United States          Commission Of The     United States Department Of
                 Environmental Protection     European Communities    Agriculture, Animal And Plant
                 Agency                           Health Inspection Service
                 Prcvsnlion. Pesticides ftnd Toxic Substances (TS-788)  EPA703-R-92-001     October 1992
\S-EPA         Methods For The Detection
                 Of Microorganisms
                 In The Environment
                                                   Printed on Recycled Paper

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                          Developed by

           The Commission of the European communities

                The US Department of Agriculture
           Animal and Plant Health Inspection Service

             The US Environmental Protection Agency
            Office of Pesticides and Toxic Substances
                 as part of the EC/US Cooperation
             in the Permanent Technical Working Group
              on Biotechnology and the Environment

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,1.    PREFACE                                            3

 2.    INTRODUCTION                                       4

 3.    OVERVIEW                                            6
      Introduction                                       6
      Direct Detection  Techniques                         6
      Detection  by Culturing  of  the Microorganism         8
      Detection  by Nucleic Acid  Hybridization            10
        General  Considerations                           10
        Nucleic  Acid Hybridization Techniques            13

      OF MICROORGANISMS                                 16

 5.    ANALYSIS OF  TECHNIQUES                             18
      Direct Detection  Techniques                        18
          Microscopy - detection of viable
          microorganisms                                18
          Microscopy - detection of total viable
          and nonviable microorganisms.                 20
          Microscopy - fluorescent antibody             22
          Summary of direct  detection techniques        23

      Detection  Techniques that  Require Culturing of the
      Microorganism                                     24
          Selective media                               24
          Differential media                           30
          Most  probable number  technique                31
          Fluorescent  antibody  - colony ELISA blots     33
          Summary of cell culture techniques            35

      Techniques to Label and Detect Nucleic Acids       39
          Nucleic acid probe construction               39
          Hybridization techniques                      47
          Probe visualization                           67
          Use of  nucleic acid hybridization
          in analyzing microbial species composition    68

 6.    REFERENCES                                        70

 7.    TABLES                                             94

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This document has been jointly developed by the Commission of the
European Communities, the Office of Pesticides and Toxic
Substances of the US Environmental Protection Agency, and the
Animal and Plant Health Inspection Service of the US Department
of Agriculture, as part of the EC/US Cooperation in the Permanent
Technical Working Group on Biotechnology and the Environment
(TWGBE).  The TWGBE was established in the framework of the EC/US
Bilateral Environmental Consultations by agreement in a plenary
session of the EC/US Bilateral Environmental Consultations in
Brussels in 1990.  The TWGBE was established with the aim of
promoting closer bilateral cooperation on technical issues
associated with evaluating and validating data to assess the
environmental risk of releases of genetically modified organisms.

The document presents an overview and summary of methods
currently in use for detecting and monitoring microorganisms in
the environment.  It is intended to be used as a reference
document for regulatory officials and scientists.  Given the
rapid developments in this area the guide may have to be updated
periodically, however an attempt was made to provide information
that was current at the time of publication.


The choice of a detection technique(s) depends greatly on the the
purpose of the intended use of the microorganism, whether there
is a need for quantitative versus qualitative data, and whether
the microorganism itself or its genetic material is to be
detected.  Thus, the purpose of a particular field test may
include assessing the efficacy of a microbial product under
commercial development, assessing safety to human health and the
environment, or to gain information as part of basic research in
microbial ecology.  In addition, the monitoring techniques
described in this document have been used for developing both
qualitative and quantitative data, as appropriate for the
purposes of the study.  Finally, studies on microbial survival, -
dissemination, and beneficial or adverse impacts in the
environment have relied classically on the detection of specific
microorganisms, but more recently methods that allow the direct
detection of nucleic acid have been developed.  These include
studies on the transfer of genetic material to other
microorganisms in the environment usually involve some method
detection of specific gene sequences.  This document attempts to
provide a source of information and references about the
expanding variety of detection techniques and their uses.

The document is divided into two principal sections and a brief
synopsis of statistical considerations in environmental
microbiology.  The principal sections are a general overview and
an analysis of individual techniques.  The Overview section is
intended to provide readers with a general interest in the topic
of microbial detection methods with a survey and discussion of
the variety of techniques used in the study of microorganisms in
the environment.

The Analysis of Techniques section provides a more detailed
examination of individual detection methods.  The discussion
proceeds from an analysis of direct detection techniques to
culture based identification and finally to identification of
specific nucleic acid sequences.

The individual analyses are designed to provide information in
five general categories: (1) specificity, (2)  sensitivity, (3)
ease of use and time necessary to execute the technique, (4)  cost
and (5) constraints on use in different environmental media.   The
five evaluation criteria may be defined as follows:

1. Specificity:  The ability of the technique to distinguish
   between the target microbe and other microbes.  Specificity
   could also include whether the technique distinguishes
   viable and non-viable organisms.

2. Sensitivity:  The minimum detection limit, e.g.,  (1) the
   number of cells needed for detection or (2) mass units of
   nucleic acid target sequence required for detection.

3. Ease of use & time necessary:  Self-explanatory, techniques
   are compared for their relative ease of use.

4. Cost:  Self-explanatory

5. Effect of environmental media:  The degree to which a given
   technique is able  to detect a microorganism when used in
   different environmental media, e.g., soil, water, plant
   tissue, air, or sediment.


The variety of techniques available to researchers in microbial
ecology has expanded greatly in recent years.  In addition to
some of the classic techniques such as direct detection
techniques and culture-based identification, the researcher now
has molecular techniques based on the identification of specific
nucleic acid sequences.  The expansion in the number of
techniques is due in part to an increased interest in the
commercial use of microorganisms in the environment for
applications such as biopesticides, symbiotic nitrogen fixation,
bioremediation, animal vaccines, feed additives for livestock and
other uses.  A further impetus has come from research in both
basic and applied microbial ecology.  As a result, the relatively
recent use of molecular techniques in the detection of
microorganisms in the environment offers the potential for
increased sensitivity.  However, the more traditional methods
such as dilution plating are still the most commonly used and
provide the simplest (and in some cases still the most sensitive)
means of enumerating a microorganism in the environment, provided
it is in a culturable state.

These advances in methodology have increased our understanding of
the manner in which microorganisms interact with their
physical/ chemical environment, and with other organisms, and the
factors that affect their environmental fate and impacts (either
beneficial or adverse) .  A concise summary of the variety of
techniques available for use in environmental microbiology is
given below.

Direct Detection Techniques

The section on direct detection techniques covers the following:
microscopy, flow cytometry and antibody capture.  Direct
detection allows the enumeration of microorganisms without
culturing the sample.  These methods do not discriminate per se
between viable and dead cells.  However, direct viable counts
(DVC) may be determined by incubating the sample with nalidixic
acid prior to counting.  Such techniques yield consistently
higher counts than those obtained with classical methods (plate
counts, most probable number, etc.) which can be hampered by the
problem of viable but nonculturable microorganisms or inefficient
recovery of the desired microorganism.

With microscopy, the sample is visualized by staining with
fluorescent dyes such as 4 ' 6-diamidino-2-phenylindole (DAPI) or
acridine orange.  Cells can then be detected using
epifluorescence microscopy.  Microscopic techniques can also be

 combined with hybridization techniques using oligonucleotide
 probes tagged with dyes  such  as  fluorescein isothiocyanate  (FITC)
 and tetrazolium isothiocyanate  (TZTC)   (Belong ej: al.,  1989,
 Amann et al., 1990a).  For example,  16S/23S oligonucleotide
 probes  (15 to 25 bases in length for jjj situ use) can be used to
 differentiate organisms  at the species level.  Antibodies raised
 against strains of microorganisms may also be used to determine
 direct counts for a  specific  subpopulation.   It is also possible
 to detect microorganisms in the  environment by means of
 antibodies using the enzyme-linked immunosorbent assay  (ELISA)
 (Morgan et al..  1989).   To eliminate background signals from
 indigenous bacteria, the antibody/oligonucleotide probe must be
 specific for the target  organism.    Environmental samples  may
 exhibit nonspecific  binding of antibodies and oligonucleotide
 probes due to the presence of phenolic compounds and organic
 matter particularly  in soils, sediments, and plant tissue.  Such
 interference can be  reduced by counterstaining with gelatin-
 rhodamine conjugants.  For these reasons, detection by  microscopy
 has been more frequently and  successfully used for microorganisms
 in aquatic environments.

 Oligonucleotide probes (Amann et al., 1990b) or antibodies
 (Saunders et al., 1990)  can be used  for enumeration of  the
 microorganisms  with  flow cytometry/fluorescence-activated cell
 sorting which allows rapid identification, sorting, and counting
 of specific microorganisms.   This technique has not been used
 extensively in  environmental  microbiology; however, it  has
 promise for future use.  Flow cytometry can be directly employed
 with aquatic samples.  Use with  soil samples would require
 extensive treatment  to remove particulates that would interfere
 with accurate enumeration of  microorganisms.

 Techniques using antibody capture require raising a monoclonal
 antibody to the  microorganism of  interest.  These antibodies can
 then be secured  to a magnetized polystyrene bead and introduced
 into the environmental sample.  Any organisms which express the
 appropriate antigen will then bind to the beads, and be retrieved
with a magnet (Saunders  et aJL./  1990).  Problems associated with
this method include nonspecific binding of the antibody and
 (potentially) low recovery of target organism, particularly in
environments with organic matter  such as soil.  These methods
should work well with aquatic samples, and find greater use in
the future.

          bv Cuno of the
Detection techniques that require culturing microorganisms
rely on the ability of the target organism to grow on or in a
suitable growth medium.  Not all microorganisms isolated from the
environment will be culturable and thus detectable by these
methods.  In addition, the accuracy of these methods (especially
dilution plate counts) may be affected by cell clumping and
inaccuracies in preparing dilutions of the sample.  Since these
methods require the growth of target microorganisms, they are
susceptible to microbial contaminants that interfere with the
detection of the desired organism, especially in environments
such as soil which contain relatively high numbers of indigenous
microorganisms.  Sensitivity can be improved by the use of
biomass concentration techniques (Herron and Wellington, 1990) .
These methods have been used in conjunction with direct counting
for the detection of nonculturable cells.

Methods for detecting specific phenotypes within a complex
community of heterogenous bacteria, such as soil, are summarized
in Tables 1 and 2.  Such methods were first developed in sterile
soil (Table 1) and many have been used for the detection of
survival and gene transfer events in nonsterile soil (Table 2) .
These tables serve to illustrate the continued predominance of
the dilution plate count technique for detection, monitoring, and
enumeration of bacteria in natural environments.  Many studies on
the use of dilution plate count techniques have been published in
the literature on gene transfer in bacteria, and Table 3 provides
a summary of these references.

Culture techniques that use selective media rely on the ability
of the selective agent to either kill or prevent the growth of
competitive microorganisms, usually on solid growth media, thus
providing relatively good sensitivity in detection.  Commonly,
the gene that encodes the trait (s) that enable the bacterium to
resist the toxic agent is introduced through genetic engineering.
Selective agents used. in media, and discussed below, include
antibiotics, heavy metals, and unique carbon sources.

(1) Resistance to Antibiotics

Markers conferring on antibiotic resistance can either be
transferred into a host from another organism or obtained by
selection.  One concern is the transfer of antibiotic resistance
determinants In situ, to pathogenic microorganisms or the
indigenous microbial population.  Efforts have been made to avoid
the use of resistance markers for clinically significant

 (2)  Heavy Metal Markers (Bale e£ al. ,  1987,  Hdfte ei al- ,  1990
 citation for Warwick meeting, November,  1990)

 The  use of heavy metal markers is basically  similar to that of
 antibiotic resistance, although potential  risks concerning
 transfer to pathogens are not as great.  However, resistance to
 heavy metals and antibiotics can often reside  on the same
 plasmid.   Theoretically heavy metal resistances could be used to
 detect microorganisms in polluted environments provided that the
 natural resistance of the indigenous population does not result
 in high background levels.

 (3)  Rare Substrates

 The  ability to grow on rare  substrates such  as pollutants can be
 found in some microorganisms, and can  be used  both  to study the
 degradative capacity of the  host organism  in the environment and
 to prevent the growth of indigenous microflora (counterselection)
 in selective media (Dwyer et al. .  1988).

 (4)  Unique Environmental Conditions

 Harsh environmental conditions,  such as  high temperature,  extreme
 pH,  or high osmotic pressure,  can be used  to select for
 microorganism that are adapted to these  conditions.  In addition
 the  ability to survive environmental extremes  can be selected in
 the  laboratory.

 Differential media exploit the unique  metabolic capacities of
 individual  species of microorganisms.  Recently, genetic markers
 have, been introduced into microorganisms in  order to provide a
 differential phenotype.   A marker (s) is chosen for  its  ability to
 confer on the host organism  a capacity to  stand out from the
 indigenous  population on isolation plates.   Some examples  of
markers used are the laeZY genes  from  Escherichjla coli. lux
 cassette  from Vibrio f iseheri .  and xvlE from Pseudomonas put Ida.
The  laeZY marker from Eseherichia  coli confers  the  ability to
metabolize  of the  chromogenic galactose derivative,  X-gal.  The
 lux  cassette encodes the enzyme,  lucif erase.  When  isolation
plates are  sprayed with an aldehyde substrate,  light is emitted
 from colonies carrying this marker (Shaw and Kado,  1986) .   The
 PROSAMO initiative (Planned Release of Selected and  Manipulated
Organisms,  Rees et ^3,. ,  1990)  is using the luxAB genes  for
monitoring  and detection in soil.  The xvlE - gene codes  for
catechol  2,3-dioxygenase and  is derived from the Pseudomonad TOL
plasmid.  Colonies  carrying this marker produce yellow  pigment on
isolation plates sprayed with catechol  (Morgan  e£ al. ,  1989).
False positives can be reduced and sensitivity optimized by using
multiple types of markers e.g., antibiotic resistance markers,
metabolic markers such as XylE. and nonexpressed markers such as

nucleotide sequences which  are targets  for nucleic  acid probe

Detection by Nucleic Acid Hybridigation

1)  General Considerations

Classical identification procedures depend upon the detection of
metabolic products, enzymatic activities, specific  cell
constituents or antigenic structures.   Nucleic acid hybridization
on the other hand  is based  on the detection of specific gene
sequences.  Hybridization is an important tool for  distinguishing
between closely related bacteria.  Once the gene coding for the
differentiating trait has been isolated, the gene can be detected
by hybridization with specific DNA probes.  With careful probe
selection, virulent strains can often be distinguished from
avirulent ones and genetically marked strains can be
distinguished from nearly isogenic strains.

The sensitivity and specificity in a given hybridization reaction
depend on probe construction and labeling, the target gene
sequence, and detection methodology.  These methods can be used
to either qualitatively or  quantitatively study microbial
populations in the environment.  However, the difficulty in
extracting sufficiently pure nucleic acids from an  environmental
sample for use in  hybidization studies  is often a problem.
Extraction of hybridizable  DNA from the environment can be
carried out by two general  procedures,  either by direct
extraction of soil (Ogram et al. . 1988; Steffan et  al. , 1988)  or
aquatic (Somerville et al. , 1989) samples or by using an indirect
method in which the bacteria are first  separated from non-
cellular material  and then  the nucleic  acids are extracted
(Holben e£ al. , 1988).
Examples of direct ONA extraction are provided in Table 4.
Direct extraction does not allow distinction between cellular and
free DNA or between bacterial, fungal, or other classes of
organisms.  The indirect method may be also prone to inaccuracies
since only approximately 30% of soil bacteria are recovered
(Holben fit al. . 1988) and thus, the extract may not represent a
truly statistical sample of the overall community, although the
recovery efficiency of this technique has been improved.

with both methods, it is necessary to confirm complete lysis of
the microbial population.  The most important considerations
governing nucleic acid yield and purity are lysis of bacterial
cells, the removal of nucleases and hybridization inhibitors, and
gently handling of DNA.  Most of the rapid lysis and purification
techniques are derived from Marmur (1961), a method originally.
developed for pure cultures of laboratory strains.  The method
may not always be applicable to microorganisms isolated from the


 Gram-negative bacteria are readily lysed by treatment with
 detergent (sodium dodecylsulfate or sarcosyl) , high pH,  cell  wall
 lytic enzymes,  or a combination of these reagents  (Maniatis,
 1982) .   Proteins and other cell components  are removed by
 proteinase digestion and  repeated phenol and chloroform
 extractions.   A variety of commercial  kits  often containing
 chromatography columns are available for nucleic acid

 The procedures outlined for the extraction  of nucleic acid from
 Gram-negative bacteria can be  applied  only  for certain Gram-
 positive bacteria.   In general,  the lysis of Gram-positive is
 more  difficult and may require treatment with cell  wall  lytic
 enzymes  (lysozyme,  lysostaphin,  mutanolysin)  often  combined with
 proteinase treatment and  detergent application  (Klinger  et al . .
 1988) .   Pretreatment of cells  with organic  solvents (acetone)
 facilitates subsequent lysis of some Gram-positive  bacteria
 (Heath et al. ,  1986) *   DNA and RNA can often be  released from
 Gram-positive  cells by hot phenol treatment of cell suspensions.
 If sufficient  lysis cannot be  achieved with these techniques,
 physical  methods such as  sonnication (Sharrock and  Rabinowitz,
 1979) or  shaking with glass beads in a cell mill (Stahl  et ajl. ,
 1988) may be required.

 Additional problems can be encountered in the preparation of
 nucleic acid  for colony hybridization.   The DNA  has to be
 released  from  cells grown or concentrated on solid  supports
 (nylon or nitrocellulose  membranes), and fixed in situ.   Gram-
 negative  cells  usually can be  lysed on filters by simple alkali
 treatment (0.5  M NaOH)  of the  cells (Grunstein and  Hogness,
 1975) .  Pretreatment of cells  with 10% sodium dodecylsulfate  at
 room temperature has been effective for  the in situ lysis of  some
 Gram-positive bacteria (Betzl  et.  al. , 1990) .  An alternative
 technique is to incubate  filter grown  colonies with detergent in
 a microwave oven (Buluwela et  al. .  1989) .
There are different approaches to the preparation of the second
hybridization component, the nucleic acid probe.  Probes can be
derived from randomly cloned DNA fragments or DNA or RNA
fragments of known sequence and function.  For the detection of a
microorganism in the environment, specific probes must be used to
differentiate it from a background in which closely related
strains are found.  Probes which identify organisms on the
species level, for example whole-cell DNA (Table 5) or ribosomal
RNA (Table 6) probes, are generally not useful for the detection
of a specific microorganism introduced to the environment.
Examples of DNA probes from randomly cloned DNA fragments and
from specific genes can be found in Tables 7 and 8, respectively.

Probes are frequently derived from the plasmid used to construct
the target microorganism.  Either the whole plasmid can be used


or a defined gene fragment can be isolated by restriction
digestion and gel purification.  A second strategy  is to use
chemically synthesized oligonucleotides.  One advantage of
oligonucleotide probes is that large quantities of  consistent
quality can be quickly prepared.  A second is the high
specificity they exhibit, since single nucleotide mismatches may
be sufficient to prevent hybridization of a short oligonucleotide
to a potential target (Wallace et al.. 1981; Ikuta  et al.. 1987).
Specificity is especially important samples derived from the
natural environment.  However, the sensitivity of oligonucleotide
probes is lower than polynucleotides because they are shorter and
thus have fewer labeling sites.  A third approach is to use
single-stranded RNA probes.  These offer the advantage of high
specific activity and no self-annealing because they are single
stranded.  Examples of these approaches with environmental
samples can be found in Tables 10 and 11.

Before hybridization, the nucleic acid probe has to be labeled to
enable detection of the resulting hybrid.  There are two
principal types of labeling in use:  direct and indirect
labeling,  with direct labeling, a label is covalently bound to
the probe.  With indirect labeling, an reporter group is attached
to the probe and is subsequently detected by a labeled binding
protein.  The label can either be measured directly (e.g.
fluorescent label) or indirectly by the reaction product formed
by an enzyme conjugated to the binding protein.

Labels can be radioactive or non-radioactive.  Originally,
radioactive isotopes were used as markers and are still preferred
in research studies where high sensitivity and low  non-specific
background are required.  Safety considerations, the lack of
stability, and waste disposal problems associated with
radioactive labels have spurred continuing efforts  to develop and
improve non-radioactive alternatives (see Table 9 for examples).

The sensitivity of non-radioactive labels is now approaching that
of radioactive methods and in certain applications  (e.g. in situ
hybridizations) is already superior.  Radioactive as well as
fluorescent-labeled nucleotide analogues can be introduced into
nucleic acid probes directly by using polymerases.  Some methods
for the incorporation of nonradioactive labels are  nick
translation (Rigby e£ al., 1977), specific or random primed
synthesis of complementary strands (Feinberg ejfe al.. 1983), in
vitro transcription for generating labeled RNA (Melton et al..
1984) and polymerase chain reaction (Saiki et al.,  1988).  These
techniques are used to label polynucleotides.

These labeling techniques can be applied to either  single
stranded RNA or ONA.  Single stranded probes can only hybridize
to the target and cannot self-anneal which makes them more
sensitive than double stranded probes.   However, their
preparation requires cloning of the fragment of interest proximal


 to a phage promoter,  which is  time-consuming.  On the  other hand,
 double stranded probes also have disadvantages.  These probes
 have to be denatured  prior to  labeling and  competition between
 probe  and target  DNA  for their respective complementary  strands
 during hybridization  decreases sensitivity.

 Oligonucleotide probes are commonly end-labeled at:  i)  5*
 terminal by using T4  kinase (Maxam and Gilbert, 1980), or ii)  3'
 terminal by using terminal transferase  (Radcliff , 1981) .  Direct
 labeling of oligonucleotides can also be achieved by attaching
 primary amino-groups  to the probe during the synthesis.  These
 linkers can be  labeled with activated fluorescent dyes or enzymes
 (Jablonski e£ ai. ,  1986; Smith et fil. , 1985; Inoue et  ai- ,  1985).
A variety of reporter groups are available for use in conjunction
with nonradioactive labelling  (indirect labeling) .  Table  9  lists
those that are available commercially,  one of the most commonly
used reporter groups is biotin.  A second reporter group that has
been gaining increased use is  digoxigenin - a steroid from
Digitalis purourea .  These reporter groups have been reviewed by
Matthews and Kricka (1988) .  Indirect labels often allow signal
amplification and may be more  versatile than direct labels.
However, some reporter groups  such as biotin exhibit nonspecific
binding to protein contaminants resulting in a decrease in
specificity.  Thus, these techniques are more difficult to apply
to complex environmental samples.

2)  Nucleic Acid Hybridization Techniques

Hybridization involves two major steps:  1) binding of the probe
to the target (hybridization); and, 2) separation of specific
probe-target hybrids from unbound probe (washing) .  The
specificity can be controlled  by the stringency of the conditions
applied (temperature, salt concentration) .

The most commonly used techniques involve a solid phase support
matrix.  The denatured target  nucleic acids are immobilized on a
solid support, such as nitrocellulose or nylon membranes.  The
most frequently used solid support, nitrocellulose, has the
advantage of relatively low levels of nonspecific hybridization
(Sambrook et al. 1989) .  Its two major limitations are:  1) it is
extremely brittle when dry and 2) it binds small fragments (less
than 200-300 nucleotides in length) poorly (Wahl et ii. , 1987).
The major advantages of nylon  are its structural stability (Wahl
fit £l*« 1987) and superior retention properties (Jagus, 1987).
Once target DMA or RNA is applied, therefore, these filters can
undergo multiple hybridizations with different probes.  With
nitrocellulose,  the nucleic acid is gradually dissociated from
the filter and thus the blot can only be reused a limited number
of times.

The following three methods employing solid phase supports  are
distinguished by the way the target nucleic acid is applied to
the solid support:

(1)  Spot/Dot/Blot Blot  (Kafatos, 1979)

DNA, isolated directly from environmental samples (Sayler and
Berkley, i987) or indirectly from pure culture isolates or
enrichments from the environment (Ezaki et al., 1989), is applied
to a defined membrane area with a slot/dot blot apparatus.
Quantitation is achieved through a densitometer or liquid
scintillation counting.  Because cultivation is not required,
culturable and non-culturable organisms may be enumerated in
environmental samples.
(2) Colony and plaque hybridization

The cells are grown directly on hybridization membranes or  cell
material is transferred from a master plate onto the  filters; in
situ lysis and DNA binding is followed by hybridization.  The
method allows the rapid screening of multiple colonies that are

(3)  Southern and Northern hybridization

Nucleic acid fragments, either DNA (Southern, 1977) or RNA
(Northern) can be transferred to a membrane from a
electrophoretic gel.  In the original method, the gel is placed
on a membrane in buffer and the fragments are deposited on  the
membrane by passive diffusion.  Modifications to this method
accelerate transfer by applying vacuum (Peferoen et al.. 1982) or
an electric current.

Since the original protocol was developed by Grunstein and
Hogness (1975), colony hybridization has been the most widely
used hybridization protocol for environmental studies (reviewed
by Sayler & Layton, 1990), particularly those that require
quantisation of specific microorganisms.  Though more labor
intensive than colony hybridizations, more detail can be obtained
with Southern and Northern analyses.  For instance, Southern
blots can be used to analyze restriction fragment length
polymorphisms (RFLPs).  This method allows the identification of
single strains; for example, parental strains and genetically
modified strains can be differentiated in soil samples (Holben e£
al.,  1988).  Also the stability and fate of specific nucleotide
sequences in microorganisms, i.e.,, rearrangements,  deletions and
gene transfer, can be detected.

The alternative to hybridization on a fixed matrix is to allow
the reaction to proceed in solution and then to capture the
probe/target duplex for quantitation.  Because the target is not
fixed and completely accessible to the probe, hybridization in
solution has the advantage of a higher rate of hybridization than


membrane techniques.  Disadvantages associated with solution
hybridization include:  1) possible self-reassociation of target
DNA;  2) difficulties in separation of probe-target hybrids from
unbound probe; and, 3) added logistical difficulties with
handling multiple samples simultaneously.

Solution reassociation hybridizations have recently been used to
estimate the genetic diversity in soil ecosystems (Torsvik et
al., 1990).  Several companies offer nucleic acid probes for the
rapid identification'of specific pathogens or environmentally
important microorganisms.  These are mostly based on rRNA-
targeted oligonucleotide probes.  However, commercially available
probes have not been widely employed for environmental studies.

There are several considerations associated with the choice of
hybridization technique when used for environmental samples.  The
Analysis of Techniques section of this document covers a variety
of hybridization techniques in detail.  A summary of nucleic acid
hybridization techniques used in environmental studies can be
found in Tables 10 and 11.  The majority of applications cited in
the literature report the use of dot/slot blots and colony
hybridizations.  The polymerase chain reaction is relatively new
and there are fewer studies in which it has been used.  These
studies are listed in Table 12.  Some permutations of the these
techniques such as multiplex amplification using the polymerase
chain reaction have yet to be applied, but are included in the
Analysis of Techniques to demonstrate possible applications.


 Much  of the  research on  the fate of microorganisms  in  the
 environment  has  relied on quantitative enumeration  of  microbial
 populations.  One  consideration when using quantitative analyses
 of data derived  from any method of detection  is the ability  to
 make  inferences  about  the microbial population of interest,  which
 requires statistical tests.  The use of most  common statistical
 tests demands certain  conditions be met by both the sample and
 the sampled  population.

 One of the first requirements  is that  the sample be taken at
 random and "representative" of the true population.  Being
 representative is  a difficult  state to define.  It  is  always
 possible that the  chosen parameter to  measure is invalid or
 skewed due to inappropriate sampling.  However, any sampling must
 be replicated to be able to derive mathematically valid
 conclusions  about  either the samples themselves or  the true
 population.  Once  replicate measures are  taken it is possible to
 examine characteristics  such as the mean  and  variability of  the
 sample population  and  infer about the  true population. Generation
 of statistics requires replication of  samples and measurements.

 Once  a mean  and  variance are generated for a  sample population it
 becomes possible to infer about the sample population  and the
 true  population.   One  of the most frequently  abused assumptions
 of the most  common statistical tests is that  the population  is
 normally distributed about the measured parameter.  This
 assumption implies that  the measured mean and variance are
 independent  and  as one increases it has no effect on the other.
 Often microbial  populations show a linkage between  the size  of
 the mean and its variance and approach a  Poisson rather than a
 normal distribution.

 Another assumption that  is frequently  found invalid is that  the
 true  population  is randomly distributed in the sampled media,
 e.g., soil, water, or  plant tissue.  This implies that recovering
 any individual in  any  one sample does  not affect the likelihood
 of finding an individual in subsequent samples.  This
 distribution results in measured means being much greater than
 their associated variance.  Populations sampled in  soil or other
 nonhomogenous media are often not randomly distributed but rather
 clustered.  Clustered  populations are  characterized by having
variances greater  than their means and more easily  fit a negative
binomial rather  than a normal distribution.

Many of the techniques discussed in this  document seem very
sensitive when the detection limits are examined.   It must be
remembered that  many have not been utilized extensively in a
variety of environments.  For example, the distribution of the
target population  in media such as animal tissues may be expected


to be more homogenous or easily localized than those found in
terrestrial or aquatic environments.  In these cases the loss of
the target population through sample processing may influence the
results more than the sensitivity of the final detection method.
Thus, the variability of the measured parameter will be more
affected by the extraction or culturing step than the final
detection method.

In the development of a detection method for quantitative
analysis of microbial populations in a new environment, it is
critical to repeat the method enough times with a given matrix to
be able to generate an accurate representation of both the
background and the minimum level of detection.  This may be
accomplished by adding known amounts of the target microorganism
to a negative and known positive environmental sample to verify
the extraction efficiency, minimum detection limit, and linearity
of response.  It is helpful to remember that a minimum detection
limit is usually defined as the measured background mean plus
2 or 3 standard deviation units of that mean.

Several of the techniques mentioned such as JLn situ and filter
hybridization involve the recognition of homology between
complementary strands of DNA.  The specificity of this reaction
can be greatly affected by the temperature and osmotic conditions
under which it is run as well as the % GC content of the
components.  This stringency can also be affected by contaminants
present in the sample.  As mentioned above it is useful if the
system is replicated for each new media tested to determine the
amount of interference and its affect on the detection limit.

In addition to stringency considerations, techniques such as
restriction endonuclease digestions and polymerase chain reaction
require purified nucleic acid preparations for proper enzyme
activity.  The purification process decreases the yield of
nucleic acid due to sample preparation and may increase -the
variability of detection.  The statistics related to data
generated from enzymatically amplified DNA targets has not been
well described for their detection limit.  It may be helpful to
generate the same sort of spiked samples for determining a
background and minimum level of detection alluded to above.



(a)   Microscopy - Detection of viable microorganisms

DESCRIPTION:   Direct count procedures consist of techniques in
which microorganisms are enumerated, often without need for
culturing or plating, generally through microscopic techniques.
For large microorganisms (e.g., protozoa, algae), counting
chambers are used, and special techniques exist for fungal counts
(modified agar film techniques).  This second direct count
procedure combines direct microscopic examination with a stain or
other technique that indicates whether the microorganism is
metabolically active.  One technique that indicates the number of
actively respiring microorganisms is the AODC method combined
with 2-(p-iodophenyl)-3*(p-nitrophenyl)-5-phenyl tetrazolium
chloride (or INT).  A second technique for use with bacteria
involves using a fluorescent stain in combination with nalidixic
acid.  Nalidixic acid (an antibiotic that inhibits cell division
through inhibition of DNA gyrase) will cause actively growing
cells supplied with a carbon source to elongate, while dormant or
dead microorganisms will retain their normal size and shape
(Kogure, et. al., 1978).  Novobiocin is an another DNA gyrase
inhibitor.  Third, autoradiography can be combined with direct
microscopic observation: bacteria are incubated with a
radiolabeled growth substrate, and actively metabolizing
microorganisms can be differentiated as those which cause
exposure of a photographic film coated over the cells.  Fourth,
there are techniques for fungal enumeration which involve the use
of an agar film combined with fluorescence microscopy using
fluorescein diacetate which only stains metabolically active
mycelia.  Techniques specific to certain species of
microorganisms have been developed, with an emphasis on detection
of aquatic microorganisms.  Recently, however, a technique has
been developed for detection of rhizobia in soil, using yeast
extract and nalidixic acid (Bottomly and Magard, 1990).

SPECIFICITY: The identification of the specific isolates is
determined by the skill of the microscopist, the unique
morphological traits of the isolate, and the ability of the dye
to discriminate between metabolically active and nonactive cells.

SENSITIVITY:  A high background fluorescence resulting from the
release of esterases which react with the fluorescein diacetate
dye may make it difficult to distinguish metabolically active
mycelia.  Exposure to nalidixic acid may not elongate some cells
if the septation mechanism involved in cell division is not
linked to DNA replication.


EASE OF USE & TIME:  Techniques that use nalidixic acid to detect
viable rhizobia from soil samples require approximately 90
minutes to recover bacteria and initiate incubation in a
substrate-antibiotic combination.  On average, the incubation
time in nalidixic acid is up to 24 hours for studies with soil
rhizobia, and, by contrast, 6 to 8 hours for aquatic studies.

COST: Similar to the costs of the direct viable count technique
described below except additional reagents are needed that are
able to discriminate between those microorganisms which are
metabolically active and those that are dormant or dead.

EFFECTS OF ENVIRONMENTAL MEDIA:  See discussion on direct viable
count techniques.

 (b)  Microscopy - Detection of total viable and nonviable

DESCRIPTION:   Direct count procedures consist of techniques  in
which microorganisms are enumerated, often without need for
culturing or plating, generally through microscopic techniques.
For large microorganisms (e.g., protozoa, algae), counting
chambers are used, and special techniques exist for fungal counts
 (modified agar film techniques).  Stains such as acridine orange
 (AODC method), 4'6-diamidino-2-phenylindole (DAPI), and
fluorescein isothiocyanate (FITC) are used to assist in
enumeration (and are detected by epifluorescence microscopy).
Often, direct count techniques are used in conjunction with
immunological techniques, such as fluorescent antibody methods,
to detect viable, and viable but nonculturable microorganisms
 (Colwell et ai., 1985 and 1988).  Other instrumentation, such as
electron microscope and particle counters (Coulter counters), has
been used in place of light microscopes.

SPECIFICITY:  The identification of specific isolates is
determined by the skill of the light microscopist and the unique
morphological characteristics of the isolate (fluorescent
antibody techniques are extremely useful for identification of
specific strains).  Stains such as acridine orange stain bacteria
and other organisms, but the intensities of the stain do not
correlate well with specific isolates (or live or dead
microorganisms).  DAPI is preferable to acridine orange for
counting small bacterial cells.

SENSITIVITY:  Counts by direct epifluorescent microscopy are
typically two orders of magnitude higher than plate count
techniques would indicate.  The AODC technique has resulted in
counts of Vibrio  cholerae that are 6 orders of magnitude higher
than plate count techniques indicate (Colwell, et al.. 1985).
However, the epifluorescent microscopy technique is not a very
sensitive one since it requires a high concentration of cells
before a single cell can be seen in a microscopic field.  The
microorganisms which are detected by direct counts (and not be
plate counts)  may be viable but nonculturable due to
insufficiencies in media, incubation time, etc.  Direct counts
are often directly proportional to the microbial biomass found in
a variety of environmental media (soil,  freshwater,  and marine

EASE OF USE £ TIME:  Microscopic examination can be tedious and
time-consuming, and is therefore impractical for a large number
of samples.  Conversion of microscopic counts to biomass
approximations can also be tedious.

COST:  This relatively low cost method does require both commonly
available equipment such as microscopes, fluorescent stains, and
possibly counting chambers and specialized equipment such for
fluorescent .  .

EFFECTS OF ENVIRONMENTAL MEDIA:  Direct counts allow enumeration
of microorganisms in a variety of habitats (marine, freshwater,
and soil) without the bias associated with plate count techniques
(see "Sensitivity").  However, underestimation of microbial
numbers may occur if high amounts of background debris exist in
the sample (such as with soil or plant tissue).

 (c)  Microscopy - fluorescent antibody

 DESCRIPTION*  Fluorescent antibody  (FA) techniques have been  used
 successfully in environmental microbiology for the detection  and
 enumeration of microorganisms at the species or even strain level
 (Bohlool and Schmidt, 1980).  The detection of microbial cells
 that have reacted with labeled antibody is usually carried out
 through direct microscopic  observation of the environmental

 SENSITIVITY AND SPECIFICITY:  The sensitivity of fluorescent
 antibody microscopy is generally not as good as standard plate
 count, most probable number, or molecular detection methods.
 This is a result of the qualitative nature of this method based  *
 on microscopic observation  of samples.  The microscopic field
 that is viewed is very small in area and only a limited quantity
 of the environmental sample can be visualized in each field.
 This necessitates that a minimum of between 10* to 106 cells per
 gram of soil or milliliter  of diluent be present in order to
 detect one cell in a microscopic field.  Aquatic samples offer
 the advantage of allowing concentration of the sample before
 microscopic analysis.  Quantitative FA techniques have been
 developed for detecting rhizobia bacteria in soil which have
 minimum detection limits approaching 10* CFU/g soil (Schmidt,
 1974).  Nevertheless, the specificity of this detection technique
 can be quite satisfactory as individual microbial strains can be
 distinguished in environmental samples. ' The degree of cross-
 reactivity between antisera prepared against the microorganism of
 interest versus reactivity  against other related strains must be
 assessed to ensure satisfactory specificity with this method.
 However, one problem with FA specificity is that no distinction
 between viable and nonviable cells is possible.  Furthermore, if
 one wants to detect a specific introduced microorganism or
 nucleic acid sequences within that microorganism, fluorescent
 antibody techniques alone are usually not sufficient.  The
 exception to this would be  a microorganism modified with novel
 DNA sequences which code for a unique antigenic product which
 allows detection with fluorescent antibodies.

Although polyclonal antisera are easier to prepare for use in
 fluorescent antibody methods, they are less specific than
monoclonal antibodies.  The use of monoclonal FA provides a
highly specific technique that has been successfully used for the
direct detection of microorganisms in the environment.


 (a)  Selective media

 {i>  Resistance to  antibiotioa

DESCRIPTION:   Resistance to antibiotics is the most frequently
used detection method  in environmental microbiology for reasons
of overall convenience and due to their successful use for over  a
decade.   The antibiotics are usually added as components of a
solid medium for plate counting of resistant colonies.  As     ''".
microbial resistance to clinically useful antibiotics is a major
health concern, efforts have been made to develop alternative
detection methods,  particularly for microorganisms introduced  to
the environment.

SENSITIVITY AND SPECIFICITY:   The sensitivity of this technique
in detecting microorganisms introduced to soil microcosms or
terrestrial environments is often in the range of 102 to 10*
CFU/g soil, although reports of detection limits as low as 1-20
CFU/g soil have been reported (Devanas and Stotzky, 1986).
Studies on microbial fate on plant surfaces (see e.g., Armstrong
et al.. 1987) or in aquatic environments (see e.g., Scanferlato
SH al*f 1989; Amy and  Hiatt, 1989) often report a greater
sensitivity - as low as 10 to 100 CPU/per unit area or volume.
Differential media  may be necessary to improve specificity and to
distinguish colonies of the introduced microorganism on selective
media containing antibiotics.  With experience an investigator
may recognize colony phenotypes.  Specificity can be improved  by
employing resistances  to more than one antibiotic, so that a
spontaneous mutation to sensitivity to one antibiotic will not
eliminate the detectability of the introduced microorganism. .
Sensitivity may also be enhanced in this way, although the
possibility of reduced recovery of  microorganisms carrying
additional resistance  markers on selective media must be checked.
Moreover, certain spontaneous antibiotic resistance mutations  may
reduce survival capabilities in situ (Compeau fit al., 1988).   All
detection techniques based on plating or culturing have an
advantage of detecting only viable microorganisms as compared  to
direct detection, e.g., serological techniques.  Sensitivity and
selectivity of the  technique (relative to detection of the
microorganism) may  be  affected since environmental influences  may
alter phenotypic expression of resistance, and resistance genes
may be transferred  from the GEM to other microorganisms.

EASE OF USES    Fluorescent antibody microscopy does not require
culturing on solid or in liquid media.  Moreover, the
physiological state of the introduced microorganism is not
critical for adequate detection in the environmental sample
unless linked to antigenic expression.  Preparation of labeled
antibodies and processing of environmental samples for microscopy
do result in this technique being more resource intensive than
plate count or MPN techniques.  However, automation of certain
steps in this procedure reduces time and expense.  Moreover, the
requirement for extensive verification of antibody specificity,
through testing of a variety of negative control microorganisms,
adds to the resource requirements.

COST:  The primary cost involves the preparation of polyclonal,
and especially monoclonal, antibodies reactive against the
lesired microorganism.

EFFECTS OF ENVIRONMENTAL MEDIA:  The specificity of fluorescent
antibody microscopy can be hampered by nonspecific binding of the
antibody to various chemical and biological components of the
environmental sample.  The natural sample may contain
microorganisms, clays, humic materials, or inorganic constituents
that are reactive with the labeled antibody.  This can be
particularly problematic when this technique is used with soil
(d)  summary of direct detection techniques

Direct detection techniques have, in theory, an unlimited
detection limit depending on how much of the environmental sample
the observer is prepared to scan with a microscope.  However, in
practice technical restraints on this detection limit are
governed by the presence and persistence of soil particles
(practical detection limit • 1-10 CFU/g).   The problems
associated with these methods are ones of contamination with
environmental material that results in false positives for direct
counts by microscopy.

EASE OF USE:   Selection of spontaneous resistance to antibiotics
is significantly easier to develop and use in environmental
microbiology than the available immunological, most probable
number, or nucleic acid hybridization procedures.  Genetic
modification of microorganisms through the addition of specific
resistance markers is becoming more routine, for example the use
of Tn£ marked microorganisms for environmental studies which
imparts both resistance to kanamycin/neomycin and provides a
convenient target DNA sequence for hybridization in the
microorganism.  However, certain antibiotics are also toxic to
humans and must be handled with appropriate precautions.

COST:  The cost of many commercially available antibiotics is
relatively low, and their use generally less expensive than
immunological, MPN, or nucleic acid hybridization detection

EFFECTS OF ENVIRONMENTAL MEDIA:  Unequal dispersal of soil,
sediment, or plant-associated microorganisms (due to associations
with soil or plant material) may lead to poor population
estimates.  Moreover, indigenous microorganisms expressing
resistance to antibiotic may have the ability to outgrow the
introduced microorganism on selective media.  This is
particularly true if the added microorganisms is better adapted
to laboratory, rather than environmental,  conditions.

 (ii) Resistance to heavy metals

DESCRIPTIONS  Resistance to heavy metals has been used to detect
microorganisms in environmental samples.  Heavy metals such as
mercury and cadmium are added to plating media to allow selection
of appropriately marked resistant microorganisms.  Resistance to .
these antimicrobials is frequently encoded by genes found on
bacterial plasmids.  It has been pointed out that the use of
microorganisms resistant to heavy metals in environmental studies
can contribute to the spread of resistance to clinically
important antibiotics as both are plasmid-borne.
SENSITIVITY AND SPECIFICITY:  The,sensitivity of techniques
employing heavy metal resistance as the selective parameter is
often in the range of 10s to 10* CFU/g  soil in terrestrial
studies.  Sensitivity can be improved up to 10 to 100 fold in
aquatic environments or in planta due to the presence of fewer
indigenous bacteria resistant to heavy metals.  Thus,
antimicrobial resistance may be less sensitive than the use of
resistance to antibiotics.  Nevertheless, Top e£ aJL. (1990)
reported a minimum detection limit of approximately 10 CFU/g soil
in nonsterile soil with microorganisms carrying resistance to the
heavy metals cadmium, zinc, and cobalt.

As discussed above for antibiotics, differential media may be
necessary to improve specificity and allow the discrimination of
colonies of the introduced microorganism on selective media
containing the heavy metal, although with experience an
investigator may recognize colony phenotypes.  Likewise,
specificity can be improved by employing resistances to more than
one heavy metal or antibiotic.  The ability to monitor
microorganisms based on plasmid-borne heavy metal resistance can
be hampered by the loss of the plasmid DNA after introduction of
the microorganism to the environment.  All detection techniques
based on plating or culturing have an advantage of detecting only
viable microorganisms as compared to direct detection, e.g.,
serological techniques.  Sensitivity and selectivity of the
technique (relative to the microorganism) may be affected since
resistance genes may be transferred from the GEM to other
microorganisms (see Tables 1 and 2).

EASE OF USE:  The toxicity of heavy metals requires that they be
handled with care when used in selective media.  Nevertheless, as
with antibiotic resistance, selection of microorganisms resistant
to heavy metals can be relatively easy and the use of this
technique in environmental microbiology more convenient than the
available immunological, most probable number, or nucleic acid
hybridization procedures.  Resistance to heavy metals is often
located on transposons which can be inserted into the genome of a
microorganism through genetic manipulation in order to provide a
means of selection in environmental studies.

COST:  As with antibiotics, the cost of many commercially
available antimicrobial agents is relatively low, and their use
generally less expensive than immunological, KPN, or nucleic acid
hybridization detection methods.

EFFECTS OF ENVIRONMENTAL MEDIA:   Unequal dispersal of soil,
sediment, or plant-associated microorganisms (due to associations
with soil or plant material) may lead to poor population
estimates.  Moreover, indigenous microorganisms expressing
resistance to antibiotic may have the ability to outgrow the
introduced microorganism on selective media.  This is
particularly true if the added microorganisms is better adapted
to laboratory, rather than environmental, conditions.

 (Ill)   Unusual carbon sources

 DESCRIPTION:  Media for the detection and differentiation of
 microorganisms based on their ability to utilize unusual carbon
 sources are an effective means for the detection, identification,
 and enumeration of microorganism in the environment.  Certain
 groups of microorganisms, e.g., the pseudomonads, are able to
 utilize carbon sources toxic to most microorganisms, such as
 toluene or 3-chlorobenzoate, which facilitates their selection on
 appropriate media.  Genetic manipulation can also be a useful,
 for example Walter et fil- (1988) cloned the xylE gene from the
 TOL plasmid of Pseudomonas aeruainosa in other genera of bacteria
 to provide a means of selection from other soil and plant
 bacteria.  In addition, expression of this gene results in a
 yellow colony which can be readily distinguished on selective
 media from indigenous bacteria.

 SENSITIVITY AND SPECIFICITY:  The sensitivity of this technique
 is a function of the environment in which it is used as the
 numbers of indigenous microorganisms capable of utilizing the
 selective carbon source is highly variable.  The use of the xvlE
 gene described above resulted in a sensitivity comparable to that
 seen with antibiotic markers in environmental studies.  The
 sensitivity was improved to 10 CFU/g soil or ml of wastewater
 when the xylE gene was used in conjunction with the selective
 antibiotic nalidixic acid.  The selective capabilities of lignin
 utilization have been exploited by Wang e-fr aJL. (1989) through the
 cloning of genes for lignin degradation into high expression
 vectors in streptomycetes used in soil studies.  All detection
 techniques based on plating or culturing have an advantage of
 detecting only viable microorganisms as compared to direct
 detection methods, e.g., serological techniques.  If expression
 of catabolic genes is inducible, their expression may be
 inhibited during primary recovery.

 EASE OF USE:  This technique requires only plating on, or growth
 in, selective media.  Thus,  overall ease of use can be
 significantly greater than immunological or nucleic acid
 hybridization methods.  If genetic manipulation of the
microorganism of interest is required to introduce the catabolic
 genes for a particular carbon source, considerable resources may
be spent obtaining satisfactory expression of this trait.

COSTS  As with antimicrobial agents discussed above, the cost of
many commercially available microbial carbon sources is
 relatively low, and their use generally less expensive than
 immunological or nucleic acid hybridization detection methods.

EFFECTS OF ENVIROHMENTAL MEDIA:    Unequal dispersal of soil,
sediment, or plant-associated microorganisms (due to associations
with soil or plant material) may lead to poor population
estimates.  The use of minimal media is often required in order
to obtain proper selection based on a sole carbon source.
However, recovery of the desired microorganism on minimal media
can be poor due to the exposure of the microorganism to
relatively harsh environmental conditions.  Furthermore, genes
for utilization of certain unusual carbon sources are often found
on plasmids or transposons which may be lost through plasmid
segregation under environmental conditions.

 (b)  Differential media

 DESCRIPTION:  Plate enumeration techniques employing differential
 media have been used extensively in environmental microbiology to
 distinguish introduced microorganisms from the indigenous
 microflora.  Often used in combination with a selective agent,
 differential media can increase sensitivity by at least an order
 of magnitude since the  microorganism being enumerated on solid
 media can be distinguished from others present in the sample.
 Examples include the detection of pigment-producing fluorescent
 pseudomonads (Drahos ei al. , 1988) , £. aeruainosa (Zechman and
 Casida, 1982) , or various enteric bacteria capable of
 metabolizing lactose.  Recently, investigators have genetically
 modified bacteria to impart a differential colony phenotype by
 the addition of genes for beta-galactosidase to non-enteric
 microorganisms (Jain e£ al« . 1988; Drahos e£ aj,.. , 1988), for
 luciferase to non-marine microorganisms  (Gutter son, 1988;
 Meighan, 1988), and for xylose utilization (Walter e£ al., 1988).
SENSITIVITY AND SPECIFICITY:  Differential media are employed in
plate enumeration of microorganisms to improve the specificity of
this detection method.  Sensitivity of detection is only as good
as the selective capability of the medium that is employed.
Since ATP is required for expression of the luciferase genes,
environmentally starved microorganisms bearing a luciferase
cassette may not be detected.

EASE OF USE:   A variety of differential media or medium
components are available commercially.  However, the majority of
microorganisms have not been studied sufficiently to design
differential media.  Thus, investigators interested in using
particular microorganisms in environmental studies have used
genetic manipulation, as discussed above, to introduce the
genetic capacity to differentiate the microorganism on selective
media .

COST:  Commercially prepared differential media or reagents for
incorporation into differential media components are often
relatively inexpensive and readily available.

EFFECTS OF ENVIRONMENTAL MEDIA:  Environmental interference
problems for differential media are similar to those seen with
selective media.

 (c)  Most probable number technique

 DESCRIPTION:   The most probable number  (MPN) method provides an
 estimate of population density without a count of single cells or
 colonies.  It  is based on a determination of the presence or
 absence of microorganisms in replicate dilutions of soil or other
 material.  Based on probability theory, tables have been
 developed that allow estimation of the numbers of microbes in an
 original sample, based on the numbers of positive and negative
 replicates which received a certain quantity of inoculum.

 MPN techniques have been developed for a number of different
 microorganisms including algae, protozoa, denitrifiers,
 nitrifiers, and rhizobia.  Often, the MPN technique is compared
 to the plate count method (or the roll-tube method for obligate
 anaerobes) in  which dilutions of samples are spread on the
 surface of, or mixed with, agar.  Both the MPN and plate count
 methods are called viable count procedures.  Viable count
 procedures suffer from the drawback that they require separation
 of microbes into individual reproductive units.

 SPECIFICITY:   This method is limited in that the specificity and
 sensitivity is dependent on the technique used to detect the
 microorganism  in the medium into which it is inoculated.  As with
 enrichment techniques for plate counts, various subsets of the
 microbial community may be enriched for through the use of
 selective growth media.

 SENSITIVITY:   This method is less precise than the plate count
 technique.  Also, both viable count procedures can be affected by
 microbial inhibitions caused by other microbes present in the
 growth medium.  Viable but nonculturable microorganisms may not
 grow under conditions of MPN incubations.  Also see limitations
 under "environmental interference" below.  The lower range of
 reported sensitivity is 10 cells per unit; however, statistical
 variability can be relatively large, for examples compared to
 plate count techniques, and may prevent detection of
 statistically  significant differences.

 EASE OF USE fi  TIME:  This is a relatively easy procedure which
 can require little time and minimal equipment, relative to
 certain direct detection or hybridization techniques discussed in
 this section.  However, it is often more time-consuming than many
 plate count procedures and can be cumbersome if statistical
tables for the number of replicates chosen are not available.

COST:  Inexpensive technique requiring only glassware,
 appropriate media, and possibly instrumentation for detection of
 isolate growth.

EFFECTS OF ENVIRONMENTAL MEDIA:   The environmental matrix is
diluted out in a 10-fold dilution series which generally stops at
10*9.  In soil, water, sediment, and plant tissue samples it may
be difficult to obtain individual reproductive units.  Also,
inhibition of the growth of the desired microorganisms by
antagonistic indigenous microorganisms may present problems.

 (d)  Fluorescent antibody - colony ELISA blots

 DESCRIPTION:   A variety of immunological methods are available
 that employ monoclonal or polyclonal  antibodies  that react with
 specific antigens on the surface of microorganisms.   Serological
 techniques have been used extensively in studies on  aquatic
 microorganisms such as the pathogen vibrio  cholerae.  and somewhat
 less frequently for soil or plant associated  microorganisms,  with
 the  exception  of the root nodulating  Rhizobium and
 Bradvrhizobium.   One of the most commonly used methods  in
 terrestrial and aquatic microbiology,  is the  fluorescent antibody
 technique.  A  technique under  development,  based on  enzyme-linked
 immunosorbant  assay methods, is  the colony  ELISA blots.

 This fluorescent antibody technique combines  the specificity  of
 antigen-antibody reactions with  plate counts  techniques  in the
 identification of individual colonies on a  solid medium  in colony
 enzyme-linked  immunosorbent assay (ELISA) blots.  In this
 relatively new technique,  the  microorganism of interest  produces
 an antigenic cellular constituent,  often an enzyme,  which can be
 detected in individual colonies  through colony blots.  Thus,
 dilutions of the environmental sample containing the antigen-
 producing microorganism,  are plated on selective media,  replica-
 plated onto nitrocellulose filters, lysed,  and detected  through
 the  use  of a fluorogenic or chromogenic label.

 SENSITIVITY AND  SPECIFICITY:   As with  any FA  technique,
 specificity can  be very good,  allowing the  detection of
 individual  microbial  strains.  An advantage of colony ELISA
 blots, when compared  to fluorescent antibody  microscopy
 techniques, is that there is less non-specific interaction of the
 antibody to components of the  environmental sample as the assay
 is carried  out on membrane filters  with isolated colonies.
 However,  colony  ELISA blots do require culturing of the
 microorganism  of interest,  and the  sensitivity of this technique
 for  environmental  samples  is only as good as  that of  the
 selective media  used  to  isolate  and enumerate the microorganism.
 Although polyclonal antisera are easier to  prepare for use in
 colony blots,  they can be  less specific than  monoclonal
 antibodies.  The use  of  labeled  monoclonal  antibodies provides  a
 highly specific  technique  that has  been used  for  the  direct
 detection of microorganisms in the  environment.

 EASE OF  USE:   Preparation  of labeled antibodies and processing  of
 environmental  samples  for microscopy do result in this technique
being more  resource intensive  than plate count or MPN techniques.
Moreover, the  requirement  for  extensive verification of antibody
specificity, through testing of  a variety of  negative control
microorganisms, adds to the resource requirements.

COST:  The primary cost involves the preparation of polyclonal,
and especially monoclonal antibodies, reactive against the
desired microorganism.  Additional costs and time is involved in
verification of the specificity of the antibody preparation.

technique can be hindered by nonspecific binding of the FA to
compounds introduced during plating of the environmental sample
on solid media for colony development.  This is largely a problem
on plates containing low sample dilutions and can result in
transfer of the contaminants to the membrane filter during
replica plating.

 (e)   Summary of cell culture techniques

 All  of the methods classified as cell  culture techniques often
 provide relatively good levels of detection with approximately
 102  CFU/g soil or plant tissue being the  accepted figure for
 traditional plate counts.   This figure can be improved in aquatic
 samples,  often through the use of biomass concentration
 techniques.  The primary practical limitation with cell culture
 techniques is one of contamination due to growth of indigenous
 microflora, either on solid media for  plate count techniques or
 in liquid media for MPN techniques.  However,  for many
 environmental studies a detection limit of 102 CPU is  sufficient
 to allow a determination that the microorganism's survival or
 dissemination is following expected patterns.   In this case,
 there is no real need to decrease limits  of detection^.  Microbial
 populations below this level could only again become significant
 if selection pressure resulted in an increase in numbers.   The
 likelihood of such selection would depend on  the microorganism.

 As the use of selective and differential  media remains the most
 common technique employed  in the detection microorganisms,  much
 experience has been gained regarding its  advantages and
 disadvantages.   Consideration of the strengths and weaknesses of
 the  technique will ensure  that the desired sensitivity and
 specificity are attained.   The considerations  discussed below are
 based on  the growing literature in microbial  ecology.   More
 detailed  discussions of these topics can  be found in reviews  by
 Grainger  and Lynch (1984),  Colwell et  al. (1985),  Sayler and
 Stacy (1986),  Stotzky si &!•  (1990).
In general, the choice of any particular selective medium
approach depends on not only the characteristics of the
microorganism, and the environment that it is introduced to, but
also the monitoring endpoints that are to be measured, e.g.,
survival, dissemination, or gene transfer.  Each of these
monitoring endpoints has unique practical problems that should be
considered in the planning of the monitoring procedures.  As an
example, resistance to an antibiotic may provide a sensitive
phenotypic marker for monitoring the survival in soil of a
microorganism, but may not be of adequate sensitivity to detect
transfer of the resistance gene to recipient cells.  Thus,
several factors cone into play in the choice of an appropriate
selective or differential medium.

(i)  Recovery of oligotrophic versus oopiotrophie microorganisms

Oligotrophic microorganisms are those that preferentially utilize
nutrients at relatively low concentrations, while copiotrophs
exhibit optimal growth at relatively high nutrient
concentrations.  When sampling an environment particular
consideration should be given to the oligotrophic or copiotrophic
nature of the microorganism to be detected.  For example, for


isolation of oligotrophic microorganisms from soil may require a.
soil extract medium while those from marine environments may
require a defined seawater medium.  These media often result  in
optimal recovery of oligotrophs compared to a more nutrient rich
medium such as those commonly used in the laboratory for
cultivation of microorganisms.

(ii) Selection of speeifie resistance to antibiotics

Resistance to antibiotics can be used with most microorganisms as
a selective technique for quantitative or qualitative monitoring
of environmental fate.  A number of practical considerations
arise in the use of this technique.

A common consideration is the stability of the resistance
genotype in the microorganism to be monitored.  Often,
chromosomally located resistance genes offer greater stability
than those located on plasmids.  Resistance to nalidixic acid and
rifampicin may be preferable for this reason (Stotzky et al.
1990), as other plasmid associated resistance markers, such as
erythromycin, are more readily lost in the absence of selection

Resistance to nalidixic acid has been reported to be advantageous
for studies of gene transfer among microorganisms (Walter, 1988}.
The presence of nalidixic acid in the plating medium used for
colony counts of transconjugants effectively prevented
artificially high gene transfer rates due to conjugal transfer
events on the agar medium, rather than in the soil microcosm.

Not all resistance phenotypes are without secondary effects.  For
example, Compeau et &1. (1988) showed that resistance to
rifampicin in certain pseudomonads conferred a selective
disadvantage to the microorganism when introduced into sterile
soil microcosms.  Resistance to tetracycline can also alter the
colony phenotype of certain microorganisms, presumably due to
alterations in cell membrane functions.

(iii)  Stability and expression of the selective or differential

As mentioned above, plasmid-borne selective markers may be lost
during incubation in the environment as a result of plasmid
segregation.  This could result in artificially low quantitative
estimates of microbial populations.  The possibility of this
occurrence can be checked in microcosm experiments in which
population estimates are compared on nonselective media and media
containing the antibiotic.

Expression of a differential phenotype on a solid medium may
allow the accurate detection of certain microorganisms, however,
this can be confounded by the presence of large numbers of


 indigenous microbial  colonies.   Competition  for  limited  nutrients
 can result in colonies of  small  size which do not express  the
 differential  phenotype,  e.g., the characteristic green colony
 produced by fluorescent pseudomonads may not be  visible  with
 small  colonies on  crowded  plates.

 (iv) Resistance to antimicrobials:  increased sensitivity
     versus reduced recovery

 The use  of more than  one antibiotic or heavy metal  in the
 selective medium will often markedly increase the sensitivity  of
 this detection method;  however,  a reduction  in efficiency  of
 recovery of the microorganism from environmental samples may also
 occur.   Reasons for reduced recovery may be  attributed to  various
 phenomena including the potential for the microorganism  to
 produce  viable but non-culturable cells, the effects of
 antimicrobials in  the selective  medium that  alter cell function
 or  physiology,  or  other stresses on the microbial cells  when
 placed in environmental  conditions.

 Available solutions include reliance on a single antimicrobial
 with shorter  incubation  times that prevent the overgrowth  of
 indigenous microorganisms.  Also, an initial plating of  the
 environmental sample  on  a  nonselective solid medium can  allow
 growth and recovery of the introduced microorganism, followed  by
 replicate plating  on  the desired selective medium for
 enumeration.   Similarly, for aquatic environments,  an initial
 growth and recovery in .nonselective liquid media may be  necessary
 although this can  result in a loss of the ability to accurately
 quantitate the microorganism.

 (v)  Viable but nonculturable or somnicells  in bacteria

 The viable but nonculturable (or somnicell)  phenomenon has been
 reported to occur  in various gram negative bacteria including
 pathogens,  and aquatic or  soil-inhabiting microorganisms (Rozak
 and Colwell,  1987;  Bottomley e£  aJL-, 1990).  Viable but  non-
 culturable microorganisms  are metabolically  active, but  not
 culturable by standard methods.  It is thought that this dormant
 stage is a survival mechanism for the microorganism allowing it
 to exist under less than optimal conditions.  Relatively few
bacteria  have been  studied for their ability to  exhibit  this
property,  so  it is not .known whether this is a general
characteristic  of bacteria that do not produce typical resistant
structures  such as endospores or conidia.  However, in those
microorganisms  in which  the production of somnicells has been
demonstrated,   it is possible that populations in the environment
can be underestimated.

(vi)  Use of selective madia for colony hybridisation

While colony hybridization techniques have been applied to the
detection of microorganisms in the environment with increasing
frequency in both microcosm and field studies, a number of
practical considerations apply to the use of this technique.

The primary consideration is that the sensitivity of detection by
colony hybridization may be the same or only slightly greater
than that obtained by selective media.  This results from the
fact that the colonies used as the source of target DNA are
either picked or replicated from selective media plates.  Thus,
the lowest dilution of the environmental sample which results in
discrete colonies on a plate determines the sensitivity of this
technique (Saylor and Layton, 1990; see also discussion on
hybridization in this paper).


 (a)  Nucleic  acid probe construction

 (i)  Labels available  for nucleic acid probes

 DESCRIPTIONS   The detection of microorganisms  in environmental
 samples by  nucleic  acid hybridization has most frequently been
 accomplished  by the use of radiolabeled probes, although
 nonradioactive probes  have been employed with  increasing
 frequency.  Radio labeling of nucleic acid probes is carried out
 by the incorporation of a radioactive nuclide,  such as 32P,  in
 the nucleotides of  the probe sequence.  Colony, slot/ dot blot, or
 Southern hybridization procedures have all been used in the
 detection of  specific  target sequences in microorganisms employed
 in microcosm  or field  studies.  Several types  of nonradioactive
 labelling procedures have been developed including nick
 translation to incorporate biotin-labelled nucleotides , nick
 translation to incorporate an antigenic sulfone group, or direct
 photolabelling with biotin.

 SENSITIVITY AND SPECIFICITY:  The specificity  and sensitivity of
 radiolabelled nucleic  acid probes have been reported to be
 significantly greater  (Forster et al. . 1985) or only slightly
 greater (Zeph et  al. .  1990) than nonradioactive probes in
 detecting target  nucleotide sequences.  It appears that optimal
 sensitivity can be  obtained with nonradioactive probes with
 Southern hybridizations or dot/slot blots (1-10 pg for single
 copy gene sequences) ;  however, background nonspecific signal
 development can at  times cause problems with colony lift
 hybridizations.   Nevertheless, Yang (1985) reported the detection
 of one bacterial  colony containing target sequences per 10s to
 106 colonies from the  environmental  community (i.e.,  food
 products) which rivals detection limits with radiolabeled
 catabolic plasmid probes (1 colony per 106 colonies from natural
 soil; see,  e.g.,  Sayler e£ aJL> , 1985).
EASE OF USE:  Nonradioactive probes are the easiest to use
because their longer storage life (up to 6 months) compared to
the common radioactive labels  (approximately 2 weeks) allows a
significantly larger number of hybridizations to be carried out.
In addition, radiolabeled probes require special handling
procedures and protective clothing.  Although certain
nonradioactive probes require relatively extensive post-
hybridization washing procedures, visualization of the
hybridization signal can often be accomplished in less than one
hour as opposed to overnight time periods typical with
radiolabeled probes.

COST:  The cost of purchasing radioactive nuclides is
significantly more than that of reagents or kits available for
nonradioactive labeling.  In addition specials materials and
equipment are necessary for handling radioactive compounds.   As
mentioned above, nonradioactive probes have a longer storage life
which also reduces their cost.

EFFECTS OF ENVIRONMENTAL MEDIA:  Non-specific hybridization can
be a problem with nonradioactive probes, particularly biotin-
labeled probes.  In addition to non-specific hybridization due to
impurities in the environmental sample, proteinaceous compounds
from the lysed cells can react with the avidin-phosphatase moiety
used to visualize the bound probe when hybridizations are
performed on membrane filters.  It is for this reason that
radiolabeled probes continue to be the method of choice in
microbial ecology for hybridization.

 (ii)  Probes  from random or defined nucleotide  fragments

 DESCRIPTION:    DNA probes  are generally derived  from two  sources.
 DNA isolated from the microorganism of interest  can be isolated
 from total genomic DNA  or  from plasmid DNA, subjected to
 restriction  digestion,  and either  an uncharacterized or random
 fragment  is  selected or a  specific or defined  fragment is used in
 the construction of a labeled probe.

 Randomly  generated DNA  fragments are screened  for the intended  .
 specificity.  Table 7 lists several cases.  These probes  can  be
 specific  for a  species  (Totten et  al.. 1983) or  only for  certain
 strains (Tannock,  1989).   The function of the  gene detected by
 the random DNA  probe is not known.  It may be  part of an
 important ubiquitously  distributed gene or of  a  DNA sequence
 without any  essential function.  Therefore nothing is known about
 its stability or genetic variability.  However,  lateral transfer
 of  these  genes  has to be ruled out before these  probes are
 considered reliable tools.

 Defined DNA  probes are  obtained through the selection of  a
 specific  restriction digestion fragment from either plasmid or
 genomic DNA.  The choice of which  restriction  fragment gives
 optimal sensitivity and specificity is based on  knowledge of  the
 genetic map  of  the microorganism and the use of  restriction
 fragments from  genes, with  relatively unique nucleotide sequences.
 Specific  target DNA sequences can  be cloned into plasmid  vectors
 for production  of large quantities of probe DNA.  For example,
 probes to the 16 S ribosomal DNA have been used  to classify
 organisms (Jain,  et al.. 1988).  For those microorganisms that
 are not well  characterized genetically, an empirical approach
 must be employed,  e.g.,  by the preparation and analysis of a
 genomic DNA library in  phage vectors.

 Nick-translation is the most widely used method  for incorporating
 labeled nucleotides into double-stranded DNA.  This procedure is
 based on  the  ability of DNA polymerase I to add  nucleotide
 residues  to the  3*-hydroxyl terminus  of a  nick while  removing
 nucleotides from the adjacent S1 - phosphoryl-terminus (Heinkoth
 and Wahl,  1987).   The nicks are generated with DNAase I.  A
 second technique is random priming.  The basis of this technique
 is  the ability of DNA polymerase I to copy single-stranded DNA
templates primed with random hexamers prepared from calf  thymus
DNA.  A second method to obtain single-stranded probe uses Ml3
phage.  The insert is cloned into M13 phage.  The plus strand  of
the phage is then  used  as  a template to synthesize the probe
using DNA polymerase I  and  labeled nucleotides.

SENSITIVITY:  Specificity and sensitivity of DNA probes
constructed front restriction fragments varies considerably
depending on the size of the probe, the amount of sequence
homology between the DNA probe and target sequence, the  labeling
method and stringency conditions that are chosen, among  other
factors.  In general, restriction fragment probes constructed
from nucleotide fragments of 200 bp or more can be highly
sensitive in DNA hybridizations.  The sensitivity is determined
by the amount of label that can be incorporated into the nucleic
acid  (Atlas and Sayler, 1988).  Oligonucleotide probes  (up to  100
nucleotides; Wallace and Hiyada, 1987) are more specific.   More
radioactivity and thus a higher specific activity can be
incorporated into longer probes (nick translation produces
fragments between 500-1500 nucleotides; Heinkoth and Wahl, 1987).
Oligonucleotides are end-labeled to incorporate one labeled
nucleotide per molecule.  Using 5' (alpha KP) ATP for example,
108 cpm/microgram DNA can be incorporated.   With random priming,
probes that have specific activities of 10* cpm/microgram DNA can
be obtained.  By preparing probes from H13 phage, specific
activities of 109 per microgram of DNA can be obtained.   Probes
are added to the hybridization filter in excess.  Double-strand
probes must first be denatured to permit single-strand probes  to
anneal to the target DNA.  During hybridization, double-stranded
probes can reanneal, thus decreasing the effective amount  of
probe available to bind to the immobilized target DNA.   With
single-strand probes, reannealing is not a problem.

By extracting bacteria from the soil and probing without pre-
culturing, Holben e£ al. (1988) were able to detect 4.3  X  10*
cells of Bradyrhizobium -iaponicum per gram of soil or 0.2
picograms of DNA per 1 microgram of total DNA using a single-
stranded 32P-DNA probe prepared from Ml3.  Other factors  that
affect sensitivity are the sequence complexity, the abundance  of
target sequences (i.e., are they multiple copies?), and  the type
of label (i.e., is it radioactive or biotinylated?).
Inaccuracies in microorganism counts can result if changes in
copy number of the target sequence occur as a result of
environmental influences.

SPECIFICITY: Specificity is increased only if the DNA (or
specific gene sequences), rather than the whole plasmid, is, used.
For example, the insert DNA can be purified by restriction
digestion and isolation of the fragment using gel
electrophoresis.  Nick translation or random priming can then  be
used to label the fragment.  Alternatively, synthesis off of a
M13 phage template primed proximal to the insert results in
insert-specific probes since synthesis does not proceed across
the entire plasmid.  A probe that is limited to a fragment of  the
gene rather than the whole gene or a whole plasmid carrying the
gene is more selective, since in the latter case considerable
cross-hybridization and non-specific reactions can occur (Sayler


       1985).  Tables 10 and 11 list examples of the use of
defined polynucleotide gene probes.

EASE OF USE £ TIME: This is dependent on the labeling procedure
and the type of label used.  Some laboratories find the use of
radioactivity problematic; however, radioactive nucleotides allow
for the maximum sensitivity.  Increase probe specificity can be
costly in terms of ease and .time.  For instance single-strand
probes require cloning into M13.  The isolation of fragments from
a plasmid is time-consuming and cumbersome but results in a more
specific probe.

COST:  The cost incurred is from the purchase of enzymes and
labeled nucleotides.


 (iii)  Oligonucleotide probes

 DESCRIPTION: Synthetic oligonucleotides are short chemically-
 synthesized nucleic acids.  The oligonucleotide corresponds to a
 defined nucleotide sequence.  It can be up to 100 nucleotides  in
 length but in practice is usually between 15 and 30.  One
 advantage of this technique is that if only the amino acid
 sequence is known, a mixture of oligonucleotides can be
 synthesized based on the deduced nucleotide sequences  (Wallace
 and Miyada, 1987).  For hybridization, the oligonucleotides are
 most commonly end-labeled with T4 polynucleotide kinase.  This
 enzyme adds gamma-32? ATP to the free 5 '-OH end of the
 oligonucleotide  (Wallace and Miyada, 1987; Berent £t al. , 1985).
 A second method  for labeling is primer extension using Klenow
 fragment (DNA polymerase I; Wallace and Miyada, 1987; Berent et
   , 1985).
8EMSITIVITY:  End-labeled oligonucleotide are 30-100 fold  less
sensitive as hybridization probes than nick-translated DNA
fragments  (Berent et al. , 1985).  The extent of the decrease  in
sensitivity varies with  the method  (dot or Southern blots) and
target nucleic acid being probed  (RNA or DNA) .   One reason for
this decrease in sensitivity is inherent in end-labeling.  In
end-labeling, only one labeled phosphate can be added per
molecule of probe whereas in nick-translation, 40 to 80 labeled
phosphates are added per molecule of probe with one of the four
nucleotides labeled.  The decreased sensitivity resulting  from
end-labelling can be alleviated to some extent with the use of
primer extension via Klenow fragment which results in labelling
of all four nucleotides.  Extrapolating from data using a
synthetic oligonucleotide to detect rat ribosomal DNA (Berent et
aJL. , 1985) , an end-labeled oligonucleotide can theoretically
detect 1 microbial genome out of 10*.  Inaccuracies in
microorganism counts can result if changes in copy number  of  the
target sequence occur as a result of environmental influences.

SPECIFICITY:  The main advantage of using an oligonucleotide
probe is specificity. Specificity is dependent on the length  of
the probe: the longer the probe is, the lower the probability
that a cross-reactive, sequence will appear with a match to the
total contiguous sequence contained in the probe.  However, this
is balanced by the higher probability of matches between small
contiguous sequences within the probe.  These partial duplexes
will be less stable than the more extensive match between  the
target sequence and the  oligonucleotide.  Thus, oligonucleotides
can be designed so that  hybridization and subsequent washing  of
the blot can be performed under very stringent conditions. Longer
probes are more stable and can be hybridized under higher
temperatures, i.e. have  a higher Td,  the temperature at which
one-half of the duplexes are dissociated (Wallace and Miyada,
1987) .   Therefore, a probe can be designed to alleviate any
problems of cross-reactivity if organisms containing sequences


closely-related to the target sequences are expected to be
isolated with the organism of interest.  Another factor that can
affect specificity is the GC content.  The higher the GC content,
the higher the T^.  However,  oligonucleotides containing high GC
contents exact other problems such as self-complementarity which
interferes with 5' labeling and purification during synthesis of
the nucleotide itself (Wallace and Miyada, 1987).

EASE OF USE ft TIME:  The oligonucleotides must be synthesized.
This can be done commercially or within the laboratory. The
synthesis is automated, but it is technical and time-consuming.
For example, the probe must be labeled (however labeling is
usually a simple procedure with few steps involved).  The use of
radioactivity as a label may be a problem for some labs.

COST:  The primary cost is due to the synthesis of the
oligonucleotide.  The cost of synthesis is dependent on the
length, i.e. the number of nucleotides that must be incorporated.
Synthesis can be done commercially or in the laboratory.
Synthesis requires an automated DNA synthesizer which is very
expensive. Therefore unless the use of oligonucleotides as probes
and primers are routine, it is more cost-effective to have the
oligonucleotide synthesized commercially.   Additional cost is
incurred with buying labeled nucleotide and enzymes that carry
out the labeling reaction.


 (iv)   Transcription probes

 DESCRIPTION:   Single-stranded RNA can be prepared for use as
 probes.   Transcripts are synthesized in yi&g. using a
 transcription vector.  The nucleotide sequence is cloned into one
 of the multiple sites in the polylinker of this specialized
 vector.   The  polylinker is 3' to the promoter so that cloning
 into one of these sites orients the insert with respect to the
 promoter.  The alignment with respect to the promoter allows the
 transcription of the DNA so that one of the two strands will be
 transcribed.   The most commonly-used promoters are from
 bacteriophage SP6, T3, and T7.  Transcription via a DNA-dependent
 RNA polymerase is performed in yJLtEfi in the presence of all four
 nucleotides,  one of which is labeled (Ford and Olson, 1988).
 Some vectors  are constructed so that the polylinker lies between
 two different promoters so that transcripts of either polarity
 can be obtained.

 SENSITIVITY:    Probes of relatively high specific activity can be
 prepared, greater than 2 X 108 cpm per microgram of RNA  (Wahl, s£
 aj,.,  1987).   In addition,  the use of asymmetric probes increases
 hybridization efficiency since the complementary strand is not
 present  in the hybridization mixture.  The ability to obtain
 large quantities of high specific activity probe and the ability
 to limit the  annealing of the probe to only the target DNA
 increases the sensitivity of RNA probes relative to double-
 stranded DNA  probes.

 SPECIFICITY:    Specificity is dependent on the extent of
 complementarity between the probe and the target DNA.
 Sensitivity and specificity are increased because the probe is
 single-stranded and does not reanneal to itself.

 EASE OF  USE § TIME:   The main disadvantage of using RNA probes
 is related to the extra cloning steps required to construct the

 COST:    The major cost incurred is from the purchase of the
 labeled  nucleotide.  A secondary cost is from the purchase of the
 RNA polymerase.   Overall,  the cost is equivalent to that incurred
.using the nick-translation procedure.


 (b)  Hybridization techniques

     These methods provide different formats which allow nucleic
 acid probes to anneal to target DNA.

 (i)  Colony Hybridisation

 DESCRIPTION:  This hybridization technique allows the screening
 of individual colonies isolated from an environmental sample.  It
 can be used for screening microorganisms when traditional
 culturing techniques are insufficient to identify the introduced
 organism, for example screening for a bacterium containing beta-
 lactamase as a marker gene in a field of "naturally-occurring"
 ampicillin resistant bacterium.

 The technique involves first culturing the microorganisms in the
 laboratory on an appropriate medium.  A solid medium is prepared
 that will support growth and allows for the selection and/or  .;
 enrichment of the microorganism and a nitrocellulose filter is
 placed on top of it thereby allowing the filter to become
 impregnated with an aliquot of the microorganisms.  The plate is
 incubated at the appropriate temperature to allow the bacteria to
 grow. The growth conditions, temperature and time, will be
 dependent on the bacteria of interest, for £. coli 8 to 10 hours
 at 37° c is recommended.  It is probably necessary to maintain the
 selection or enrichment at this step even if the original
 culturing of the organism contained it.

 This technique was developed to screen libraries in £. coli. a
 rapidly growing organism.  Most organisms introduced into the
 environment will be relatively slow growing and if the
 nitrocellulose is not presterilized, contaminants may be
 introduced via handling the filters and may grow out if a long
 time span is required to grow the bacterium.  After the
 appropriate incubation time,, the filters are removed and replica
 plated onto other hybridization membranes.  The master copy is
 then reincubated on fresh media to regenerate the colonies.  This
 plate is the master plate to which the results from hybridization
 of the replicas can be referred (Vogeli and Kaytes, 1987).

 Depending on the efficiency of transfer and the amount of
 replicas made from each master, it may be necessary to incubate
 the replicas on fresh media to reach a cell density high enough
to obtain a signal sufficient for the detection of the nucleotide
sequence of interest.  Before hybridization the cells must be
lysed.  Again these techniques have been developed for £. soli.
and may have to be adjusted for the organisms of interest,  van
Elsas eJt al. (1989) found that by adding an extra lysozyme step
to the standard procedure, they were able to improve the lysis
efficiency of soil isolates of Pseudomonas fluorescens.  The
membranes are pretreated with SDS to remove debris that can

 interfere with hybridization.  The cells are then lysed in  situ
 and the DNA  is denatured in an alkali solution.  The alkali
 solution also destroys the RNA which can interfere with
 hybridization.  The cells are placed in a neutralizing solution.
 If nitrocellulose is used, the membranes are baked to fix the
 DNA. In order to remove remaining debris, the baking is done in
 an EDTA and  SDS solution.  If a nylon filter is used, baking is

 The probe .can be composed of either RNA or DNA; an isolated DNA
 fragment, the whole plasmid, or a synthetic oligonucleotide; and
 be radiolabeled or labeled with a nonradioactive marker.  The
 conditions of the hybridization will depend on the probe used,
 the TD of the duplex,  the complexity of the nucleotide sequence,
 the match of the probe to the target sequence, and the cation
 concentration (Wahl and Berger, 1987).  The stringency of the
 wash is dependent on the specificity of the probe for the target
 sequence; i.e. the extent of match to the target sequence versus
 the presence of cross-reacting sequences in any of the background

 SPECIFICITY:  The specificity of this technique is dependent on
 the ability  of the probe to differentiate between the target
 sequence and cross-reacting sequences in background
 microorganisms.  Because of carry-over of components of culture
 media on the hybridization membrane such as salts and impurities
 from cell lysis, i.e., hybridization is in situ and the DNA is
 not purified away from the cellular debris, there are artifactual
 spots that arise that can be misinterpreted as false positives if
 they coincide with colonies (Amy and Hiatt, 1989).  It is
 therefore necessary to hybridize, at the minimum, duplicate
 copies to identify artifacts, i.e. a true signal will be show up
 on both copies.

 One advantage of the technique is that it detects only live
 organisms and there is a confirmatory method of identification,
 i.e., biochemical or immunological.  Thus, the target DNA can be
 positively associated with a given strain and/or species and it
 can be determined if conjugation or transduction into new strains
 or species had occurred.  The technique is inexact because  of the
 impurity of  the target DNA and therefore is limited to screening.
 If a more exact procedure is needed, the potential positives can
be subjected to a more involved analysis such as hybridization of
Southern blotted DNA for verification.

 SENSITIVITY:  The sensitivity of the technique is limited because
 of the need to culture out the organism.  Thus this step is
 limited by the ease in which the bacteria can be grown under
 laboratory conditions.  Organisms that are hard to grow may be
underrepresented in this type of study.  Also, the technique is
 limited by the inability of most cultivation methods to isolate
but a small percentage of the total microbial population at the


time of sampling  (Jain et a1.f 1988; Steffen et al-,  1989).   For
those organisms that can be grown  in culture, the method has  the
potential to be 4 to 5 times more  sensitive than identification
using traditional culturing techniques  (Jain et al.,  1988).   Also
because it is sometimes difficult  to reisolate cells  on a
selective/differential media, the  cells can be isolated on an
enriched medium and then identified.

It has been shown that detection of 1 cell in 106 colonies is
possible with this technique  (Jain et al.. 1988).  Thus, this
technique has the potential to allow for  a more sensitive
quantitation than traditional plate counts.  In binary cultures
of Pseudomonas putida and £. coli. Savior e£ al. (1985) were  able
to detect 1 colony of £. putida in 10* nonhomologous cells using
a nick translated KP-DNA probe specific for the TOL catabolic
plasmid.  In a fresh water microcosm containing Alcaliaenes A5
and Pseudomonas cepacia. Steffen e£ &1. (1989b) found the  limits
of detection for the bacteria to be 102 cells/ml using either a
whole plasmid (Alcaligenes) or a species  -specific  (£. cepacia)
radiolabeled probe.  The limit of  detection for Tn£-marked
Azospirilum lipoferum in soil was  found to be 103 colonies/ gram
of soil using a radiolabeled plasmid probe containing the  npt II
gene (Bentjen et al., 1989).                                     .

EASE OF USE & TIME:  Of all the hybridization techniques,  this
methods can generally be the easiest, fastest, and least
expensive (Sayler and Layton, 1990).  The speed of this method is
limited by the growth rate of the  organism.  Large numbers of
colonies can be screened simultaneously,  thus saving  time.
Another time saving factor is the  small amount of sample
preparation once the organism is grown, i.e. ONA does not have to
be isolated and purified.

COST:  The cost is the same as traditional culture methods plus
the cost of probe preparation (see above  discussions).

EFFECTS OF ENVIRONMENTAL MEDIA:  The environmental limitation of
this technique is the same as that encountered with traditional
isolation techniques, i.e. the growth of the organism on solid
media .under laboratory conditions.  The ease of lysis of the
bacterium could also be a limiting factor with those organisms
that are difficult to lyse because of the presence of a capsule,
slime layer etc.  Moreover, the ability of the bacterium to
adhere to the membrane support because of an extracellular layer
may be a factor.
Colony hybridization was designed to screen high density
libraries contained in a pure culture (Sayler and Layton,  1990).
Screening environmental samples represents a different
application.  One major problem encountered is colony density
(Sayler and Layton,  1990).   The probe must be able to select  a
specific colony in a dense heterogeneous background.  A problem
can be encountered with environmental samples such as soil in


which their is an abundance of microorganisms and the target
organism is a small percentage of the total.

Conversely, in dilute aqueous environments the same problem
arises because a concentration step must be included.  Steffen
(1989a) found that if the target organism is less than 0.1% of
the total viable population than the plates are totally overgrown
by non-target organisms.   For hybridization analysis, the ideal
number of cells per plate is between 30 and 300. Dilutions to
obtain this range will exclude the target organism if the total
population is three orders of magnitude higher than the target
organism (Steffen et al. 1989b).   Often the colonial morphology
and size of environmentally isolated colonies limit the
application of this technique (Sayler and Layton, 1990). In a
study in which genetically-engineered £. co}i was introduced into
lake water and allowed to compete with the indigenous organisms,
it was found that after a few days the indigenous organisms
overwhelmed the introduced one that was barely visible on the
plate.  A combination of greater cell volumes and selective
pressure can alleviate this problem (Amy and Hiatt, 1989; Steffen
ejt ai. I989b).  Some organisms or a percentage of the introduced
organism may be non-culturable (Sayler and Layton, 1990).  In
some cases, high cell densities are needed for cross-feeding in
the initial isolation thereby hindering the ability to achieve
colony counts low enough for hybridization (Mancini, 1987).

 (ii)  in situ Hybridization

 DESCRIPTION:  This technique takes advantage of the observation
 that  fixing whole cells makes them permeable to oligonucleotide
 probes. Identification of single microbial cells can then  be
 achieved with radiolabelled, rRNA targeted, group-specific
 oligonucleotide probes (Giovannoni e£ fli. , 1988) that
 specifically hybridize to rRNA in situ.   Identification can then
 be achieved without the need for cell lysis or nucleic acid
 extraction.  The method is comparable to  staining with
 fluorescent dyes (e.g. DAPl, Acridine Orange) or the
 immunofluorescence approach, in that the  visualization of  the
 results require an epi fluorescence microscope or a flow cytometer
 (Amann et al. , 1990b) .  Flow cytometry has the potential for
 rapid identification, counting and sorting of specific
SENSITIVITY MID SPECIFICITY:  De Long fit al.  (1989) could
demonstrate that fluorescently labelled oligonucleotides offer
not only all the advantages of a non-radioactive label but are
even more sensitive than radioactive probes.  In situ detection
of intestinal microorganisms has been shown in the extremely
complex cecum content as well as differentiation at the genus, :
species and subspecies level (Amann fit aj,. , 1990a) .  The method
has not been extensively used for environmental samples so far,  '
but has several promising aspects:  (a)  cultivation of the
organism of interest is not required for probe design and
detection, (b) probe specificity is not restricted to strains or
species but also larger phylogenetic groups of organisms may be
studied (e.g. all eubacteria, SRBs) , (c) physiological activity
is correlated to the rRNA content (De Long s£ at. , 1989) and
therefore to the amount of bound probe per cell, and (d) the
probe is synthesized chemically, quickly and inexpensively.
Problems in the use of fluorescent, rRNA-targeted oligonucleotide
probes are:  auto- fluorescent background in some environments and
low sensitivity (10* cells per g of soil are required for
detection) .'

EFFECTS OF ENVIRONMENTAL MEDIA:  In some environmental matrices,
background auto- fluorescence may interfere with identification.

 (ii)  Dot/slot blot

 DESCRIPTION:  This, procedure involves the immobilization of
 nucleic acid onto  a solid support matrix such as nitrocellulose
 or nylon.  Like the colony lift technique, it is a screening
 procedure for the  detection of the inserted DHA.  It does not
 require isolated cells but rather the DNA can be extracted either
 directly from the  environmental sample itself or from batch
 culture grown from the environmental sample.  RNA can also be
 immobilized though it is more unstable than DNA and not likely to
 be isolated directly from the environment.  For direct detection,
 the DNA can be isolated and enriched from the environmental
 sample using known techniques such as that published by Sayler
 and Barkley (1987).  Alternatively, cells can be lysed and DNA
 partially purified from bacteria separated out of the
 environmental sample, i.e., without laboratory culturing (Holben
 et aJL., 1988).

 The DNA must be made single stranded for it to adhere to the
 support matrix and thus the first step must be either a
 denaturing step or if a covalently-closed circular plasmid
 contains the target sequence, digestion to a linear form and then
 denaturation (Costanzi and Gillespie, 1987).  If enrichment for
 the insert has been performed by culturing techniques and a few
 microliters of sample is sufficient to verify the presence of the
 microorganism, purified denatured DNA can be applied directly to
 the filter.  Under most circumstances, a much larger quantity of
 sample must be applied and it is necessary to use a
 minifiltration device such as a minifold or slot blotter which
 allows the application  of the sample under vacuum.  With
 nitrocellulose, the blot is baked to fix the DNA before
 hybridization. It  is unnecessary to bake nylon filters prior to

 All types of probes can be used with this technique, either RNA
 or DNA, random or  defined nucleic acid fragments, whole plasmids
 or synthetic oligonucleotides, and radiolabeled or .labeled with a
 nonradioactive marker.  The conditions of the hybridization will
 depend on the probe used, the T_ of the duplex,  the complexity of
 the nucleotide sequence, the match of the probe to the target
 sequence, and the  cation concentration (Wahl and Berger, 1987).
 The stringency of  the wash is dependent on the specificity of the
 probe for the target sequence; i.e. the extent of match to the
 target sequence versus the presence of cross-reacting sequences
 in any of the background microorganism.

 SPECIFICITY:  The  specificity of the technique is dependent on
 the probe used and the nature of the target DNA.  If the DNA is
 isolated directly  either out of the soil or from cells separated
 from the soil, it  is not possible to determine whether any
 positive signals are from viable cells or from extraneous DNA.
Thus, it is not possible under these conditions to associate a


 positive signal with the presence of  the  introduced genetically
 engineered microorganism.  It is  likewise not possible to
 determine if the DNA of interest  is still contained in the
 introduced organism or has  been transferred via conjugation for
 example to another strain or species.

 The amount of target DNA sequence is  low  in relation to the
 background,  especially if the DNA is  probed directly from the
 environmental sample and an enrichment step for the recombinant
 organism has not been performed.   Thus, a large amount of sample
 must be loaded  onto the hybridization membrane.  This  can result
 in  non-specific hybridization and false positives.   Also,  since
 the sample is loaded in bulk,  cross-reactive sequences from
 indigenous microorganisms cannot  be differentiated  from the
 sequences of interest.   Washing the membrane under  conditions  of
 high stringency helps reduce background caused by cross-reactive
 sequences.   The probe is extremely important in this regard.   The
 sequences chosen should be  unique to  the  target DNA and there
 should  be a perfect match,  if possible, between the probe and  the
 target  sequence.

 The stability of the duplex is dependent  on the extent of
 matching sequences between  the hybridizing nucleic  acid
 molecules.   Thus the higher the complementarity between the
 molecules the higher the TB/ and the more stable the duplex at
 higher  temperatures.   Salt  helps  to stabilize duplexes thus
 reducing the salt concentration will  destabilize imperfect
 matches.   If the probe is perfectly matched to unique  insert
 sequences,  the  wash can be  performed  under high stringency
 conditions,  high temperature/  low salt, thus destabilizing any
 duplexes formed by hybridization  of the probe with  similar but
 non-identical sequences from non-target organisms.

 SENSITIVITY:  The sensitivity of  the  technique is dependent on
 the probe and also the way  the DNA is isolated.  One advantage of
 the technique is that many  bacteria do not survive  the transfer
 from the environment to laboratory media.  By direct probing,
 higher  numbers  of bacteria  can be sampled.  By extracting
 bacteria from the soil  and  probing without pre-culturing,  Holben
 et  al.  (1988) were able to  detect 4.3 X 10*  cells of fi.
per gram of soil or 0.2 picograms of DNA per 1 microgram of total
DNA using a single-stranded RP-DNA probe.  They reported that
their technique can possibly detect as few as 10* cells per gram
of soil.  Steffen and Atlas (1988) reported the detection of 103
cells of £. ceoacia per gram of sediment using a nick- translated
species-specific "P-labeled isolated DNA fragment as a probe
(repeat sequence, 15-20 copies/ cell, RS-1100-1 that is located
on plasmids and chromosomally) .  To detect £. celi and  £. putida
containing a xylE marker in lake water, Morgan et al. (1989) ,
used an 822 base-pair fragment labeled .with [32P]dCTP by random
priming.  The limit of detection using this fragment for dot blot


analysis was 10* CFU/ml.  In the same study,  the limit of
detection in an ELXSA analysis was 10s CFU/ml.

A major limiting factor is the amount of OKA that can be bonded
onto the nitrocellulose before the membrane is saturated (Steffen
and Atlas, 1988).  Since the amount of DNA attributable to the
introduced microorganism is low compared to background, it may
not be possible to load enough material to exceed the limits of
detection.  In many instances, the low sensitivity will be a
problem if there is a need to determine die-back, i.e., survival
in low numbers (Morgan  et al.. 1989).  For example if there is a
need to determine survival in reservoirs or overwintering of
plant pathogens.

EASE OF USE & TIME:  This technique is relatively easy however it
does involve processing of the environmental sample before
hybridization.  The ease of the technique is dependent on the
complexity of the DNA extraction procedure and/or the cell
separation procedure used.  In those cases where the cells are
first cultured, the DNA must still be isolated however extensive
DNA purification such as CsCl gradients is usually not necessary.
The other procedure that is factored into the complexity is probe

COST:  It allows multiple samples to be tested at the same time,
which makes this method cost-effective.  The major cost factor is
incurred with probe labeling.

EFFECTS OF ENVIRONMENTAL MEDIA:  It is advisable to use an
isolation or enrichment protocol for the DNA or cells in order to
obtain enough DNA to be within the limits of detection and to
prevent adding extraneous contaminants such as salts and acids
that can interfere with hybridization.  A major problem with
isolating DNA from soil samples is purity (Sayler and Layton,
1990).  Steffen et aj..  (1988) found that high levels of humic
acid inhibited the detection of target DNA by dot blot analysis.
However, low amounts of humic acid and other contaminants such as
clay did not interfere with detection by dot blot analysis.
False positives can be  obtained by non-specific binding to soil
particles (Frederickson et fll., 1989).  Filtration methods need
to be applied to aqueous matrices.  However even so, the
sensitivity of the technique may not be sufficient to detect
organisms in water where bacteria are maintained in low
concentrations (Saylor  and Layton, 1990).

If a sufficient concentration of DNA can be isolated, DNA from
water samples is usually of high-purity and large-fragment size
(Sayler and Layton, 1990).  One environmental factor that all
direct DNA detection methods circumvent is the problem with
organisms that are either non-culturable or difficult to isolate
(Sayler and Layton, 1990).


 (ill)  Most-Probable  Number-Hybridization

 DESCRIPTION:   Frederickson  et  al.  (1988)  developed methodology to
 enumerate  the  number  of  colonies by dot blot analysis  by
 combining  it with the most-probable-number technique.   Batch
 cultures are grown  in either selective or non-selective media  and
 diluted out to infinity  in  microtiter plate.  The dilutions are
 then transferred by aid  of  a filtration manifold onto
 nitrocellulose or nylon  filters, lysed in situ, and hybridized.
 This technique allows cell  enumeration without colony  counts.
SENSITIVITY & SPECIFICITY:  Frederickson fit al.  (1989)  found  that
the limit of detection of an introduced £. putida and biovars of
£. lecruminosarum containing Tn5 was 10 and 10Z , respectively ,
cells per gram of soil using selective medium  and probing with a
32P-labeled plasmid probe.   The specificity is  dependent on  the
probe .

EASE AND COST:  Comparable to dot blot analysis  (see above
discussion) .

EFFECTS OF ENVIRONMENTAL MEDIA:  The guantitation depends both on
the ability to recover viable cells in culture and the
interactions of DNA with environmental contaminants
(Frederickson, ejfe al. , 1989) .  These workers found that over  time
enumeration of cells  being recovered from soil by MPN-
hybridization was lower than the counts determined by florescent
antibody enumeration.  Moreover, using £. putida marked with  Tnj>,
they found that cell  counts  were inversely proportional to clay
and organic matter content.  Steffen et al. (I989b) found this
technique to give highly variable results in their monitoring of
£. ceuacia introduced into a freshwater microcosm.

 (iv)  Solution Hybridisation

DESCRIPTION:  This technique involves the hybridization in
solution of DNA with a specific probe.  It is a screening
procedure.  As in the dot blot procedure, DNA is extracted
directly from the environmental sample or from batch cultures
thus avoiding the need to obtain isolated colonies.  The DNA is
isolated from the sample directly (Steffen and Atlas, 1990; Jain
et al.. 1988) or after enrichment steps for bacterial cells
 (Holben e£ al.. 1988); or from a culture(s) grown from
environmental samples.  The DNA is then mixed with the probe and
allowed to hybridize under standard conditions required for the
type of probe used and the DNA being targeted (eg. 12 h\50° C).
At the end  of the hybridization period, the probe must be
removed.  This can be done with a combination of RNasc and SI
nuclease to remove single stranded nucleic acid (if the probe is
RNA) or hydroxyapatite chromatography (separates single from
double stranded nucleic acid).  The duplex DNA is recovered by
ethanol precipitation and any free nucleotides are removed with a
Sephadex 6-25 column.  The detection of positive cells is
dependent on how the probe was labeled eg. measuring the amount
of radioactivity in a scintillation counter.

SPECIFICITY:  As in other hybridization techniques, the
specificity of the technique is mainly dependent on the probe
used and the nature of the target DNA.  This technique does not
detect viable cells unless a preculturing step is included.
Like, the dot blot it is not possible to determine whether any
positive signals are from viable cells or from extraneous DNA if
this preculturing step is not included.  Thus, it is not possible
under these conditions to associate a positive signal with the
presence of a specific microorganism.  It is likewise not
possible to determine if DNA sequences of interest are still
contained in the introduced organism or has been transferred via
conjugation for example to another strain or species.

Non-specific binding of the probe to sequences with some
percentage of matching base pairs as a result of omission of the
high stringency wash steps used with membrane hybridization
procedure may give false positives.  The purpose of the high
stringency washes is to remove any  duplexes formed by cross-
reaction of the probe to similar sequences in DNA from non-target
organisms.  This procedure does not correct for this cross-
reactivity. The specificity may be helped somewhat because there
will be no non-specific binding of the DNA to the membrane itself
or to an overloaded spot of DNA on the membrane.

 SENSITIVITY:   Since the amount of DNA present  in the  introduced
 microorganism is low in relation to  the background, a
 concentration of total  must be used  that  is within the  limits  of
 detection.  Therefore,  more DNA can  be used in this procedure
 than in procedures that utilize DNA  binding to a matrix support
 system.   Steffen and Atlas  (1990)  reported detection  limits  in
 the range of  10Z to 103  cells of £. ceoacia per gram of  sediment
 using a T7-generated radiolabeled RNA probe.   This is
 approximately 10-fold more  sensitive than that reported for  the
 dot blot procedure.

 EASE OF USE £ TIME:   This technique  is relatively easy  however it
 does involve  processing of  the environmental sample before
 hybridization.   The ease of the technique is dependent  on the
 complexity of the DNA extraction procedure and/or the cell
 separation procedure used.   In those cases where the  cells are
 first grown-up in the laboratory,  the DNA must still  be isolated
 however extensive DNA purification such as CsCl gradients is
 usually not necessary..

 The other procedure that must  be factored into complexity is
 probe preparation.   The types  of probes that can be used is  more
 limited.  Either an RNA probe  generated by transcription from  a
 vector designed for in  vitro transcription or  a single-strand  DNA
 probe generated from a  H13  phage system must be used.   The reason
 single-stranded probes  must be used  is that the step  to remove
 the labeled probe  so that it will  not interfere with  the
 detection of  true  hybrids depends on the  separation of  single-
 stranded nucleic acids  from duplexes.  Nick-translated  DNA probes
 rehybridize during the  hybridization step thus forming  duplexes
 that can not  be differentiated in solution from the target duplex
 by  procedures such as nuclease digestion  or hydroxyapatite
 chromatography.  Thus,  this procedure introduces the  necessity
 for an extra  cloning  step to prepare the  vector.  Column
 chromatography steps  to replace the  washing of the hybridization
 membrane  can  be more  cumbersome.   Since the kinetics  of
 hybridization are  faster in solution, this procedure  can be
 adapted to yield more rapid assays than those  using a solid-
 support matrix (Sayler  and  Layton, 1989).

 COST:  In addition to the cost of  probe preparation,   expensive
 equipment must be used  such as a liquid scintillation counter or
 fluorometer since the procedure does not  involve visual  detection
 of  label  on a solid-support matrix or an  autoradiogram.  Also
 column chromatography can add  to the expense.

EFFECTS OF ENVIRONMENTAL MEDIA:  Extraneous material such as
acids, alkalis, and salts can  interfere with hybridization
therefore it  is necessary to isolate and purify the DNA.  The
major limitation in this regard is the ability to purify the DNA


from a given environmental medium or the ability to transfer
bacteria out of that medium and into a laboratory medium.

 (v)   southern blot analysis

 DESCRIPTION:   This technique  also uses hybridization with  a
 specific probe to detect DMA  fragments that have been  immobilized
 on a matrix support such as nitrocellulose.  This procedure
 involves digesting the  DNA with a restriction enzyme to  obtain
 fragments of  various sizes which can be  fractionated by  gel
 electrophoresis.   The DNA in  the gel is  than transferred to  a
 nitrocellulose or nylon filter for hybridization with  the
 appropriate probe.   The electrophoresis  is usually done  in an
 agarose  gel and the DNA is transferred to the hybridization
 membrane by passive diffusion by denaturing the DNA in situ  with
 alkali and neutralizing.   The hybridization membrane is  placed on
 the  treated gel and a circuit of transfer buffer is created  to
 allow the flow of buffer from the gel through the filter.  The
 DNA  that is carried in  the buffer is trapped on the filter.
 Alternatively for small fragments that must be fractionated  by
 poly acryl amide gel  electrophoresis,  the transfer is achieved by
 applying current  through the  system.  After the DNA is deposited
 on the membrane,  it is  denatured and in  the case of
 nitrocellulose, fixed onto the filter by baking (Wahl  et al.
 1987; Jain et al. ,  1988).
The probe can be either RNA or DNA, an isolated DNA fragment, the
whole plasmid, or a synthetic oligonucleotide; radiolabeled or
nonradioactive.  The conditions of the hybridization will depend
on the probe used, the TR of the duplex,  the complexity of the
nucleotide sequence, the match of the probe to the target
sequence, and the cation concentration (Wahl and Berger, 1987} .
The stringency of the wash is dependent on the specificity of the
probe to the target sequence; i.e. the extent of match to the
target sequence versus the presence of cross-reacting sequences
in any of the background microorganism.

SPECIFICITY:  The specificity of the technique is mainly
dependent on the probe used and the nature of the target DNA.
The restriction digestion and fractionation adds to the
specificity of the analysis.  The length of the fragment
containing the foreign DNA is predictive for the  restriction
enzyme used.  If the cut is internal in the DNA molecule inserted
into the host, the length of the fragment isolated from any
strain is expected to be the same as in the original plasmid
molecule.  Thus, the original plasmid is digested and
electrophoresed along side of the isolated DNA as a reference.
Because this procedure is more complex than solution
hybridization or dot blots it is usually used for diagnosis and
verification rather than screening.

False positives can be eliminated because a fragment of a
predicted size could not be detected with the probe.  The one
exception would be if the insert had undergone a deletion or
insertion, the fragment length will have changed.  However, false


positives can usually be distinguished by the intensity of the
band after reacting with the probe.  Often false positives are
not the result of one piece of ONA reacting but rather since the
dot blot detects batch DNA, many closely-related sequences
reacting.  Once these sequences are fractionated out, the signal
from the probe is dispersed and therefore much less intense and
often can no longer be detected.  An insertion or deletion does
not usually affect the intensity unless it is very large and
falls within the sequence that is complementary to the probe.  If
the DNA is isolated directly either out of the soil or from cells
separated from the soil, it is not possible to determine whether
any positive signals are from viable cells or from extraneous
DNA.  Thus, it is not possible under these conditions to
associate a positive fragment with the presence of the introduced
genetically engineered microorganism.

It is likewise not possible to determine if the DNA sequence of
interest is still contained in the introduced organism or has
been transferred (via conjugation) for example to another strain
or species.  However, if the cells can be cultured in the
laboratory, Southern blot analysis can be used in conjunction
with cell isolation procedures to verify the presence of the
microorganism or the transfer of the DNA to a new organism.  In
the absence of being able to culture out the organisms, the
movement of the DNA of interest can be discerned.  By using a
restriction enzyme that cuts once within the nucleotide sequence
of interest, restriction length polymorphisms can be detected and
can be one indicator of gene transfer.  Restriction length
polymorphisms can also indicate if the specific sequences have
moved from the chromosome to a resident plasmid or from an
introduced plasmid into the chromosome.   Polymorphisms can also
distinguish between two identically engineered organisms that
have been co-introduced into the environment.

Jansson ft al. (1989) could distinguish between two Pseudomonas
spp. inoculated into a soil microcosm by the mobility of a Clal
restriction fragment containing the engineered nptll gene.
Changes in restriction fragment mobilities can indicate a
deletion or insertion in the target fragment.  Using a marker
gene, Jansson et. al (1989) were able to demonstrate by Southern
hybridization a deletion in their engineered DNA sequence had
occurred in cells after inoculation into soil.  They used a KP
single-stranded DNA probe to marker gene fnot ££) inserted into
Pseudomonas sp. strain B8.  In addition to the major target
fragment, an anomalous band that had a faster mobility than
predicted was detected in Southern blots.

 SENSITIVITY:   Sensitivity is dependent on the 1)  the probe used
 and 2)  the efficiency of transfer.   The efficiency of transfer is
 dependent on  the size of the fragment and the gel concentration.
 Frequently, an acid depurination step is added prior to
 denaturing the ONA in the gel.   The purpose of this step is to
 hydrolyze the DNA and thus increase the efficiency of transfer
 (Maniatis,  1984).   Conversely,  smaller fragments  bind the
 nitrocellulose inefficiently.   To detect a single copy gene in
 mammalian DNA, a minimum of 10  micrograms of DNA  must be applied
 to the  well.   The mammalian haploid genome is 3 X 109 base pairs.
 The bacterial genome is  in the  range of 4 X 106 base pairs.   A
 comparable quantity of DNA represents approximately 1000
 bacterial cells; therefore,  theoretically 1 microorganism in 1000
 cells can be  detected.   In practice Holben et al.  (1988)  found
 that the  hybridization of Southern  blots was 5-fold less
 sensitive than dot blots,  0.02  picograms of the £.  laponicum
 insert  sequence per microgram of total DNA can be detected in dot
 blots using a 32P single  -stranded DNA probe as compared to 0.1
 picograms of  the sequence in Southern analysis using the same
 probe.  The reduction in sensitivity was attributed to the
 inefficiency  of transfer from the gel to the blot.

'EASE OF USE & TIME:   It  is more time consuming and technically
 involved  because it includes a  digestion step(s),
 electrophoresis,  and transfer (usually overnight)  in addition to
 probe preparation and hybridization.

 COST:   Southern analysis is  more costly than dot blots due to the
 restriction enzymes.   In addition,  expense is incurred due to the
 enzymes and labeled nucleotides required for probe preparation.

 EFFECTS OF ENVIRONMENTAL MEDIA:   Purity of DNA is  very important
 in  this technique.   Restriction enzymes are very  sensitive to
 impurities.   Contaminants can alter the sequence  specificity or
 inhibit cutting.   Enzymes are sensitive to glycerol, magnesium
 concentration,  and salts for example.   Steffen s£ fll.  (1988)
 found that humic acid and montmorillonite clay inhibited
 restriction enzyme digestion.   These investigators  also showed
 that trace contaminants  can  also inhibit digestion.  DNA
 preparations  isolated form soil and sediment that were highly
 purified  as determined by the A260/28Q ratios inhibited the
 digestion of  target DNA  by some restriction enzymes.
 Methylation of the DNA can inhibit  cutting.

 (vi)  Polymerase chain reaction

 DESCRIPTION:  Table 12 lists studies in which PCR techniques have
 been used with environmental samples.  This technique  is used  to
 amplify a targeted segment of DNA.  Amplification is accomplished
 by repeated cycles of DNA synthesis primed at opposite ends of
 each complementary strand of the DNA duplex.  The DNA  between  the
 these ends is replicated.   Primers are prepared that  are
 complementary to a short sequence at the 3' ends of each strand.
        5' 	 3'   strand  1
                                      3* 	 5'   primer  1

       3*  	 5«   strand  2
       5'  	 31                                 primer  2

The duplex DNA is denatured and allowed to anneal to the single
stranded primers.  DNA synthesis proceeds off of each target
template via the annealed primers.  The nucleotides are added to
the 3'-OH of the primers, i.e., 5* to 3' chain elongation. The
newly synthesized strand is then removed form its complement by
denaturation and a new cycle of annealing and synthesis begins,
now with double the number of templates so that amplification is

These cycles can be repeated with one reaction mixture 25  or more
times to amplify a targeted sequence 4 X 106 times (Lewin,  1990).
One reaction mixture is prepared for the  25 or more cycles.
Included in this reaction mixture are the nucleotides needed for
polymerization, the DNA preparation, the buffer, the specific
primers, and the DNA polymerase.

The DNA polymerase used in this procedure is isolated from
Thermus aquations f a thermophilic bacterium and is stable  at
temperatures up to 95° c,  the temperature at which denaturation
of the two strands is performed (Gelfand, 1989).  Its optimal
activity is in the range of 75-80° c.  Typically,  the DNA is
denatured at 90-95° C,  annealing of the  primers proceeds at 40-
60° C,  and polymerization is carried out at 72° C.   Heating and
cooling intervals during the 25 cycles is controlled by a
computerized temperature block. The primer length is around 20
base pairs and the fragment of DNA to be amplified can be  up to 2
kilobases though for the purposes of diagnosis one considerable
shorter is adequate.

Recently, two other thermostable polymerases have been identified
that could be used for polymerase chain reaction amplification of
DNA.  The two are the VENT™ DNA polymerase, isolated from  the
thermophile Thermococcus litoralis which inhabits thermal vents
on the ocean floor (Neuner e£ al., 1990), and the PjEa DNA
polymerase from the thermophilic archaebacterium Pyrococeus
furlosis (Bergseid et al., 1991).  These two enzymes, unlike


 DNA polymerase, possess both the 5' to 3' DNA polymerase  and  3'
 to 5*  exonuclease-dependent proofreading activities.  The
 proofreading  activity will excise mismatched 3' terminal
 nucleotides from  a primer:template complex and correctly
 incorporate nucleotides complementary to the template strand, a
 feature which enhances the fidelity of these DNA polymerases
 (Eckert and Kunkel,  1991).

 SPECIFICITY:  Specificity is determined by the primers. Sequences
 unique to the insert should be chosen for primer synthesis.   The
 requirement that  two separate sequences for each strand be
 complementary contributes to stringency of the technique.
 Stringency can be added by using a high annealing temperature.
 The kinetics  of binding should also favor the primer binding  to
 the targeted  sequence over a competing sequence with imperfect
 match  to the  primers.  Initially, primers will bind to the
 imperfect as  well as the perfect matched sequences and the extent
 of the interference  from cross-reacting sequences will depend on
 their  molar concentration as compared to the target sequence  and
 the amount of matched base pairs with the primer.  As cycling and
 amplification proceeds, the target sequence will increase in
 concentration and should effectively out-compete any cross-
 reacting sequences.

 MgCl, is an important component of the buffer that can
 significantly affect specificity.  The optimal MgCl-
 concentration varies with the sequences being amplified and the
 primer.  The  concentration of primer can also affect specificity.
 Excessive primer  concentration can amplify non-target cross-
 reacting sequences.  Excessive concentrations of the Tag
 polymerase can also  increase the non-specific products of the
 reaction (Saiki,  1990).  Once optimization is achieved the
 reaction should be able to amplify a DNA sequence of a predicted
 length.  The  products of PCR can be electrophoresed in a  sizing
 gel to identify the  fragment.  For reference, purified plasmid
 containing the insert DNA can be amplified in parallel and
 electrophoresed.  It is highly improbable that cross-reacting
 non-target sequences even if they interfered, and competed with
 the reaction  would yield an identical fragment.

 The availability  of  a restriction map of the fragment can improve
 specificity,  i.e., the restriction map should match the
 restriction map of the insert in the reference plasmid.  Lastly,
 any of the fragments can be hybridized to a specific probe using
 a Southern or dot blot  By adding this step the identity of a
 specific DNA  fragment is demonstrated by the complementarity of
 the two primers and  a probe that matches a sequence unique from
the primers.

 Chaudhry et al.  (1989) used this approach to study survival of
bacteria in sterile  lake water and sewage.   A 0.3 kb sequence
 from napier grass was inserted into £. coli via pBR322 and three


complementary oligonucleotides were made,, two to serve as primers
and a third to serve as a probe in dot blot analysis.  They  found
that the organism was undetectable by 6 days using plate counts
on a selective medium, however could still be detected up to  14
days by PCR combined with dot blot analysis.  Since this
technique utilizes isolated DNA it does not necessarily identify
whether the DNA was isolated from a viable organism nor does it
identify the organism that contained the DNA.  These studies
could not determine if the increased survival was an artifact of
DNA from cell lysis or was a result of the detection of viable
cells by the more sensitive method of PCR.  Culturing techniques
can be used in conjunction with PCR (provided the number of  cells
is in the range of detection by culturing methodology), either in
isolating the DNA or to identify the source of specific DNA

SENSITIVITY:  This technique can amplify DNA 1 x 106 times.
Using PCR to amplify and dot blots with a 32P-nick translated DNA
probe, Steffen and Atlas (1988) were able to detect 1 cell of £.
ceoacia in 1 gram of sediment in a background of 1011 diverse
organisms.  This is a 103-fold increase in detection over
hybridization procedures on non-amplified DNA (Steffen and Atlas,
1988).  By using PCR it is possible to use the number of
amplification cycles to calculate back to the original amount of
DNA present and therefore, the number of cells in the sample.

EASE or USE ft TIME:  The amplification is extremely easy to
perform because it involves a computerized incubation system.
The more technical aspects of the reaction are selecting the
primers and optimizing the reaction.

COST:  The expense is incurred in buying the cycler and
synthesizing the target-specific primers.  The reaction
components (except for the primers) are sold as a kit and are
fairly costly.

EFFECTS OF ENVIRONMENTAL MEDIA:  The DNA can be fairly impure.
However, contaminants from environmental samples may inhibit the
polymerase (Chaudhry et fll., 1989).  Any requirements for purity
that subsequent manipulations require, such as restriction
digests, are bypassed because the sample is amplified.

 (vii)  Multiplex amplification

 DESCRIPTION:  A modification of the polymerase chain reaction,
 multiplex amplification,  allows the simultaneous amplification  of
 multiple genetic loci.  Within one reaction mix, multiple  sets  of
 primers  are used to amplify unique sequences to identify
 corresponding genes within a mixed DNA population.  The technique
 was developed by Chamberlain et al. (1988) to detect multiple
 deletion mutations within different sites of the Duchenne
 muscular dystrophy locus  but it should be readily adaptable to
 environmental analysis.   For instance, it can be used to follow
 the fate of organisms in  a field test involving a multi-species
 release  or the  fate of multiple genes in a field test involving
 one organism with multiple insertions.

 The basic parameters in the use of multiplex amplification are  as
 described above for the polymerase chain reaction.  An increase
 in reagents including the Taq polymerase must be included  in
 order to accommodate the  increase in target DNA.  Dot blots,
 reverse  dot blots, or gel electrophoresis can be used to detect
 the diagnostic  fragment.  If identification is based on size
 fractionation,  the primers must amplify corresponding fragments
 of unique lengths that can be differentiated in gel

 SPECIFICITY i SENSITIVITY:  Optimization of conditions may be
 necessary for each set of primers since not all primer-target
 duplexes in the mix are expected to have identical melting

 EASE OF  USE « TIME:  This adaptation of PCR simplifies the
 analysis of complex field releases involving multiple organisms
 or multiple gene insertions in one organism.  Using standard
 hybridization reactions or standard PCR, multiple probes or
 primers  have to be used and this involves a corresponding  number
 or reaction mixtures, blots, sample preparations etc.  This
 technique especially when used in conjunction with reverse dot
 blot analysis reduces the number of steps and the amount of time
 required for detection.   Chamberlain et al. (1988) reported a
 time of  5 hours for the total analysis.  With the reduction in
 time and complexity of the analysis, a corresponding reduction  in
 cost is  achieved.

 EFFECTS OF ENVIRONMENTAL MEDIA:  Contamination from extraneous
 DNA could be a problem.   If the reaction was limited to 25
cycles. Chamberlain et al. found that 3 * 5% contamination could
be tolerated.

 (viii)  Reverse dot blot

 DESCRIPTION:  Reverse dot blot analysis is a detection method  for
 polymerase chain reaction products.  A modified nucleoside
 triphosphate, e.g. biotinylated uracil, is added to the reaction
 mix.  During chain elongation the modified nucleoside
 triphosphate is incorporated into the growing chain resulting  in
 an amplified product that is labeled.  Alternatively, a labeled
 oligonucleotide can be used as a primer.  In this case the label
 is incorporated in the 5' end of the product rather than
 dispersed through the extended portion of the chain.  For
 example, biotin or fluorescent dyes such as fluorescein or
 rhodamine can be used to either end-label the 5' end of the
 primer or be incorporated into the oligonucleotide during
 synthesis.  In either case, the amplified fragment is labeled
 during the reaction.

 The PCR product can then be "captured" with a nonlabeled probe
 that has been immobilized on a solid-support such as
 nitrocellulose.  Immobilization is accomplished by the addition
 of & polythymidine tail to each oligonucleotide by terminal
 deoxyribonucleotidyltransferase.  Exposure to UV light causes
 covalent coupling of the thymidine residues to the nylon
 membrane. Detection of the product on the nitrocellulose membrane
 is via the fluorescent dye that remains after the appropriate
 wash has been performed or in the case of biotin through an
 enzymatic reaction via a streptavidin-enzyme complex.  For
 example, Saiki et al. (1989) used a streptavidin-horseradish
 peroxidase conjugate to detect PCR fragments hybridized to probe.
 A third variation on this technique is to label the PCR products
 directly using biotin during first strands synthesis.
 Streptavidin is then used to "capture" a fluorescent probe-target

 SPECIFICITY & SENSITIVITY:  Hybridizations that employ short
 oligonucleotide probes are highly specific (see discussion above
 on nucleic acid probe construction).  Under stringent
 hybridization conditions, a single base pair mismatch will
 destabilize the. complex and prevent the formation of a probe-
 target duplex.  One major difficulty encountered with the use  of
 multiple probes is that not all the probes may be sequence-
 specific under identical hybridization conditions leading to
 inconclusive results.  The length, choice of sequence, and/or
 concentration of the probe applied to the filters can be adjusted
 to meet uniform hybridization requirements.  Another
 consideration, is that the length of the poly dT tail and the
 amount of UV exposure can effect the hybridization efficiency.

The main advantage of this technique is flexibility.  For
multiple gene sequences that require different primers and
probes, reverse dot blots can be used to detect sequences
synthesized via multiplex amplification.  Alternatively if a


 family  of genes are  inserted  into a microbe, they can be followed
 simultaneously by amplifying  the polymorphic region of the genes
 using one set  of primers specific to common flanking sequences.
 Probes  that  incorporate the variation can be used to screen the
 environmental  sample.  Likewise, polymorphisms in a common
 sequence can be used to detect an introduced organism in a dense
 background or  to differentiate between organism that have been
 released into  the environment simultaneously.

 Using species  specific probes to 'a common gene, unrelated
 microbes can be monitored concomitantly.  This technique can also
 be adapted to  detect predicted changes in an inserted sequence.
 It can  be used in conjunction with culturing techniques for
 screening species into which  horizontal transfer of an introduced
 sequence has occurred.  For instance if during plate
 hybridization  it is  determined that the insert has been
 transferred  to an unidentified species, positive colonies can be
 pooled  and DNA isolated.  PCR amplification of a ribosomal gene,
 for example, followed by screening using a panel of immobilized
 species-specific probes can be used to screen possible recipients
 of the  genetic exchange.

 The sensitivity of the amplification can be decreased in
 protocols using one  set of primers because of competition.
 Sensitivity  could also be reduced as a result of the adjustments
 in the  hybridization conditions that may be needed to accommodate
 differences  in the oligonucleotide probes.  Sensitivity can also
 be decreased if label is incorporated via 5* end-labeling of the

 BASE OF USE  i  TIME:  This technique consolidates a number of
 separate procedures.  The labeling is done during the
 amplification  thereby eliminating the need for a separate
 labeling step.  For  those screening protocols requiring multiple
 probes, the  immobilization of probe rather than target DNA
 eliminates the need  for multiple hybridization reactions and
 subsequent filters to be washed.  The use of a non-radioactive
 probe also eliminates the complexity inherent in the precautions
 required with  the use of radioactivity.

 Once conditions are  optimized, the amplification, hybridization,
 and color development can be accomplished in as little as 3 to 4
 hours.  Used in conjunction with multiplex amplification, it
permits the concurrent analysis of multiple genetic loci thereby
 readily allowing the simultaneous detection of organisms or

(e)  Probe visualisation                   •

DESCRIPTION:  Liquid scintillation is used in procedures that
involve solution hybridization.  Hybridization of DNA attached to
a solid matrix routinely involves the use of autoradiography or
some fora of fluorography*  Autoradiography is the exposure of
film by radioactive particles whereas fluorography is the
exposure of the film by light particles generated from the
interaction of radioactive particles with added fluors.  The type
of fluor used is dependent on the energy and range of the
emissions from the isotope.  Gels or blots obtained using 35S
radiolabelled probes are treated with a solution containing 2,5
diphenyloxasole (PPO) or a commercially prepared fluor.  32P has
a longer range and therefore, an external CaWO4 intensifying
screen can be placed next to the film (Bonner, 1987).
Autoradiography is used to quantitate the amount of target DNA
since intensity is linear up to absorbances of 1 unit.

SENSITIVITY AND SPECIFICITY:   Fluorography is commonly used to
enhance the sensitivity.- For instance, the use of an
intensifying screen (s) can increase the sensitivity of detection
with a 3ZP-labeled probe 5 to 20 fold.  Pretreating the blot or
gel with a fluor can increase the sensitivity of detection using
a 35S-labeled probe 5 to 15 fold.  The most sensitive method is
the use of a sandwich composed of a screen-film-blot (dried gel)-
screen arrangement.  The second screen acts by increasing the
backscatter of the beta rays from the ^P from the blot.  Longer
exposure increases the sensitivity and allows detection of weak
positives.   However, this is accompanied by a decrease in
resolution (Bonner, 1987).  If the sample is very hot,
autoradiography is used.  Some combination of autoradiography
followed by reexposure using fluorography can be used to obtain
the best combination of resolution and sensitivity.  In addition,
exposure time can be varied to optimize resolution and
sensitivity.        .                              '

 (d)  Use of nucleic acid hybridization in analysing microbial
 species composition

 DNA-DNA hybridization studies  are  an  important  tool for
 distinguishing between closely related bacteria and can be used
 for  their identification.   However, the  classical  DNA
 hybridization  methods are  too  cumbersome to be  used to identify a
 large number of strains.   DNA  must be purified  from each of the
 strains tested.   Furthermore,  the  various DNA hybridization
 techniques are relatively  tedious  and time-consuming (Schleifer
 and  Stackebrandt,  1983).   Recent studies have shown that the
 blotting of whole cells onto membrane filters,  followed by lysis
 and  hybridization with labelled chromosomal DNA from reference
 strains,  is a  quick method of  identifying bacterial isolates
 (Roberts et al..  1984,  1987; Moromoti et al. 1988).   Examples of
 whole cell DNA probes are  given in Table 5.  Ezaki et al.  (1989)
 are  using photobiotin-labelled whole-cell-probes to hybridize
 reference DNAs bound  to microdilution plates.   For identification
 of an unknown  strain  it is then sufficient to extract its DNA
.from 1  to 3 ml of overnight culture,  to  label it with photobiotin
 and  to  probe the immobilized reference DNA.

 The  advantage  of the  whole-cell DNA probe method is that it is
 simple,  quick  and that a large number of strains can be handled .
 at the  same time.   Moreover, specifically designed probes  are not
 necessary.   Disadvantages  are  mainly  the lack of specificity and
 the  fact  that  only culturable  organisms  can be  identified.
 Cross-hybridization of whole cell  DNA probes with  other related
 bacteria  is rather common  (Grimont e£ aJU, 1985; Hyypia et al.,   .
 1985) and stringency  has to be carefully controlled.

Another approach is the use of comparative sequence analysis of
 16S  and 23S ribosomal RNA  (or  any  gene of interest)  in the
elucidation of phylogenetic relationships in prokaryotes (Woese,
 1987).  The rRNA molecules contain regions of highly conserved
sequences interrupted by more  variable sequences.   Because of the
differences, in the degree  of conservation within the molecule,
rRNA can  be used to design probes  with specificity ranging from
species to kingdom level.   The use of rRNA as target nucleic acid
has  even  more  advantages.   First,  rRNAs  are present  in very high
copy numbers (about 104 molecules  per £.  coli cell).   Thus,  a
considerable increase of sensitivity  (at least thousand times)
can  be achieved by targeting rRNA  instead of DNA.  Another
advantage is the single strandedness  of  rRNA which avoids
problems with  target  renaturation.  In contrast  to mRNA, rRNA is
quite stable and rigid methods assuring  high yields,  can be used
for  target  extraction without  any  damage to rRNA (chromosomal DNA
would be  considerably degraded).   The  use of probes  with
different specificities allows rapid  classification  of an  unknown
isolate  (Regensburger fit al.,  1988).  As  first step,  a universal
probe is applied as a control  for  bound target.  By  repeated use


of probes with increasing specificity unknown isolates are
characterized with a few steps.

These polynucleotide probes are sensitive and are not affected by
minor nucleotide mismatches.  The first defined DNA probes were
derived from cloned DNA.  Therefore, it was rather time consuming
and difficult to prepare these probes in large quantities and at
constant quality.  Moreover, care must be taken to prepare probes
free of vector sequences in order.to avoid unspecific
hybridizations.  More recently, the use of polymerase chain
reaction methodologies allows preparation of vector-free nucleic
acid probe in a relatively short amount of time.


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Weinberg &
Stotzky, (1972)
Graham &
Istock, (1978
& 1979)
Trevors &
Oddie, (1986)
van Elsas et
Krasovsky  &
Stotzky,  (1987)
Intraspeciflc crosses
between E. coli
auxotrophic recipient
& prototrophic donor.

Intraspecific crosses
between B. Subtilis in
autoclaved potting

Intraspecific crosses
between E. coli in
amended and unanended
autoclaved soil.

Intergeneric crosses
between B. cereus and
B. subtilis.  Gamma
irradiated soil, 20-22%
moisture (60% MHC).

Intraspecific crosses
between prototrophic
& auxotrophic E. coli
in autoclaved amended
& unamended soil.
Moisture -33kPa.
Conjugal transfer of
chromosomal genes,
genetic recombination.
Transformation of linked
chromosomal genes.
Conjugal R-plasmid
Conjugal plasmid
transfer (pFT30).
Conjugal transfer of
chromosomal DNA, and
genetic recombination.
Selection of prototrophs /
Resistance of recipients to Sm.
Selection of auxotrophs /
Resistance of recipients to
Sp & Lm.
Selection of transconjugants
by expression of plasmid
encoded Tcr / Resistance of
recipients to NX.

Selection of transconjugants
by expression of plasmid
encoded Tcr / Resistance of
recipients to Em.
Selection of prototrophic
recombinants on minimal agar
/ Resistance of recipients to

TABLE 1.  (Continued)
Rafii &
Lorenz et al
al..  (1988)
Zeph et al..
Inter- & Intraspecific
crosses between
Autoclaved soil,
moisture 60% MHC.
Competent Bf subtilis
(auxotrophic, TrpC2)
attached to sterile
sand grains.
Conjugal piasmid
transfer & piasmid
Transformation of naked
DNA Isolated from
B. subtilis 168
Inter- & Intraspecific   Conjugal piasmid
crosses between strains  transfer (pIJ673).
of streptomycetes.
Autoclaved soil, amended
& unamended, moisture
40% MHC.
Colnoculation of phage
PI lysates and E. coli
recipients, inoculation
of lysogenic E. coli
donor and non-lysogenic
recipient.  Autoclaved
soil, moisture 21.1-
24.4% (wt./wt.).
PI phage infection,
formation of lysogens.
Selection of transconjugants
by expression of piasmid
encoded Tsr or Nmr, and
production of melanin/

Resistance  of recipients  to
various antibiotics*

Transformants selected on
modified minimal salts medium
(Aardema et alP. 1983)
                          Selection of transconjugants
                          by expression of piasmid
                          encoded Tsr and Nmv Resistence
                          of recipients to Sm.
Selection of lysogens by the
expression of phage Cmr & Hgr
genes / Counterselection of
auxotrophic E. Coli lysogenic
donor using MM.

TABLE 1.  (Continued)
Richaume et
al.. (1989)
Herron &
Top et alt.
Intergeneric crosses
between E. coli donor
and Rhizobium freudii.
autoclaved soil, various
soil conditions.
Co-inoculation of KC301
phage and s. lividans.
soil amended with 1%
soluble starch, 1%

Intergeneric crosses
between E. coli donor
and A. eutroohus.
autoclaved soils,
moisture 75% field
Conjugal plasmid
transfer (CpRK2073:Tn5)
Actinophage infection,
formation of lysogens.
Conjugal plasmid
transfer, and plasmid
Selection of transconjugants
by expression of antibiotic
resistance genes / Counter-
selection of donor by the use
of a selective carbon source
and  resistance of  recipient
to NX.

Selection of lysogens by
expression of phage Tsr
Selection of transconjugants
by expression of plasmid borne
heavy metal resistance to Co,
Cd & Zn / Counterselection of
donor due to non-expression of
heavy metal plasmid markers in
donor E. coll
Abbreviations: Cd; Cadmium.  Cm; Chloramphenicol.   Co; Colbalt.   Em; Erythromycin.  Hg; Mercury.  Lra;
               Lincomycin.  MM; Minimal medium.  NX; Nalidixic Acid.  Nm; Neomycin.  Sp; Spectlnomycin.
               Sm; Streptomycin.  Tc; Tetracycllne.  Ts; Thiostrepton. Zn; Zinc.
 (Table reproduced from, Cresswell & Wellington, 1991)

                                       RECIPIENT SELECTION AND DONOR
Schilf &
van Elsas et
Krasovsky &
Stotzky, (1987)
Trevors &
Zeph et al

van Elsas
al.. (1988)
Crosses with E. coll
donor to artificially
raised numbers of
indigenous bacteria
ca. 109 CFU/g soil.

As in Table 1.
As in Table 1.
Conjugal R-plasmid
As in Table 1.

As in Table 1.
Intraspecific crosses    Conjugal R-plasmid
between E. coli strains, transfer.
Various soil conditions.
As in Table 1.
Intraspecific crosses
between Pseudomonads.
Rhizosphere, bulk
& amended bulk soil,
moisture 20% (wt./wt.)
As in Table 1.
Conjugal R-plasmid
Selection of transconjugants
by selection of plasmid encoded
antibiotic resistance genes.
No counterselection, no transfer

As in Table 1.
Additional requirement for
selection of auxotrophic
recombinants with PC 8|ig/ml.

Selection of transconjugants
by expression of plasmid
encoded Tcr / Resistance of
recipients to NX.

As in Table 1.
Selection of transconjugants
using Kings B medium and
expression of plasmid encoded
Kmr and Tcr / Resistance of
recipient to Rp.

TABLE 2.  (Continued)
Germida &
Co-inoculation of        Transduction by phage
transducing Pi phage     Pi.
lysates and auxotrophic
E. coli K12-GK401, soil
As in Table 1.
As in Table 1.
Herron &
As in Table 1.
Soil unattended.
As in Table 1.
Top et al,
As in Table 1.
Soil unamended &
As in Table 1.
Selection of recipient E. coli
using EMB medium and Smr,
selection of transductant
phenotypes using MM, Tc and
leucine or threonine.

Selection as in Table 1,
use of a streptomycete
selective medium 'RASS1
to aid the counterselection of
indigenous organisms.

Selection of S. lividans
recipient by resistance to
Sm.  Use of streptomycete
selective medium 'RASS*.  No
lysogens detected (Tsr Smr).
Phage survived for 39 days.

Selection of transconjugants
on Tris azelate medium and
expression of plasmid encoded
heavy metal resistance genes
to Zn2"1" & Cd2+.  Counter-
selection of indigenous
bacteria by replica plating
onto 2nd medium containing;
Co2"1" and T Tc.

TABLE 2.  (Continued)
Henscke &
Schmidt, (1990)
Smit & van
Elsas, (1990)
E. coli SM10 plasmid
donor, study of
transfer to indigenous
Conjugal plasmid
P. fluorescens plasmid
donor (R2F RP4;;pat
Rpr), study of transfer
to indigenous microflora.
Conjugal RP4 plasmid
Selection of transconjugants
by the expression of Tbr &
growth in the presence of
IPTG / Counterselection of
donor facilitated by the poor
survival; rapid decline after
25 days.

Selection of transconjugants
by expression of plasmid encoded
Tcr / Deselection of donor
facilitated by the use of a
donor specific virulent phage.
Abbreviations: Cd; Cadmium.   Co; Cobalt.   EMB;  Eosin methylene blue  medium.   IPTG; Isopropyl-p-D-
               thiogalactoside.   Km;  Kanamycin.   MM;  Minimal medium.   Nm;  Nalidixic acid.   PC;
               Penicillin.  RASS; Reduced arginine starch medium.  Rp;  Rifampicin.  Sm; Streptomycin.
               Tc; Tetracycllne.  Tb; Tobramycin.  Zn; Zinc.
(Table reproduced from, Cressvell & Wellington, 1991).

TABLE 3.  Plasmid transfer rates in laboratory, microcosm, and in situ soil and aquatic systems.
          Rates given in terms of transconjugants per donor cell (D) where possible; other rates are
          given in terms of the transconjugants per recipient cell  (R).
pBC16 conjugative
RP4 conjugative
8a conjugative
R68.45 conjugative
fi. subtilus
fi. subtilus/
fi. 1 icheni f ormis
fi. subtilus/
B. meaaterium
lab: broth
lab: broth
lab: broth
fi. coli/ lab: filter
Thiobacillus novel lus mating
Thiobacillus novel lus/
£• coli
£• coli/
!• colj.

£. aeruainosa/
£. aeruainosa

lab: combined
spread plate
lab: broth '
lab: broth
lab: sterile
lake water
lab: sterile
m _ «_ _ _ . *.
Koehler and Thorne (1987)

Davidson and Summers

Walter et a_l. (1987)

O'Morchoe et aJL. (1988)

lake water
plus filter

field: non-
sterile lake
TABLE 3.   (Continued)
PMQ1 conjugative


£. aeruqinosa/
£. aeruqinosa
pBR325    nobilizable    £.  coli/E.  coli


           conjugative    Bacillus cereus/
                          B.  subtilis
                                              lab:  broth     10'1R
                                              with  sterile
                                              stone &  filter

                                              lab:  sterile   10'2R
                                              river water,

                                              field: unencl.      1(T*R
                                              river water,
                                              sterile  stone
                                              &  filter
      Bale et al.  (1987)
                    field: unencl.
                    water, nonsterile
                    stone  ,  & filter

                    lab: broth
                    & mobilizer

                    lab: raw,sterile
                    wastewater  &
                    mobilizer plasmid

                    lab: filter
                                              lab: sterile        10"7D
                                                                            McPherson and Gealt (1986)
          Van Elsas  (1987)

TABLE 3.  (Continued)
R plasmids conjugative
pRTrSa conjugative
lab: nonsterile OD
lab: nonsterile 10"7D
soil & bentonite
Klebaiella/ lab: nonsterile 10'6D
Klebaiella radish rhizosphere
Rhizobium trifolii/ lab: in situ ?
B. leauminsarura

Talbot et al. (1980)
Hooykaas et al.

TABLE 4.  DMA extraction from the environment
Abbot et al.
et al. (1988)
Bej et al.
et al. (1986)

Deflaun &
Paul  (1989)

et al. (1988)
et al. (1988)
Hay et al.
Holben et al.
Kniaht et al.
Kuritza et al,
Human T-cells
Fresh water/

50ng DNA
(2X107 cfu)

1-10 fg
DNA or 1-5
E. coll

Cells  isolated,
DNA extracted,
FCR amplified.

Indirect lysis,
Indirect lysis,
PCR,    hybridisa
Indirect lysis
Hoechst 33258.
167 fg Herpes  Indirect lysis,
DNA in E. coll hybridisation.
10-100 cfu/g
E. colil
25-50% of
Enrichment, in
direct lysis,

Indirect lysis,
agarose gel
     Hoechst 33258.

Direct lysis,
PCR, hybridisation.
0.2 pg DNA/104 Indirect lysis,
cfu/g          hybridisation.

7-15 ng        Indirect lysis,
DNA/2.6 x 103  hybridisation.
               Indirect lysis,

 Kuritza  &
 Salyers  (1985)
et al.  (1986
Ogram et al.

Paul &
Carlson (1984)

Paul & Myers

Preston et al.

et al. (1989)
Steffan &
Atlas  (1988)
Steffan et al.
Torsvik &
Goksoyr (1977)

Torsvik (1980)
Torsvik et al.





     Waste  water



2% of popula-


40-91% total
95-100% total

104 pfu

1-3 cfu/ml
100 cfu/ioog
(Pseudo monas1
 Indirect lysis,

 Indirect lysis,

 Direct  lysis,

 Indirect lysis,
 Hoechst 33258.

 Indirect lysis,
 Hoechst 33258.

 Indirect lysis,

 Indirect lysis,
 agarose gel

 Indirect lysis,
 PCR, hybridisation.

 Direct  lysis/
 Indirect lysis,
 agarose gel,

 Indirect lysis,

 Indirect lysis,

 Indirect lysis,
Tm analysis.
  Also see reviews by Hazen & Jiminez  (1988); Holben & Tiedje (1988) ;
Ogram & Sayler (1988); Lidstrom  (1989); Salyers  (1989); Trevors  &  Van
Elsas (1989).

TABLE 5:  Examples of whole cell ONA probes
Ac inet obacter spp.
Bacteroides spp.
Bacteroides spp.
Campy 1 obacter spp.
Chlarovdia tracho-
Mobiluncus spp.
whole-cell dot
whole-cell dot
whole-cell dot
dot blot
dot blot
dot blot
whole-cell dot
whole-cell dot
dot blot
                                                     Tjernberg et  al.,

                                                     Roberts et al.,

                                                     Horotomi et al.,

                                                     Wetherall et  al.,

                                                     Chevrier et al.,
                                                    Hyypia  et  al.,

                                                    Tjernberg  et al.,

                                                    Roberts et al.,

                                                    Athwal  et  al.,

 TABLE 6:   Examples of DNA probes derived from 16 or 23S rRNA targets
 Type of Probe
 Cloned fragment

 Cloned fragment
 Of 23S rRNA

 Cloned fragment

 Cloned fragment
 Cloned fragment
 (not specified)

 Cloned rRNA gene


 (16  and 23S)
 (not specified)

 all  organisms

 all  organisms
                     Giovannoni et al.,
                     et al.,  1988





Micrococcus -




                     Giovannoni et al.,
                     Giovannoni et al.,
                     et al.,  1988
                     Chen et  al.,  1989

                     DeLong et al., 1989
                     et al.,  1988

                     Gobel  and
                     Stanbridge,  1984
                     Festl et al. ,1986

                     Haun and Gobel,
                     Enns,  1988
Leqionella pneu-
Mvcoplasma pneu-
Proteus species
                    Grimont  et  al.,
                    Gobel  et al.,
                    Haun and Gobel,
Species associated  Chubaetal., 1988
with human perio-
Baeteroj.de§         Stahl  et al.,
succinooenes. La-   1988
chnosoira multioarus
Clostriditim diffi-  Wilson et al.,
eile                1988
Neisseria gonorr-   Rossau et al.,
hoeae               1989
Mycoplasma pneump-  Enns,  1988
Mvcobacteriuro avium Drake  et al.,
complex             1987
Vibrio ancmillarum  Rehnstam et al.,
                    Stahl et al.,

TABLE 7:  Examples  of  DNA probes  derived from randomly cloned DNA
Genomic  DNA

Genomic  DNA

Genomic  DNA

Genomic  DNA

(not specified)
Genomic  DNA
Genomic  DNA

Genomic  DNA

Genomic  DNA

Genomic  DNA
Genomic  DNA
Genomic  DNA

Genomic  DNA

Cryptic  plasmid
Genomic  DNA

Genomic  DNA

Genomic  DNA

Genomic  DNA


Genomic  DNA
Salmonella spp.
Spiroplasma spp.

Bacteroj.des thetaio-
Bacteroides vulcraris

Bacteroides fraoilis

Campvlobacter jeiuni

Chlamydia trachomatis
CorvnebacteriuBi sepe—
                              MvcobacteriuM leprae

                              Mvcoplaama hvorhinis
                              Mycoplasma pneumonias
                              Mvcoplasma aallisep-
Mvcoplasma-like .
Neisseria aonorrhoeae
Rickettsia prowazekii

Streptococcus oral is

Thiobaeillus Spp.

Strain of Bacteroides
                              Strains of Laetoba-
                              cillus delbrueckii,
                              and L* reuteri
                              Strains of Thiobaeil-
                              lus ferrooxidans
Fitts et al., 1983
Nur et al., 1986

Salyers  et al.,
Kuritza   and
Salyers,  1986
Kuritza   et  al.,
Bryan et al,, 1986

Palva, 1985
Verreault et al.,
Eisenach et al.,
Clark-Curtiss and
Docherty, 1989
Taylor et al., 1985
Hyman et al., 1987
Santha et al., 1987
Lee et al., 1988

Totten et al., 1983
Regnery  et  al.,
Schmidhuber et al.,
Yates et al., 1986

Attwood  et  al.,
Tannock, 1989
                         Yates et al., 1986

TABLE 8:  Examples of DMA probes  from genes
Strains of
Gene coding  for
Delayed hyper-      Listeria strains
sensitivity factor

               Different anti-
               biotic resistance
               Elongation factor
               Pili and outer
               membrane proteins
               ciated plasmid
                    £. coli and
                    Pseudomonas aeru-
                    various gram-nega-
                    tive eubacteria
                    various eubacte-

                    Mycoplasma spp.

                    Neisseria spp.

                    Vibrio parahaemo-
                    lyticus. V.
                    Yersinia spp.
                    Notermans et al.,

                    Cooksey et al.,

                    Huovinen et al.,
                    Halbert, 1988
                    Yogev et al.,
                    Aho et al.,  1987

                    Nishibuchi et al.,
                    1985; 1986

                    Gemski et al.,
                    1987; Rabins-Browne
                    et al., 1989
Surface protein


Capsular antigen
Anaolasma mar-
                                   Listeria mono-
                                   Salmonella tvpfai
Eriks et al., 1989

Datta et al., 1988

Rubin et al., 1985
Liebl et al., 1987

Pozzi et al., 1989

TABLE 8 (continued)
Gene coding for
Strains   Delta-endotoxin

          Heat-labile and

          Invasion process
          Shiga-like toxi
          Capsular anti-

          Toluene degrada-



                    Bacillus thuringensis

                    £• coli strains
                    £• coli strains
                    £• coli and
                    Shioella spp.

                    £• coM strains

                    £• SSSUL strains

                    £• eoli strains
                    putida strains

                    aureus strains

                    Yersinia entero-

                    genome (water)
                         Prefontaine et al.,

                         Groot Obbink
                         et al., 1985

                         Hoseley et al.,
                         1982; Sommerfelt
                         et al., 1988a,b

                         Wood et al., 1986;
                         Gomes et al., 1987

                         Bohnert et al.,
                         Meyer et al., 1989

                         Nataroetal., 1985
                         Roberts et al.,

                         Sayler et al,,

                         Rifal et al., 1989
                         Jagow and Hill,

                         Barkay et al.,

TABUS 9:  Non-radioactive reporter groups for indirect detection of
Reporter group

Labelled avidin,

Labelled antibody

Labelled antibody

Labelled antibody

Labelled antibody

Labelled antibody
Langer et al., 1981;
Leary et al., 1983;
Kumar et al., 1988
Tchen et al., 1984;
Landegent et al., 1984
Verdlov et al., 1974;
Syvanen et al., 1986
Vincent et al., 1982;
Keller et al.,1989
Heiles et al., 1988

Table 10.  Applications of hybridization methods in environmental studies.
PI Cm cts;tTnSOl
Streptomyces sp.2
 Pseudomonas sp.3
 ?. ceoacia
                      plasmid, pIJ303
insertion of
vector sequences
from suicde
dot blot on DNA
extracted from
clonies isolated
on selective media
using biotinylated
probe to repA

with selective
media using a
nick translated
probe made from
the whole plasmid

hybridization on
DNA isloated from
Southern analysis
on total DNA
using a 32P-
labeled single-
stranded DNA
probe to notII
sterile and non-
sterile soil
                                           sterile soil
non-sterile soil
                     mobilization of
                     plasmid in
                     Streptomvces sp.
                                                                                      verify the
                                                                                      presence of the
                                                                                      plasmid and
                                                                                      modifications in
                                                                                      new host by RFLP.

                                                                                      use restrction
                                                                                      polymorphisms to
                                                                                      detect plasmid
                                                                                      sequence deletion
                                                                                      as a result, of a
                                                                                      double cross-over

Table 10.  (Continued)
P. cepacia4
Pseudomonas sp.
notll chromosomal
P. nutida5
Tnfi chromsomal
Colony blot
analysis with
probe made to 32P-
labeled pRL425

slot blots on
total DNA using a
single- stranded
DNA probe
soil microcosm
Dot blots on DNA
from isolated
colonies using
nick translated
32P-labeled vector
containing Tn£ as
sterile soil
                                          Dot blots on
                                          cells grown in
                                          selective medium
                                          and diluted for
                                          MPN analysis
                                           sterile and non-
                                           sterile soil
                                                                                      verify the .
                                                                                      absence of vector
                                                                                      sequence in kanr
follow the
movement, and
leaching of

determine if Tn£
insert is stable
in soil
                                            recovery of and
                                            follow growth of
                                            organisms over

Table 10.  (Continued)
£. fluorescens6
plasmid RP4
                      pBR322 modified
                      with Drosphila
E. coli8
Rhizobium fredi,
with selective
media using a
nick translated
32P-labeled probe
made from whole

with selective
and non-selective
media using a 32P-
labeled probe to
Drosophila insert

Southern analysis
on individual
colonies grown in
selective media
using a probe to
Tn5 made by nick
translation of a
containing Tnj>
non-sterile soil
detect plasmid
transfer to
                                           sterile and non-
                                           sterile soil
sterile soil
                     stability of
                     plasmid and
                     foreign DNA
transfer of
plasmid from £.
coli and fi.

Table 10.  (Continued)
E .coli9
£• putida10
Alcaligenes AS11
non-conj uga t1ve
 containing herpes
 simplex thymidine
 kinase (tkl gene
 plasnid RK2 and
 plasmid TOL
 encoding the xvl
 operon colony.
 plasnid, pSSSO
 allows the
 degradation of 4-
 genes involved in
 acid degredation
 with selective
 media using a
 nick translated
 32p-labeled 850 bp
 tk fragment as a

 with non-
 selective media
 using nick-
 tanslated 32P-
 labeled probes to
 RK2 and xvlR gene
 on TOL plasmid

 with selective or
 media and dot
 blots on
 extracted DNA
 using 32P-labeled
 probes, to RS-
 1100-1, 1.3 KD
 repeat fragment
 (15-20 copies/cell)
laboratory waste
 aqifer material:
 and contaminated
 mobilization of
 non-conj ugative
 plasmid in a
 mating with
 recepient cells
 stability and
 maintenance of
 strain and
 monitor growth
 and perslstance
 of organisms

Table 10.  (Continued)
£• coll12
£. putida13
hybrid plastnid
between pBR325
and pEML159 from
Alcallaienes sp.
encoding mercury
resistance and
acid degredatlon

non-conj ugatlve
IncQ broad-host
range plasmid In
which xvlE gene
has been Inserted.
chromosomally and
on plasmlds In £.
cepacia or to
pSSSO for

with selective
and non-selective
media using a
nick translated
probe to whole

Dot blots of DNA
isolated from
filtered cells
lysed in situ on
membranes using a
0.6 kb xylE
fragment randomly
primed and
labeled with
filtered and
unfiltered lake
survival and
monitoring study
filtered and
unfiltered lake
survival and
monitoring study

Table 10.  (Continued)
insertion using
insertion vector
gene and
Streptococca1 tet
1Zeph et al.  1988
fRafii and Crawford.  1988
3Jansson et al.  1989
4Jansson et al.  1989
5Fredrickson  et  al. 1989
6van Elsas et al.  1989
'Devanas and  Stotzky. 1986
8Richaume et  al. 1989
9Mancini et al.  1987
Colony blots from
MPN dilutions
with selective
media using an
(30 base pair)
probe to nptll
end-labeled with

blots on colonies
grown in
selective and
transferred to
and lysed ip situ;
probed with
isolated delta-
entotoxin and tet
genes labeled by
random priming with
corn seeds coated
with &. lipoferum
germinated in
corn plants
planted in the
field for a
determine levels
in rhizosphere,
and xylem
determine extent
of leaching into
study gene loss
and segregation
rates of inserts
in corn;
determine rate of
survival in plant
debris post-
harvest; monitor
into trap plants

Table 10.  (Continued)
10Jain et al. 1987
"Steffen et al. 1989
12Amy and Hiatt. 1989
13Morgan et al. 1989
14Bentjen et al. 1989
15Turner et al. USDA submission 90-016-01

TABLE 11.  Hybridization techniques for detection of microorganisms introduced into the environment.


j aponicum*

E. coli5
£• outida
TOL plasmid


repeat 1.3KB


TOL plasmid
nptll sequence
containing 1
KB of RS1100-I
stranded DNA

822 base-pair
hybridization .

dot blot

dot blot

dot blot

sediment 1 colony in
soil 103colonies/
gram of soil

sediment 103 cells/
gram of

soil 4 X 104/9rara
of soil or
0.02 picograms
total DNA
lake water 104colonies/ml

£• cepacia6

product of
cells/gram of

TABLE 11.  (Continued)
Rhizobiun          Tn£
stranded DMA
                plasmid DNA
                containing Tn£
Southern blot
0.1 picogram
total DNA

102 cells/gran
of soil
£* putida
                                                                       10 cells/ gram
                                                                       of soil
lSaylor et al. 1985.
2Bentjen et al. 1989
'steffen and Atlas. 1988
iHolben et al. 1988.
'Morgan et al. 1989
'Steffen and Atlas. 1990.
'Holben et al. 1988.
'predrcikson et al. 1988

TABLE 12.  Detection of bacteria in environmental  samples by total community DNA extraction and polymeras
           chain reaction (PCR) enhancement
(lake, sew.)

* Detection
of target
JH: infec
Strain Seq. Copy Remarks/
detected arnpl. no. problems
Pseudomonas 0.7 kb 1-3 Persistent impurities;
fluorescens pj£ 1:10 dilution needed
£. f3,uor. 0.7 kb 1-3 Idem above
Frankia sp. nifH 1? Non-target amplifi-
fFrankia) cation; (booster)
Frankia sp. 0.2 kb 1? Dilution (1:4/1:9)
nifJH needed for PCR
G. araminis 0.29/0.19 1? Nested primers; 2
kb fragm. rounds of
£. ceoacia 1.0 kb 15-20 Primer selection
Coliform LacZ/ 1?
bacteria LamB
£• coli 0.2 kb multiple
Leaionella 0.65 kb 1
pneumophila mip fr.



of microorganisms in clinical samples or food are not listed here.
Copy, no..: number of copies of the target sequence per cell; detection
organisms or amount of target DNA per ml. or g. of
:, fragment; kb: kilobases; G. araminis; Gaeumannomvces
tive units.
sample still
qraminis; cfu;
Van Elsas et al., 199:
Cresswell et al., 199]
Myrold et al., 1990
Simonet et al., 1990
Schesser et al., 1991
Steffan & Atlas, 1988

Chaudry et al . , 1990
Bej et al., 1991
Mahbubani et al . , 1990
Starnbach et al . , 1989
Seer, amol.: sequence
limit: minimum number
detectable after PCR;
colony- forming units;

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