Matching the position
commercially available replicators that have pins spaced to
lation of hundreds of cultures per incubation, with minimal
labor involved. Further modifications of the ichip may make
Seawater; ichip
Seawater; Petri dish
Soil; ichip
Soil; Petri dish
9 1-94 <91
New New
families orders and above
 1. New Approaches to Microbial Isolation 9

it suitable to grow previously uncultivated microorganisms, 1.4. METHOD 3: MICROBIAL TRAP

not only from natural environments, but also from the hu-
man microbiome. For example, miniaturization of the ichip
design opens a possibility of its incubation in the oral cavity.
In this application, microorganisms from a dental plaque
can be loaded into a mini-ichip (Fig. 5A and B), which is
then incubated on a retainer inside the volunteer mouth
(Fig. 5C).This is important because >50% of oral microflora
has not been cultivated yet, and the uncultivated fraction
contains species implicated in oral diseases (1, 23). Equally
simple modifications can make the ichip useful to grow
microorganisms from other parts of the human microbiome.
1.4.1. Principles of the Method
Several types of microorganisms that are particularly im-
portant for drug discovery, microscopic fungi and actinomy-
cetes, grow by forming filaments capable of penetrating soft
substrates. Considering that actinomycetes can pass through
filters with pores as small as 0.2 km, we hypothesized that
the reverse use of the diffusion chamber (Fig. 1A) could se-
lectively enrich for these microorganisms by capitalizing on
their unique abilities (trap method, Fig. 1B [15]). Visually,
the trap is indistinguishable from the diffusion chamber, but

FIGURE 5 Prospective ichip to grow oral microorganisms. (A) The assembly of the device is
similar to the ichip currently in use (Fig. 3): the central plate is loaded with target (dental plaque)
microorganisms; two additional panels with matching through holes press the 0.03-pm-pore-size
membranes against the central plate, creating multiple diffusion minichambers. (B) Relative size of
the prospective ichip for oral microflora cultivation. (C) Assembled ichip is fastened in the opening
of a mold of the volunteer’s upper palate, to be incubated in the mouth in touch with the molar
used to sample the plaque.
10 ISOLATION AND SCREENING FOR SECONDARY METABOLITES
the main principle of its application is very different. The
trap is built from similar components except the membranes
have larger pores, and it is not inoculated with environ-
mental cells (contrast Fig. I A and B). Instead, the trap is
incubated in the environment with sterile agar inside, with
the expectation that diffusion through membranes will
establish conditions inside the trap that closely mimic the
natural conditions. The pore size above 0.2 pm should allow
filamentous organisms active at the time of the experiment
to penetrate the membranes, grow into the agar inside the
trap, and establish colonies there. Though selected motile
microorganisms could in principle cross the membranes
and grow inside the trap as well, we hypothesized that the
method should enrich for target (filamentous) organisms.
These could be further purified by subculturing in diffusion
chamber or petri dishes. The test study described below sup-
ported these expectations.
1.4.2. Trap Design
Traps are built using 0.2- to 0.6-pm-pore-size polycarbonate
membranes, glued to the bottom of washers as in the diffu-
sion chamber method. The trap is filled with 3 ml of sterile
1% agar or 1.2% gellan gum, with or without vitamin sup-
plement. After the medium solidifies, the top polycarbonate
membrane, with a 0.03-pm pore size, is glued to the washer,
sealing the trap. After the glue dries, the traps are placed on
top of the moist soil or marine sediment, ensuring that the
bottom filter (with larger pores) is in good contact with the
substrate. The small pore size of the upper membranes pre-
vents contamination from the air. In aquatic application,
the two membranes can be the same, with 0.2- to 0.6-pm
pore sizes, allowing penetration by filamentous organisms
from both sides.
1.4.3. Trap Application
We tested the trap performance (15) by incubating these
devices on top of garden soil kept in large petri dishes,
sealed with Parafilm to prevent evaporation. After 14 to
21 days of incubation at room temperature in the dark,
the traps were opened and the slabs of solid agar or gellan
a b
FIGURE 6 Bacterial colonies and fungal hyphae (a) and actinomycetes-like colonies (b; scale,
0.1 mm) grown in a trap after 2 weeks of incubation in garden soil.
gum were removed, inverted, and placed into a sterile
petri dish. The solid disks were examined for growth un-
der a stereomicroscope at 20 to 1OOX magnification, and
visible microcolonies were sampled with sterile needles.
In several experiments, the agar slabs were incubated un-
disturbed for 5 to 7 days prior to colony sampling to allow
actinomycetes to form aerial mycelia to facilitate colony
picking. All sampled material was streaked on plates with
agar or gellan gum medium supplemented with 0.1% Casa-
mino Acids and 0.1% nutrient broth (BD Difco, Detroit,
MI), or Actinomycete Isolation Agar (BD Difco, Detroit,
MI). Because the experiment aimed to isolate actinomy-
cetes, nystatin (50 pg/ml) and cycloheximide (100 pg/ml)
were added to the media in petri dishes to prevent growth
of fungi. Subcultivation was repeated to obtain pure cul-
tures. For comparative purposes, we also prepared parallel
conventional petri dishes by inoculating them with the
air-dried soil material directly, followed by 2-week-long
incubation in the laboratory. Similarly to subculturing
trap-derived material, conventional petri dishes were es-
tablished with agar or gellan gum and were supplemented
with the same nutrient additives. Upon incubation and
purification, 90 colonies from traps and 90 colonies from
conventional petri dishes were identified by sequencing
their 16s rRNA genes.
Visual inspection of traps revealed an abundance of
actinobacterial colonies (Fig. 6). Traps with 0.4- to 0.6-pm-
pore-size bottom membranes contained a significant
number of fungal filaments, but cutting the pore size
to 0.2 pm effectively excluded fungi. The majority of
organisms grown in the traps proved to be actinomy-
cetes, some of which represented rare and unusual spe-
cies from the genera Dactilosporangium, Catellatospora,
Catenulispora, Lentzea, and Streptacidiphilus. In general,
gellan gum-based traps appeared to have captured more
diverse assemblage of actinomycetes; conventional petri
dishes produced more nonfilamentous actinobacteria.
We concluded that the trap method allows for a selective
capture of filamentous actinomycetes enriched for new
and rare species.

1.5. CONCLUSIONS
There is a growing appreciation of microbial cultivation
because microbial culture remains central not only to
the study and utilization of microbial properties and ca-
pabilities, but also to the placement of molecular surveys
and metagenomics data into a larger context. With the
majority of microbial species remaining uncultivated, there
is an urgent need to develop novel methodologies to grow
previously uncultivated species from the environment and
human and animal microbiomes. One way to gain access
to “missing” species is to grow environmental cells in their
natural habitat, capitalizing on a naturally occurring suite
of growth factors. This can be accomplished by placing
target cells in diffusion chambers incubated back in the
cells’ habitat. We developed several variants of such dif-
fusion chambers, each with its own set of advantages. The
diffusion chamber method is a simple and inexpensive tool
to grow mixes of novel environmental microorganisms. The
ichip design allows for massively parallel, high-throughput
isolation of novel microorganisms that may be difficult or
impossible to grow by use of conventional techniques. Min-
iaturization of the ichip makes it suitable to grow presently
uncultivated microorganisms from human microbiome. A
reverse use of the diffusion chamber leads to a different type
of cultivation (trap method), which selectively enriches for
new and rare filamentous actinomycetes. This makes the
trap method particularly useful for the discovery of novel
secondary metabolites.
REFERENCES
1. Aas, J. A,, B. J. Paster, L. N. Stokes, I. Olsen, and F. E.
Dewhirst. 2005. Defining the normal bacterial flora of the
oral cavity. J. Clin. Microbiol. 43:5721-5732.
2. Amann, J. 1911. Die direkte Zahlung der Wasserbak-
terien mittels des Ultramikroskops. Zentmlbl. Bakteriol.
29:381-384.
3. Aoi, Y., T. Kinoshita, T. Hata, H. Ohta, H. Obokata,
and S. Tsuneda. 2009. Hollow-fiber membrane chamber
as a device for in situ environmental cultivation. Appl.
Environ. Microbiol. 75 :3826-3833.
4. Beja, O., L. Aravind, E. V. Koonin, M. T. Suzuki, A.
Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates,
R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F.
DeLong. 2000. Bacterial rhodopsin: evidence for a new
type of phototrophy in the sea. Science 289:1902-1906.
5. Bollmann, A., K. Lewis, and S. S. Epstein. 2007. Incu-
bation of environmental samples in a diffusion chamber
increases the diversity of recovered isolates. Appl. Environ.
Microbiol. 73:6386-6390.
6. Bruns, A., H. Cypionka, and J. Overmann. 2002. Cyclic
AMP and acyl homoserine lactones increase the cultiva-
tion efficiency of heterotrophic bacteria from the central
Baltic Sea. Appl. Enoiron. Microbiol. 68:3978-3987.
7. Cholodny, N. 1929. Zur Methodik der quantitativen Er-
forschung des bakteriellen Planktons. Zencralbl. Bakteriol.
Parasitenid. Infektionskr. Hyg. A 77:179-193.
8. Connon, S. A., and S. J. Giovannoni. 2002. High-
throughput methods for culturing microorganisms in
very-low-nutrient media yield diverse new marine isolates.
Appl. Environ. Microbiol. 68:3 878-3 885.
9. Davis, K. E., S. J. Joseph, and P. H. Janssen. 2005. Ef-
fects of growth medium, inoculum size, and incubation
time on culturability and isolation of soil bacteria. Appl.
Environ. Microbiol. 7 1:826-834.
10. DeLong, E. F. 1992. Archaea in coastal marine environ-
ments. Proc. Natl. Acud. Sci. USA 895685-5689.
1. New Approaches to Microbial isolation 11
11. DeLong, E. F. 2005. Microbial community genomics in
the ocean. Nut. Rev. Microbiol. 3:459469.
12. Dojka, M. A,, J. K. Harris, and N. R. Pace. 2000. Ex-
panding the known diversity and environmental distribu-
tion of an uncultured phylogenetic division of bacteria.
Appl. Enuiron. Microbiol. 66:1617-1621.
13. Ferrari, B. C., S. J. Binnerup, and M. Gillings. 2005.
Microcolony cultivation on a soil substrate membrane
system selects for previously uncultured soil bacteria. Appl.
Enwiron. Microbiol. 71:8714-8720.
14. Fuhrman, J. A., K. McCallum, and A. A. Davis. 1992.
Novel major archaebacterial group from marine plankton.
Nature 356:148-149.
15. Gavrish, E., A. Bollmann, S. Epstein, and K. Lewis.
2008. A trap for in situ cultivation of filamentous actino-
bacteria. 1.Microbiol. Methods 72:257-262.
16. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and
K. G . Field. 1990. Genetic diversity in Sargasso Sea
bacterioplankton. Nature 345:60-63.
17. Handelsman, J. 2004. Metagenomics: application of
genomics to uncultured microorganisms. Microbiol. Mol.
Bid. Rew. 68:669-685.
18. Huber, J. A., D. B. Mark Welch, H. G. Morrison, S. M.
Huse, P. R. Neal, D. A. Butterfield, and M. L. Sogin.
2007. Microbial population structures in the deep marine
biosphere. Science 3 18:97-100.
19. Hurst, C. J. 2005. Divining the future of microbiology.
ASM News 71:262-263.
20. Jannasch, H. W., and G. E. Jones. 1959. Bacterial popu-
lations in seawater as determined by different methods of
enumeration. Limnol. Oceanogr. 4: 128-139.
21. Kaeberlein, T., K. Lewis, and S. S. Epstein. 2002. Isolat-
ing “uncultivable” microorganisms in pure culture in a
simulated natural environment. Science 296: 1127-1 129.
22. Liesack, W., and E. Stackebrandt. 1992. Occurrence of
nove! groups of the domain Bacteria as revealed by analysis
of genetic material isolated from an Australian terrestrial
environment. J . Bacteriol. 174: 5072-5078.
23. Marsh, P. D. 2003. Are dental diseases examples of eco-
logical catastrophes?Microbiology 149:279-294.
24. Nichols, D., K. Lewis, J. Orjala, S. Mo, R. Ortenberg,
P. O’Connor, C. Zhao, P. Vouros, T. Kaeberlein, and S.
S. Epstein. 2008. Short peptide induces an “uncultivable”
microorganism to grow in vitro. Appl. Environ. Microbiol.
74:48894897.
25. Olsen, G. J., D. J. Lane, S. J. Giovannoni, N. R. Pace, and
D. A. Stahl. 1986. Microbial ecology and evolution: a ribo-
somal RNA approach. Annu. Rev. Microbiol. 40:337-365.
26. Rappe, M. S., S. A. Connon, K. L. Vergin, and S. J.
Giovannoni. 2002. Cultivation of the ubiquitous SARll
marine bacterioplankton clade. Nature 418:630-633.
27. Rappe, M. S., and S. J. Giovannoni. 2003. The uncultured
microbial majority. Annu. Rev. Microbiol. 57:369-394.
28. Schloss, P. D., and J. Handelsman. 2004. Status of the
microbial census. Microbiol. Mol. Biol. Rev. 68:686-691.
29. Sogin, M. L., H. G. Morrison, J. A. Huber, D. Mark
Welch, S. M. Huse, .?I R. Neal, J. M. Arrieta, and G.
J. Herndl. 2006. Microbial diversity in the deep sea and
the underexplored “rare biosphere.” Proc. Natl. Acud. Sci.
USA 103:12115-1 2 120.
30. Staley, J. T., and A. Konopka. 1985. Measurement of in situ
activities of nonphotosynthetic microorganisms in aquatic
and terrestrial habitats. Annu. Rev. Microbiol. 39:321-346.
31. Stevenson, B. S., S. A. Eichorst, J. T. Wertz, T. M.
Schmidt, and J. A. Breznak. 2004. New strategies for cul-
tivation and detection of previously uncultured microbes.
Appl. Enwiron. Microbiol. 704748-4755.
32. Waksman, S. A., and M. Hotchkiss. 1937. Viability of
bacteria in sea water. J. Bacteriol. 33:389-400.
12 1 ISOLATION A N D SCREENING FOR SECONDARY METABOLITES
33. Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16s
rRNA sequences reveal numerous uncultured microorgan-
isms in a natural community. Nature 345:63-65.
34. Wilson, G. S. 1922. The proportion of viable bacteria in
young cultures with especial reference to the technique
employed in counting. J. Bacteriol. 7405-446.
35. Window, C.-E. A., and G. E. Willcomb. 1905. Tests of a
method for the direct microscopic enumeration of bacte-
ria. J. Infect. Dis.Suppl. 1:273-283.
36. Winterberg, H. 1898. Zur Methodik der Bakterienzah-
lung. Zeitschr. Hyg. 29:75-93.
37. Young, P. 1997. Major microbial diversity initiative rec-
ommended. ASM News 63:417421.
38. Zengler, K., G. Toledo, M. Rappe, J. Elkins, E. J. Mathur,
J. M. Short, and M. Keller. 2002. Cultivating the uncul-
tured. Proc. Natl. Acad. Sci. USA 99:15681-15686.
39. ZoBell, C. E. 1946. Marine Microbiology: a Monograph on
Hydrobacteriology. Chronica Botanica Co., Waltham, MA.