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
-------
This Page Intentionally Blank
-------
METHODS FOR THE DETECTION OF MICROORGANISMS IN THE ENVIRONMENT
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
-------
This Page Intentionally Blank
-------
METHODS FOR THE DETECTION OF MICROORGANISMS IN THE ENVIRONMENT
,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
4. USE OF STATISTICS IN THE DETECTION
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
-------
This Page Intentionally Blank
-------
1. PREFACE
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.
-------
2. INTRODUCTION
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.
-------
3*
Introduction
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
antibiotics.
-------
(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
hybridization.
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
environment.
10
-------
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
purification.
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
11
-------
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
12
-------
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.
13
-------
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
culturable.
(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
14
-------
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.
15
-------
4« USE Of STATISTICS IN THE DETEpTION OffMICROORGANISMS
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
16
-------
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.
17
-------
4* ANALYSIS OP TECHNIQUES •
DIRECT DETECTION TECHNIQUES
(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.
18
-------
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.
19
-------
(b) Microscopy - Detection of total viable and nonviable
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). 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
habitats).
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.
20
-------
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
sample.
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.
22
-------
DETECTION TECHNIQUES THAT REQUIRE CULTURING OF THE MICROORGANISM
(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.
24
-------
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
samples.
(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.
23
-------
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
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.
25
-------
(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.
26
-------
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.
27
-------
(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.
28
-------
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.
29
-------
(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.
30
-------
(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.
31
-------
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.
32
-------
(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.
33
-------
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.
EFFECTS OF ENVIRONMENTAL MEDIA: The specificity of FA-ELISA
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.
34
-------
(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
35
-------
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
pressure.
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
phenotype
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
36
-------
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.
37
-------
(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).
38
-------
TECHNIQUES TO LABEL AND DETECT NUCLEIC ACIDS
(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.
39
-------
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.
40
-------
(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
42
-------
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.
EFFECTS OF ENVIROHHENTAL MEDIA: Not applicable
43
-------
(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
44
-------
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.
EFFECTS OF ENVIRONMENTAL MEDIA: Not applicable
45
-------
(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
vector.
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.
EFFECTS OF ENVIRONMENTAL MEDIA: Not applicable
-------
(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
unnecessary.
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
microorganisms.
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
48
-------
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
49
-------
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).
50
-------
(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
microorganisms.
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.
51
-------
(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
hybridizing.
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
52
-------
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
53
-------
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
preparation.
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).
54
-------
(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.
55
-------
(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.
56
-------
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
57
-------
from a given environmental medium or the ability to transfer
bacteria out of that medium and into a laboratory medium.
58
-------
(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
59
-------
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.
61
-------
(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
exponential.
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
62
-------
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
63
-------
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
sequences.
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.
64
-------
(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
electrophoresis.
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
temperatures.
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.
65
-------
(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
duplex.
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
66
-------
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
primer.
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
insertions.
67
-------
(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
69
-------
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.
70
-------
REFERENCES
Aardema, B.W., Lorenz, M.G., and Krumblien, W.E. 1983.
Protection of sediment-adsorbed transforming DNA against enzymic
inactivation. ADD!.Environ. Microbiol.. 46:417-420.
Abbot, M.A., Poiesz, B.J., Byrne, B.C., Kwok, S., Sninsky, J.J.,
and Ehrlich, G.D. 1988. Enzymatic gene amplification:
Qualitative and Quantitative methods for detecting proviral DNA
amplified in vitro. J. Infect. Pis. 158:1158-1169.
Aho, E.L., Murphy, G.L. and Cannon, J.G. 1987. Distribution of
specific DNA sequences among pathogenic and commensal Neisseria
speices. Infec. Immun. 55:1009-1013.
Amann, R.I., Krumholz, L., and Stahl, D.A. 1990a. Fluorescent
oligonucleotide probing of whole cells for determinative
phylogenetic and environmental studies in microbiology. J.
Bacteriol. 172:762-770.
Amann, R.I., Binder, B.J., Olsen, G.J., Sallie, W., Chisolm,
R.D., and Stahl, D.A. 1990b. Combination of 16s rRNA targeted
oligonucleotide probes with flow cytometry for analysis of mixed
microbial populations. APP!. Environ. Mierobiol. 56:1919-1925.
Amy, P.S., and H.D. Hiatt. 1989. Survival and detection of
bacteria in an aquatic environment. ADO!. Environ. Microbiol.
55:788-793.
Armstrong, J.L., Knudsen, G.R., and Seidler, R.J. 1987.
Microcosm method to assess survival of recombinant bacteria
associated with plants and herbivorous insects. Curr. Microbiol.
15:229-232.
Arnheim, A. and Levenson, C. 1990. Polymerase Chain Reaction.
Chem. Enar. News. 68:36-57.
Athwal, R.S., Deo, S.S. and Zmaeda, T. 1984. DNA relatedness
among Mvcobacterium leprae Mvcobacterium lepraemurj^up and
selected bacteria by dot blot and spectrophotometric DNA
hybridization assays. Int. J. Svst. Bacteriol. 34:371-375.
Atlas, R. and Bartha, R. 1987. Microbial Ecology.
Benjamin/Cummings Pub. Co., Inc., Menlo Park, CA.
Atlas, R., and Sayler, G. 1988. Tracking microorganisms and
genes in the environment, pp. 31-45. In.: Omenn, G. (ed.)
Environmental biotechnology: Reducing risks from environmental
chemicals through biotechnology. Plenum Press, NY.
71
-------
Attwood, G.T., Lockington, R.A., Xue, G., & Brooker, J.O. 1988.
Use of a unique gene sequence as a probe to enumerate a train of
Bacteroides rqff^niC"?!8 introduced into the rumen. Appl. Environ.
Microbiol. 54:534-539.
Bale, M.J., Fry, J.C., and Day, M.J. 1987. Plasmid transfer
between strains of Pseudoaonas aerucrinesa on membrane filters
attached to river stones. J. Gen. Microbiol. 133:3099-3107.
Barkay, T., Liebert, C., and Gillman, M. 1989. Hybridization of
DNA probes with whole-community genome for detection of
genes that encode microbial responses to pollutants. Appl.
Environ. Microbiol. 55:1574-1577.
Bej, A.K., Steffan, R.J., Dicesare, J., Haff, L., and Atlas, R.M.
1990. Detection of coliform bacteria in water by polymerase
chain reaction and gene probes. APP!. Envir. Mierobiol. 56:307-
314.
Bej, A.K., Mahbubani, M.H., and Atlas, R.M. 1991. Detection of
viable Leaionella pneuroophila in water by polymerase chain
reaction and gene probe methods. Appl. Environ. Microbiol. 57:
597-600.
Bentjen, S.A., Frederickson, J.K., Van Voris, P., and Li, S.W.
1989. Intact soil-core microcosms for evaluating the fate and
ecological impact of the release of genetically engineered
microorganisms. Appl. Environ. Microbiol. 55:198-202.
Berent, S., Massoud, M., Troczynski, R., Bragg, P., and Bollon,
A. 1985. Comparison of oligonucleotide and long DNA fragments as
probes in DNA and RNA dot. Southern, Northern, colony, and plaque
hybridizations. Biotechniques 3:208-220.
Bergseid, M., Scott, B., Mathur, S., Nielson, K., Shoemaker, D.
and Mathur, E. 1991. A high fidelity thermostable DNA
Polymerase isolated from Pvrococeus furiosus. strategies in
Molecular Biology Newsletter 4:34-35.
Betzl, D., Ludwig, W. and Schleifer, K.H. 1990. Identification
of lactococci and enterococci by colony hybridization with 23s
ribosomal RNA-targeted oligonucleotide probes. Appl. EnvironT
Mierobiol. 56:2927-2929.
Bohlool, B.B., and Schmidt, E.L. 1980. The immunofluorescence
approach in microbial ecology. Adv. Microbial Ecology 4:203-241.
Bohnert, M.G., D'Hauteville, H.M..and Sasnonetti, P.J. 1988.
Detection of enteric, pathotypes of Eseheriehia cjali by
hybridization using six DNA probes. Ann. Inst. Pasteur 139t189-
202.
72
-------
Bonner, W. 1987. Autoradiograms of 35S and MP. pp. 55-61. In;
(Berger, S., Kimmel, A., eds.) Methods in Enzymology Vol. 152,
Guide to Molecular Cloning Techniques, Academic Press, New York,
NY.
Bottomly, P. and Haggard, S. 1990. Determination of viability
within serotypes of a soil population of Rhizobium leouminosarum
bv. trifolli. Aonl. Environ. Mierobiol. 56:533-540.
Bryan, R.N., Ruth, J.L., Smith, R.O. and Le Bon, J.M. 1986.
Diagnosis of clinical samples with synthetic oligonucleotide
hybridization probes, pp. 113-116. In; Microbiology - 1986
(L. Leive, ed.), American Society for Microbiology, Washington,
DC.
Buluwela, L., Forster,A., Boehm, T., and Rabbitts, T.H. 1989. A
rapid procedure for colony screening using nylon filters. Nucleic
Acid Res. 17:452.
Burg, J.L., C.M. Grover, P. Pouletty and J.C. Boothroyd. 1989.
Direct and sensitive detection of a pathogenic protozoan,
Toxoulasma aondii. by polymerase chain reaction. J. Clin.
Mierobiol. XX:1787-1792.
Castle, D.L. and Hulm, S.c. 1989. Microbial activity in relation
to pesticide degradation, pp. 523-528, In: (T. Hattori, Y.
Ishida, Y. Maruyama, R.Y. Morita and A. Uchida, eds.) Recent
advances in microbial ecology. Japan Scientific Societies Press.
Chadwick, E.G., R. Yogev, S.Y. Kwok, J.J. Sninsky, D.E. Kellogg ,;
and S.M. Wolinsky. 1989. Enzymatic amplification of the human
immunodeficiency virus in peripheral blood mononuclear cells from
pediatric patients. J. Infect. Pis. 160:954-959.
Chamberlain, J., Gibbs, R., Ranier, J., Nguyen, P., and Caskey,
C. 1988. Deletion screening of the Duchenne muscular dystrophy
locus cia multiplex DNA amplification. Nucleic Acids Res.
23:11141-11156.
Chaudhry, G., Toranzos, G., and Bhatti, A. 1989. Novel method
for monitoring genetically engineered microorganisms in the
environment. Appl. Environ. Microbiol. 55:1301-1304.
Chen, K., Weimark, H. and Steinman, C.R. 1989. Broad range DNA
probes for detecting and amplifying eubacterial nucleic acids.
FEMS Mierobiol. Letters 57:19-24.
Chevrier, D., Larzul, D., Megrand, F. and Guesdon, J.-L. 1989.
Identification and classification of Campvlobacter strains by
using nonradioactive DNA probes. J. Clin. Microbiol. 27:321-
326.
73
-------
Chollet, A. and Kawashima, E.H. 1985. DNA containing the base
analogue 2 aminoadenine preparation use as hybridization probes
and cleavage by restriction endonucleases. Nucleic Acids Res.
13:1529-1541.
Chu, B.C.F. & Orgel, L.E. 1985. Detection of specific DNA
sequences with short, biotin-labeled probes. Dflft 4:327-331.
Chuba, P.J., Pelz, K., Krekeler, 6., De Isele, T.S. & Gobel, U.
1988. Synthetic oligodeoxynucleotide probes for the rapid
detection of bacteria associated with human periodontis. J. Gen.
Microbiol. 134:1931-1938.
Clark-Curtiss, J.E. & Docherty, M.A. 1989. A species-specific
repetitive sequence in Mvcobagteriuyi leprae DNA. J. Infect. Pis.
159:7-15.
Collins, M.L. & Hunsacker, W.R. 1985. Improved hybridization
assays employing tailed oligonucleotide probes: a direct
comparison with 5'-end-labeled oligonucleotide probes and nick
-translated plasmid probes. Anal. Biochem. 151:211-224.
Colwell, R., Brayton, P., Grimes, D., Roszak, D., Huq, S., and
Palmer, L. 1985. viable but non-culturable yj.br3,° cholerae and
related pathogens in the environment: implications for release of
genetically engineered microorganisms. Bio/Technoloav 3:817-820.
Colwell, R., Sommerville, C., Knight, I., and Straube, W. 1988.
Detection and Monitoring of Genetically-engineered Microo-
organisms. pp. XX-XX In: (M. Sussman, C. Collins, F. Skinner,
and D. Stewart-Tull, eds.), The Release of Genetically-engineered
Microp-organisms, Academic Press, London.
Compeau, G., B.J. Al-Achi, E. Platsouka, and S.B. Levy. 1988.
Survival of rifampin-resistant mutants of Pseudomonas fluorescens
and Pseudomonas putida in soil systems. Appl. Environ.
Microbiol. 54:2432-2438.
Conner,. B.J., Reyes, A.A., Korin, C., Itakura, R., Teplitz, R.L.
& Wallace, R.B. 1983. Detection of sickle cell betaS-globin
allele by hybridization with synthetic oligonucleotides. Progj
Natl. Acad. Sci. USA 80:278-282.
Cooksey, R.C., Clark, W.c. & Thornsberry, C. 1985. A gene probe
for TEN type beta—lactamases. Antimicrob. Agents Chemother.
28:154-156.
Costanzi, C. and Gillespie, D. 1987. Fast blots:
Immobilization of DNA and RNA from cells. Methods Enxvaol.
152:582-587.
74
-------
Cresswell, N. and Wellington, E.M.H. 1991. Detection of genetic
exchange in the terrestrial environment. In: (E.M.H. Wellington
and J.D. van Elsas, eds.) Genetic interactions between
microorganisms in the microenvironment, Manchester University
Press (In Press).
Datta, A.R., Wentz, B.A., Shook, D. & Trucksess, M.W. 1988.
Synthetic oligodeoxyribonucleotide probes for detection of
Listeria monocytocrenes. Applt Environ. Microbiol. 54:2933-2937.
Davidson, M. and Summers, A. 1983. wide-host range plasmids
function in genus Thiobacillus. Apol. Environ. Microbiol.
46:565-572.
DeFlaun, M.F., & Paul, J.H. 1989. Detection of exogenous gene
sequences in dissolved DNA from aquatic environments. Microbial
ecology 18:21-28.
DeFlaun, M.F., Paul, J.H., & Davis, D. 1986. Simplified method
for dissolved DNA determination in aquatic environments. Appl. '
Environ. Mierobiol. 52:654-659.
Delidow, B.C., J.J. Peluso & B.A. White. 1989. Quantitative
measurement of mRNAs by polymerase chain reaction. Gene Anal.
Techn. 6:120-124.
Delong, E.F., Wickham, G.S., and Pace, N.R. 1990. Phylogenetic
stains: rRNA based probes for the identification of single
cells. Science 243:1360-1363.
Denhardt, D.T. 1966. A membrane-filter technique for the
detection of complementary DNA. Biochem. Biophys. Res. Commun.
23:641-646.
Devanas, M.A. and Stotzky, G. 1986. Fate in soil of a
recombinant plasmid carrying a Drosophila gene. Curr. Microbiol.
13:279-283.
Drahos, D.J., Barry, G.F., Hemming, B.C., Brandt, E.J., Skipper/
H.D., Kline, E.L., Kluepfel, D.A., Jughes, T.A., and Gooden, D.T.
1988. Pre-release testing procedures: US field test of a lacZY-
engineered bacterium, pp.181-191. In: (Sussman, M., Collins,
C.H., Skinner, F.A., and Stewart-Tull, D.E., eds.) Release of
Genetically-engineered Microorganisms, Academic Press, London.
Drake, T.A., Hindier, J.A., Berlin, O.G.W. & Bruckner, D.A. 1987.
Rapid identification of Mycobacterium avium complex in culture
using DNA probes. J. Clin. Microbiol. 25:1442-1445.
Draper, D.E. 1984. Attachment of reporter groups to specific,
cytidine residues in RNA using bisulfite-catalyzed transamination
reaction. Nucleic Acids Res. 12:989-1002.
75
-------
Dwyer, D.F., Rojo, F.r and Timmis, K.N. 1988. Fate and behaviour
in an activated sludge microcosm of a genetically engineered
microorganism designed to degrade substituted aromatic compounds.
pp. 77-88. In; (H. Sussman, C.H. Collins, F.A. Skinner and D.E.
Stewart-Tull, eds.) Release of Genetically- engineered
Microorganisms, Academic Press, London.
Eckert, K.A. and Kunkel, T.A. 1991. DNA polymerase fidelity and
the polymerase chain reaction. PCR Methods and Applications
1:17-24.
Eisenach, K.D., Crawford, J.T. and Bates, J.H. 1988. Repetitive
DNA sequences as probes for Mvcobacterium tuberculosis. J. Clin.
Microbiol. 26:2240-2245.
Enns, U.K. 1988. ONA probes - an overview and comparison with
current methods. Laboratory Medicine 19:295-300.
Eriks, I.S.,, Palmer, G.H., Mcguire, T.C., Allred, D.R. and
Allred, D.R. 1989. Detection and guantitation of Anaplasma
marainale in carrier cattle by using a nucleic acid probe. J.
Clin. Microbiol. 27:279-284.
Ezaki, T., Hashimoto, Y. and Yabuuchi, E. 1989. Fluoremetic
DNA-DNA hybridization in microdilution wells as an alternative
to membrane filter hybridization in which radioisotopes are
used to determine genetic relatedness among bacterial strains.
Int. J. Svst. Bacteriol. 39:224-229.
Feinberg, A.P. and Vogelstein, B. 1983. A technique for
radiolabeling DNA restriction endonuclease fragments to high
specific activity. Anal. Biochem. 132:6-13.
Festl, H., Ludwig, W. and Schleifer, K.H. 1986. DNA
hybridization probes for the Pseudomonas fluoreseens group.
APPl. Environ. Microbiol. 52:1190-1194.
Fitts, R., Diamond, M., Hamilton, C. and Neri, M. 1983. DNA-DNA
hybridization assay for detection of Salmonella spp. in foods.
APPl. Environ. Microbiol. 46:1146-1151.
Ford, S., and Olson, B. 1988. Methods for detecting genetically
engineered microorganisms in the environment. Advances in
Microbial Ecol. 10:45-79.
Forster, A.C., Mcinnes, J.L., Skingle, D.C. and Symons, R.H.
1985. Nonradioactive hybridization probes prepared by the
chemical labelling of DNA and RNA with a novel reagent,
photobiotin. Nucleic Acids Res. 13:745-761.
-------
Fredrickson, J.K., Bezdicek, D.F., Brockman, F.J., and Li, S.W.
1988. Enumeration of Tn£ mutant bacteria in soil by using a
most-probable-number-DNA hybridisation procedure and antibiotic
resistance. AppL. Environ. Microbiol. 54:446-453.
Frederickson, J.K., S.A. Bentjen, H. Bolton, Jr., S.W. Li, and P.
Van Voris. 1989. Fate of Tn£ mutants of root growth-inhibiting
Pseudomonas sp. in intact soil-core microcosms. Can. J.
Microbiol. 35:867-873.
Fuhrman, J.E., Comeau, D.E., Hagstrom, A., and Chan, A.M. 1988.
Extraction from natural planktonic microorganisms of DNA suitable
for molecular biological studies. Aopl. Environ. Microbiol.
54:1426-1429.
Gebeyehu, 6., Rao, P.Y., Soochan, P., Simmes D.A. and Klevan, L.
1987. Novel biotinylated nucleotide: analogs for labeling and
colorimetric detection of DNA. Nucleic Acids Res. 15:4513-4534.
Gelfand, D. 1989. Thermus aouaticus DNA polymerase. pp. 11-18
In: (H. Erlich, R. Gibbs, and R. Kazazian, eds.), Polymerase
Chain Reaction. Cold Spring Harbor Laboratory Press, New York.
Gemski, P., Sodd, M.A., Neill, R.J., Seguin, M.C. and Williams,
J.E. 1987. Cloning and use of Vwa plasmid DNA as gene probes for
virulent Yersiniae. Contrib. Microbiol. Immunol. 9:296-303.
Germida, J.J. and Khachatourians, G.G. 1988. Transduction of
Escherichia coli in soil. Can. J. Microbiol. 34:190-193.
Giovannoni, S.J., Delong, E.F., Olsen, G.J., and Pace, N.R. 1988.
Phylogenic group specific oligodeoxynucleotide probes for
identification of single microbial cells. J. Bacteriol. 170:720-
726.
Gobel, U. and Stanbridge, E.J. 1984. Cloned Mvcoplasma ribosomal
RNA genes for the detection of Mvcoplasma contamination in tissue
cultures. Science 226:1211-1213.
Gobel, U., Geiser, A., and Stanbridge, E.J. 1987.
Oligonucleotide probes complementary to variable regions of
ribosomal RNA discriminate between Mvcoplasma species. J. Gen.
Microbiol. 133:1969-1974.
Gomes, T.A.T., Toledo, M.R.F., Trabulsi, L.R., Wood, P.K. and
Morris, J.G. Jr. 1987. DNA probes for identification of
enteroinvasive Eseherichia coli. J. Clin. Microbiol. 25:2025-
2027.
Graham, J.B. and Istock, C.A. 1978. Genetic exchange in Bacillus
subtilis in soil. Mol. Gen. Genetics 166:287-290.
77
-------
Graham, J.B. and Istock, C.A. 1979. Gene exchange and natural
selection cause Bacillus subtilis to evolve.in soil culture.
Science. 204:637-639.
Grainger, J.M. and J.M. Lynch. 1984. Microbiological Methods for
Environmental Biotechnology. Academic Press Inc., London.
Grimont, P.A.D., Grimont, F., Desplaces, N. and Tchen, P. 1985.
DNA probe specific for Legionella pneumophila* J. Clin.
Microbiol. 21:431-437.
Groot Obbink, D.J. Ritchie, L.J., Cameron, F.H., Mattick, J.S.
and Ackerman, V.P. 1985. Construction of a gentamicin resistance
gene probe for epidemiological studies. Antimicrob. Agents
Chenother. 28:96-102.
Grunstein, M. and Hogness, D.S. 1975. Colony hybridization: A
method for the isolation of cloned DNA that contains a specific
gene. Proc. Natl. Acad. Sci. USA 72:3961-3965.
Gutterson, N. 1988. pp.198-201 In: (Sussman, M., Collins,
C.H., Skinner, F.A., and Stewart-Tull, D.E., eds.), Release of
Genetically—engineered Microorganisms. Academic Press, London.
Hahn, D., Kester, R., Starrenburg, M.J.C., and Acker-mans, A.D.L.
1990. Extraction of rRNA from soil for detection of Frankia
strains with oligonucleotide probes. Arch.Microbiol. 154:329-
335
Halbert, D.N. 1988. DNA probes for the detection of antibiotic
resistance genes. Clin. Microbiol. Newsletters 10:33-37.
Hance, A.J., B. Grandchamp, V. Levcy-Frebault, D. Lecossier 6 J.
Rauzier. 1989. Detection and identification of mycobacteria by
amplification of mycobacterial DNA. Mol. Mierobiol. 3:843-849.
Haun, G. and Gobel, II. 1987. Oligonucleotide probes for genus,
species, and subspecies specific identification of
representatives of the genus Proteus. FEMS Microbiol. Lett.
43:187-193,
Hay, P.E., Clarke, J.R., strugnall, R.A., Robinson-Taylor, D.,
and Goldmeir, D. 1990. Use of the polymerase chain reaction to
detect DNA sequences specific to pathogenic treponemes in
cerebrospinal fluid. FEMS Microbiology letters 68:233-238.
Hazen, T.C., and Jiminez, L. 1988. Enumeration and
identification of bacteria from environmental samples using
nucleic.acid probes. Microbiological Sciences 5:340-343.
78
-------
Heath, L.S., Sloan, 6.L. and Heath, H.E. 1986. A simple and
generally applicabel procedure for releasing DNA from bacterial
cells. Apol. Environ. Microbiol. 51:1138-1140.
Heiles, B.J., Genersch, E., Kessler, C. , Neumann, R. and Eggers,
H.J. 1988. In situ hybridization with digoxigenen- labeled DNA of
human papillomaviruses HPV 16-18 in HeLa and SiHa cells.
Biotechnioues 6:978-981.
Henscke, R.B. and Schmidt, R.J. 1990. Plasmid mobilization from
genetically engineered bacteria to members of the indigenous
microflora in situ. Curr. Microbiol. 20:105-110.
Herron, P.R. and Wellington, E.M.H. 1990. New method for the
detection of Streptomvces spores from soil and application to the
study of lysogeny in sterile amended and nonsterile soil. Aopl.
Environ. Microbiol. 56:1406-1412.
Herron, P.R, and Wellington, E.M.H. 1990. New method for
extraction of Streptomycete spores from soil and application to ,
the study of lysogeny in sterile amended and nonsterile soil.
Aopl. Environ. Mierobiol. 56:1406-1412.
Holben, W.E., and Tiedje, J.M. 1988. Application of nucleic
acid hybridisation in microbial ecology. Ecology 69:561-568.
Holben, W. , Jansson, J., Chelm, B. , and Tiedje, J. 1988. DNA
probe method for the detection of specific microorganisms in the
soil bacterial community. ADD!. Environ. Microbiol. 54:703-711.
Hooykaas, P., Van Brussel, A., den Dulk-Ras, H. , van Slogteren,
G. , and Schilperoot, R. 1981. Sym plasmid of Rhizobium trifolii
expressed in different rhizobial species and Aarobacterium
tumefaciens. Nature 291:351-353.
Huovinen, S., Huovinen, P. and Jacoby, 6. A. 1988. Detection of
plasmid-mediated beta lactamases with DNA probes. Antimicrob.
Agents Chemother. 32:175-179.
Hymah, H.C., Yoger, D. and Razin, S. 1987. DNA probes for
detection and identification of Mvcoplasma pneumoniae and
Mvcoplasma aen^taliinB- J. Clin. Mierobiol. 25:726-728.
Hyypia, T. 1985. Detection of adenovirus in nasopharyngeal
specimens by radioactive and nonradioactive DNA probes. J. Clin
Microbiol. 21:730-733.
Hyypia, T., Jalava, A., Larsen, S.H., Terho, P. and Hukkanen, V.
1985. Detection of Chlamydia trachomatis in clinical specimens
by nucleic acid spot hybridization. J. Gen. Microbiol. 131:975-
978.
-------
Ikuta, S., Takagi, K., Bruce, R. and Itakura, K. 1987.
Dissociation kinetics of 19 base paired oligonucleotide-DNA
duplexes containing different single mismateched base pairs.
Nucleic Acids Res. 15:797-811.
Inoue, H., Imura, A. and Ohtsuku, E. 1985. Synthesis and
hybridization of dodecadeoxyribonucleotides containing a
fluorescent pyridopyrimidine deoxynucleoside. Nucleic Acids Res.
13:7119-7128.
•
Jablonski, E., Moomaw, E.W., Tullis, R.H. and Ruth, J.L. 1986.
Preparation of oligodeoxynucleotide-alkaline phosphatase
conjugates and their use as hybridization probes. Nucleic Acids
Res. 14:6115-6128.
Jagow, J. and Hill, W.E. 1986. Enumeration by DNA colony
hybridization of virulent Yersinia enterocolitica colonies in
artificially contaminated food. Appl. Environ. Microbiol.
51:441-443.
Jain, R., Burlage, R., and Sayler, 6. 1988. Methods for
detecting recombinant DNA in the environment. CRC Critical
Reviews in Biotechnology 8:33-84.
Kafatos, F.C., Jones, C.W. and Efstratiadis, A. 1979.
Determination of nucleic acid sequence homologies and relative
concentrations by a dot hybridization procedure. Nucleic Acids
Res. 7:1541-1552.
Keller, G.H., Huang, D.P. and Manak, H.H. 1989. Labelling of
DNA probes with a photoactivatable hapten. Anal. Biochem.
177:392-395.
Kempe, T., Sundquist, W.J., Chow, F. and Hu, S.L. 1985.
Chemical and enzymatic biotin-labeling of
oligodeoxyribonucleotides. Nucleic Acids Res. 13:45-57.
Koehler, T. and Thome, C. 1987. Bacillus subtilus fnattol
plasmid pLS20 mediates interspecies plasmid transfer. J.
Bacteriol. 169:5271-5278.
Klinger, J.D., Johnson, A., Groan, D., Flynn, P., Whippie, K.,
Kimball, M., Laurie, J., and Curiale, H. 1988. Comparative
studies of nucleic acid hybridization assay for Listeria in
foods. J. Assoc Anal. Chen. .71:669-673.
Knight , I.T., Shults, s., Kaspar, C.W., and Colwell, R.R. 1990.
Direct detection of Salmonella SPP. in estuaries using a DNA
probe. Appl. Environ. Microbiol. 56:1059-1066.
Kogure, K., Simidu, U., and Taga, N. 1976. A tentative direct
microscopic method for counting living marine bacteria. Can. J.
Microbiol. 25:415-420.
80
-------
Krasovsky, V.N. and Stotzky, G. 1987. Conjugation and genetic
recombination in E. coli in sterile and non-sterile soil. Soil
Biol. Biochem. 19:631-638.
Kumar, A., Tchen, P., Roullet, F. and Cohen, J. 1988.
Nonradioactive labeling, of synthetic oligonucleotide probes with
terminal deoxynucleotidyltransferase. Anal. Biochem. 169:376-
382.
Kuritza, A.P., and Salyers, A.A. 1985. Use of a species-
specific DNA hybridisation probe for enumerating Bacteroides
vulaatus in human faeces. ADP!. Environ. Microbiol. 50:958-964.
Kuritza, A.P., Shaughnessy, P., and Salyers, A.A. 1986.
Enumeration of polysaccharide-degrading Bacteroides species in
human faeces by using species-specific DNA probes. Apol.
Environ. Microbiol 51:385-390.
Langer, P.R., Waldrop, A.A. and Ward, D.C. 1981. Enzymatic
synthesis of biotin labeled poly nucleotides novel nucleic-acid
affinity probes. Proc. Natl. Acad. Sei. PSA 78:6633-6637.
Lathe, R. 1985. Synthetic oligonucleotide probes deduced from
amino-acid sequence data theoretical and practical
considerations. J. Mol. Biol. 183:1-12.
Leary, J.J., Brigati, D.J. and Ward, D.C. 1983. Rapid and
sensitive colcrimetrie method for visualizing biotin labeled
DNA probes hybridized to DNA or RNA immobilized on nitrocellulose
bio-blots. Proc. Natl. Acad. Sci. USA 80:4045-4049.
Lee, I.-M., Davis, R.E., Hammond, R. and Kirkpatrick, B. 1988.
Cloned riboprobe for detection of a mycoplasmalike organism.
Biochem. Bioohvs. Res. go|fliiyiin.- 155:443—448* • '
Lewin, B. 1990. The extraordinary power of DNA technology, .pp.
451-465. In: Genes IV. Oxford University Press, Oxford.
Lidstrom, H.E. 1989. Molecular approaches to the problems of
biogeochemical cycling. Antonie van Leeuwenhoek 55:7-14.
Liebl, W., Rosenstein, R., Gotz, F. and Schleifer, K.H. Use of
staphylococcal nuclease gene as DNA probe for staphvloeoeeus
aureus. 1987. FEMS Microbiol. Letters 44:179-184.
Lorenz, M.G., Aardema, B.W., and Wackernagel, W. 1988. Highly
efficient genetic transformation of Bacillus subtilis attached to
sand grains. J. Gen. Microbiol. 134:107-112.
Mahbubani, M.H., A.K., Bej, R. Hiller, L. Haff, J. Dicesare and
R.M. Atlas. 1990. Detection of Leaionella with polymerase chain
reaction and gene probe methods. Mol. Cell Probes 4:175-187.'
81
-------
Mahbubani, M., A.K. Bej, Dicesare, R. Miller, L. Haff and R.M.
Atlas. 1990. Detection of bacterial inRNA using PCR.
Biotechnioues 10:48-49.
Mancini, P., S. Fertels, D. Nave, and M.A. Gealt. 1987.
Mobilization of plasmid pHSV106 from £ coli HB101 in a laboratory
scale waste treatment facility. ADD!. Environ. Microbiol.
53:665-671.
Maniatis, T., Fritsch, E.F. and Sambrock, J. 1982. Molecular
cloning. A laboratory manual. Cold Spring Harbor, New York.
Maniatis, T., Fritsch, E., and Sambrook, J. 1984. Southern
transfer: Transfer of DNA from agarose gels to nitrocellulose.
pp. 382-387. In: Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Laboratory Press, New York.
Marmur, J. 1961. A procedure for the isolation of
deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-
218.
Matthews, J.A. and Kricka, L.J. 1988. Analytical strategies for
the use of DNA probes. Anal. Biochem. 169:1-25.
Maxam, A. and Gilbert, W. 1980. Sequencing end-labelled DNA with
base-specific chemical cleavages. Methods of Enzymology 65:499-
560.
McPherson, P. and Gealt, M. 1986. Isolation of indigenous
wastewater bacterial strains capable of mobilizing plasmid
PBR325. Appl. and Environ. Microbiol. 51:904-909.
Meighan, E. 1988. Enzymes and genes from the lux operons of
bioluminescent bacteria. Annu. Rev. Microbiol. 42:151-176.
Meinkoth, J. and Wahl, G. 1984. Hybridization of nucleic acids
immobilized on solid supports. Anal. Biochem. 138:267-271.
Melton, D.A., Krieg, P.A., Rebagliati, M.R., Bitzan, M.,
Maniatis, T., Zinn, K. and Green, M.R. 1984. Efficient in vitro
synthesis of biologically active RNA and RNA hybridization probes
from plasmids containing a bacteriophage SP6 promoter. Nucleic
Acids Res. 12:7035-7056.
Meyer, T., Bitzan, M., Sandkamp, O. and Karch, H. 1989.
Synthetic oligodeoxyribonucleotide probes to detect
verocytotoxin-producing Escherichia coli in diseased pigs. FEMS
Microbiol. letters 57:247-252.
Miliotis, M.D., Galen, J.D., Kaper, J.B. and Morris, Jr., J.G.
1989. Development and testing of a synthetic oligonucleotide
probe for the detection of pathogenic Yersinia strains. J. Clin.
Mierobiol. 27:1667-1670.
82
-------
Morgan, J.A.W., Winstanley, C., Pickup, R.W., Jones, J.G., and
Saunders, J.R. 1989. Direct phenotypic and genotypic detection
of a recombinant pseudomonad population released into lake water.
APPl. Environ. Microbiol. ,55:2537-2544.
Morotomi, M., Ohno, T. and Mutai, M. 1988. Rapid and correct '
identification of intestinal Bacteroides spp. with
chromosomal DNA probes by whole-cell dot blot hybridization.
Appl. Environ. Mierobiol. 54:1158-1162.
Morrissey, D.V. and Collins, H.L. 1989. Nucleic acid
hybridization assays employing dA-tailed capture probes, .single
capture methods. Mol. Cell. Probes 3:189-207.
Morrissey, D.V., Lombardo, M., Eldredge, J.K., Kearney, K.R.,
Groody, E.P. and Collins, H.L. 1989. Nucleic acid hybridization
assays employing d-a-tailed capture probes. I. Multiple capture
methods. Anal. Biochem. 182:000-000.
Moseley, S.L., Echeverria, P., Seriwatana, J., Tiripat, C.,
Chaicumpa, W., Sakuldaipeara, T. and Falkow, S. 1982.
Identification of enterotoxigenic Escherichia coli by colony
hybridization using three enterotoxin gene probes. J. Infect.
Pis. 145:863-869.
Murasugi, A. and Wallace, R.B. 1984. Biotin-labeled
oligonucleotides enzymatic synthesis and use as hybridization
probes. DNA 3:269-277.
Myrold, D.D., A.B. Hilger and S.H. Strauss. 1990. Detecting
Frankly in soil using PCR. p. 429. In: (Greshoff, Roth, Stacey
& Newton, eds.), Nitrogen Fixation: Achievements and Objectives,
Chapman & Hall, London. .
Nannipieri, P., Ciardi, C., Badalucco, L., and Casella, S. 1986.
A method to determine soil DNA and RNA. Soil Biol.Biochem.
18:275-281.
Nataro, J.P., Baldini, M.H., Kaper, J.B., Black, R.E., Bravo, N.
and Levine, M.M. 1985. Detection of an adherence factor of
enteropathogenic Escherichia coj.i with a DNA probe. J. Infect.
Pis. 152:560-565.
Nesme, X., M.C. Leclerc and R. Bardin. 1989. PCR detection of
an original endosymbiont: the Ti plasmid of Aarobacterium
tumefaclens. Endocvtobiol. 4:47-50.
Neuner, A., Jannasch, H.W., Belkin, S. and Stetter, K. O. 1990.
Thermococcus literalis sp. nov.: a new species of extremely
thermophilic marine archaebacteria. Arch. Microbiol. 153: 205-
207. . . '
83
-------
Nishibuchi, M., Ishibashi, M., Takeda, Y. and Kaper, J.B. 1985.
Detection of the thermostable direct hemolysin gene and related
DNA sequences in Vibrio parahaemolvticus and other Vibrio spp. by
the OKA colony hybridization test. Infec. Immin. 49:481-486.
Nishibuchi, M., Hill, W.E., Zon, G., Payne, W.L. and Kaper, J.B.
1986. Synthetic oligodeoxyribonucleotide probes to detect
kanagawa phenomenon-positive Vibrio parahaemolvticus. J. Clin.
Microbiol. 23:1091-1095.
Notermans, S., Heuvelman, K.J. and Wernars, K. 1988. Synthetic
Enterotoxin B DNA probes for detection of enterotoxigenic
Staohylococcus aureus strains. Apnl. Environ. Microbiol. 54:531-
535.
Notermans, S., Chakraborty, T., Leimeister- Wachter, M.,
Dufrenne, J., Heuvelman, K.J., Maas, H., Jansen, W., Wernars, K.
and Guinee, p. 1989. Specific gene probe for detection of
biotyped and serotyped Listeria strains. ADD!. Environ.
Mierobiol. 55:902-906.
Nur, I., Bove, J.M., Saillard, C., Rotten, S., Whitcomb, R.H. and
Razin, S. 1986. DMA probes in detection of spiroplasmas and
mycoplasma-like organisms in plants and insects. FEMS Microbiol,
Letters 35:157-162.
O'Morchoe, S., Ogunseitan, O., Sayler, 6. and Miller, R. 1988.
Conjugal transfer of R68.45 and FP5 between Pseudoponas
aeruainosa strains in a freshwater environment. Appj,. Environ.
Microbiol. 54:1923-1929.
Ogram, A., Sayler, G.S., and Barkay, T. 1988. The extraction and
purification of microbial DNA from sediments. J. Microbiological
Methods. 7:57-66.
Ogram, A.V., Sayler, G.S., Gustin, D. and Lewis, R.J. 1987. DNA
adsorption to soils and sediments. Environ. Sci. Tech. 22:982-
984.
Page, A.L. (ed.). 1982. Methods of Soil Analysis. Part 2:
Chemical.and Microbiological Properties. American Society for
Agronomy Madison, WI.
Palva, A. 1985. Nucleic-acid spot hybridization for detection
of Chlamvdia forachomatis. FEMS Microbiol. Letters 28:85-91.
Paul, J.H., and Carlson, D.J. 1984. Genetic material in the
marine environment: implication for bacterial DNA. Limnology
and Oceanography 29:1091-1097.
Paul, J.H., and Myres, B. 1982. Fluorometric determination of
DNA in aquatic microorganisms by use of Hoechst 33258. Appl.
Environ. Mierobiol. 43:1393-1399.
84
-------
Peferoen, H. 1982. Vacuum blotting a new simple and efficient
transfer of proteins from sodium dodecyl sulfate polyacrylamide
gels to nitro cellulose. FEBS Lett. 145:369-372.
Pozzi, G., Oggioni, M. and Tomasz, A. 1989. DNA probe for
identification of streptococcus pneumontae. J. Clin. Microbiol.
27:370-37.
Prefontaine, G., Fast, P., Lau, P.C.K., Hefford, M.A., Hanna, Z.
and Brousseau, R. 1987. Use of oligonucleotide probes to study
the relatedness of delta endotoxin genes among Bacillus
thurincriensis subspecies and strains. ADD!. Environ. Microbiol.
53:2808-2814.
Preston, D.R.,. Chaudhry, G.R., and Farrah, S.R. 1990. Detection
and identification of poliovirus in environmental samples using
nucleic acid hybridisation. Can. J. Mierobiol. 36:664-669.
Ratliff, R. 1981. Terminal deoxynucleotidyltransferase, pp.
105-118. In; The Enzymes/ Volume 14A, (ed. P. Boyer), Academic
Press, Inc., New York, NY, USA.
Rafii, F. and Crawford, D.L. 1988. Transfer of conjugative
plasmids and mobilization of a non-conjugative plasmid between
Streptomyces strains'in soil. Appl. Environ. Microbiol. 54:1334-
1340.
Rees. M., Kohn, D., Hails, R., Crawley, M., and Malcolm, S.
1990. An ecological perspective to risk assessment. Proc. Intl.
Sym. on the Biosafety Results of Field Tests of Genetically
Modified Plants and Microorganisms. Agricultural Research
Institute, Bethesda, Maryland, USA.
Regensburger, A., Ludwig, W. and Schleifer, K.H. 1988. DNA
probes with different specificities from a cloned 23s ribosomal
RNA gene of Microcoecus luteus. J. Gen. Microbiol. 134:1197- .
1204.
Regnery, R.L., Fu, Z.Y. and Spruill, C.L. 1986. Flying
squirrel-associated Rickettsia prowazekii epidemic typhus
rickettsiae characterized by a specific DNA fragment produced by
restriction endonuclease digestion. J. Clin. Microbiol. 23:189-
191.
Rehstam, A.-S., Norqvist, A., Wolf-Watz, H. and Hagstrom, A.
1989. Identification of Vibrio anguillarum in fish by using
partial 16s recombinant rna sequences and a specific 16s
recombinant RNA oligonucleotide probe. APP!. Environ. Microbiol.
55:1907-1910.
Richaume, A., Angle, J.S., and Sadowsky, M.J. 1989. Influence of
soil variables on in situ plasmid transfer from Escherichia coli
to Rhizobium freudii. APP!. Environ. Mierobiol. 55:1730-1734.
85
-------
Rifai, 8., Barbancon, V., Prevost, G. and Piemont, Y. 1989.
Synthetic exfoliative toxin A and B DNA probes for detection of
toxigenie Staohvloeoccus aureus strains. J. Clin. Microbiol.
27:504-506.
Rigby, P.W., Dieckmann, M., Rhodes, C. and Berg, P. 1977.
Labeling ONA to high specific activity in-vitro by nick
translation with DNA polymerase I. J. Mol. Biol. 113:237-251.
Roberts, M.., Roberts, I., Korhonen, T.K., Jann, K., Bitter-
Suermann, D., Boulnois, 6.J. and Williams, P.H. 1988. DNA
probes for K-antigen capsule typing of Escherichia sali. £±.
Clin. Mierobiol. 26:385-387.
Roberts, H.C., Killer, S.L., Schoenknecht, F.D. and Holmes, K.K.
1984. Nitrocellulose filter blots for species identification of
Mobiluncus curtisij, and Mobiluncus pulieris. J. Clin. Microbiol.
20:826-827.
Roberts, M.C., Koutsky, L.A., Holmes, K.K., LeBlanc, D.J. and
Kenny, G.E. 1985. Tetracycline-resistant Mycoplasma hominis
strains contain streptococcal tet-m sequences. Antipicrob.
Agents Chemother. 28:141-147.
Roberts, M.C., Moncla, B. and Kenny, G.E. 1987. Chromosomal DNA
probes for the identification of Bacteroides sp. J. Gen.
Microbiol. 133:1423-1430.
Robins-Browne, R.M., Milioits, M.D., Cianciosi, S., Miller, V.L.,
Falkow, S. and Morris, J.G. Jr. 1989. Evaluation of DNA colony
hybridization and other techniques for detection of virulence in
Yersinia spp. J. Clin. Microbiol. 27:644-650.
Rossau, R., Van Mechelen, E., De Ley, J. and Van Heuverswijn, H.
1989. Specific Neisseria gonorrhoeas DNA-probes derived from
ribosomal RNA. J. Gen. Microbiol. 135:1735-1745.
Rozak, D.B. and R.R. Colwell. 1987. Survival strategies of
bacteria in the natural environment. Microbiol. Rev. 51:365-379.
Rubin, F.A., Kopecko, D.J., Noon, K.F. and Baron, L.S. 1985.
Development of a DNA probe to detect Salmonella £ypj&. J. Clin.
Microbiol. 22:600-605.
Saiki, R. 1989. Optimization of the polymerase chain reaction.
pp.25-30. in; (H. Erlich, R. Gibbs, and R. Kazazian, eds.),
Polymerase Chain Reaction. Cold Spring Harbor Laboratory Press,
New York, NY.
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Riguchi,
R., Horn, G.T., Mullis, K.B. & Erlich, H.A. 1988.
Primer-directed enzymatic amplification of DNA with a
thermostable DNA polymerase. Science 239:487-491.
86
-------
Saiki, R.K. , Scharf, S.f Faloona, F., Mull is, K.B., Horn, 6.T.,
Erlich, H.A. & Arnehim, N. 1985. Enzymatic amplification of
beta globin genomic sequences and restriction site analysis for
diagnosis of sickle cell anemia. Science 230:1350-1354.
Saiki, R., Walsh, S., Levenson, C., Erlich, A. 1989. Genetic
analysis of amplified DNA with immobilized sequence-specific
oligonucleotide probes. Proc. Natl. Aead. Sei. 86:6230-6234.
Saiki, R.K. 1990. Amplification of genomic DNA. pp. 13-20.
In; PCR Protocols: A guide to methods and applications.
Academic Press, Inc. , San Diego, CA, USA.
Saluz, H. and Jost, J.P. 1986. Optimized genomic sequencing as
a tool for the study of cytosine methylation in the regulatory
region of the chicken vitellogenin II gene. Gene 42:151-157.
Salyers, A. A., Lynn, S.P. and Gardner, J-F. 1983. Use of
randomly cloned DNA fragments for identification of Bacteroides
thetaiotaom jlcron . J t_Baeteriol . 154:287-293.
Salyers, A. A. 1989. Molecular and biochemical approaches to
determine what bacteria are doing in^givo. Antonie van
Leeuwenhoek 55:33-38.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular
cloning: a laboratory manual, cold Spring Harbor Laboratory
Press, Cold Spring Harbor, USA.
Santha, M. , Burg, K. , Rasko, I. and Stipkovits, L. 1987. A
species-specific DNA probe for the detection Mvcoolasma
era 1 1 ;Lsept icum . Inf ec . Immun . 55:2857-2859.
Saris, P.E.J., Paulin, L.G. and Uhlenhlen, M. 1990. Direct
amplification of DNA from colonies of Bacillus subti,^j.s and
Escherichia coll by the polymerase chain reaction, j.
Methods 11:121-126.
Saunders, N.A. , Harrison, T.G., Kachwalla, N. and Taylor, A.G.
1988. Identification of species of the genus Leoionella using a
cloned ribosomal RNA gene from Lecrionella pneumophila. J. Gen.
Mierobiol. 134:2363-2374.
/
Saunders, J.R., Morgan, J.A.W., Winstanley, C., Raitt, F.C.,
Carter, J.P. , Pickup, R.H. , Jones, J.G., and Saunders, V.A. 1990.
pp. 3-21. in: (J.C. Fry and M.J. Day, eds.) Bacterial genetics
in natural. environments. Chapman and Hall, UK. ;
Sayler, G.S. and Layton, A.C. 1990. Environmental application
of nucleic acid hybridization. Ann. Rev. Mierobiol. 44:625-648.
87
-------
Sayler, G.S., Harris, C.r Pettigrew, C., Pacia, D., Breen, A.,
and Sirotkin, K.M. 1987. Evaluating the maintenance and effects
of genetically engineered microorganisms. Dev. Ind. Microbiol.
27:135-149.
Sayler, G.S., Shields, M.S., Breen, A., Tedford, E.T., Hooper,
S., Sirotkin, K.M., and Davis, J.W. 1985. Application of
DNA:DNA colony hybridization to the detection of catabolic
genotypes in environmental samples. Appl. Environ. Microbiol.
49:1295.
Scanferlato, V.S., Orvos, D.R., Cairns, J., Jr., and Lacy, G.H.
1989. Genetically engineered Erwinia carotovora in aquatic
microcosms: survival and effects on functional groups of
indigenous bacteria. Appl. Environ. Microbiol. 55:1477-1482.
Schesser, K., A. Luder and J.M. Henson. 1991. Use of polymerase
chain reaction to detect the take-all fungus, Gaeumannomvces
araminis in infected wheat plants. ADD!. Environ. Microbiol.
57:553-556.
Schilf,. W. and Klingmuller, w. 1983. Experiments on the
dispersal of plasmids in environmental samples. Recoiab. DNA
Tech. Bull. 6:101-102.
Schleifer, K.H. and Stackebrandt, E. 1983. Molecular
systematics of prokaryotes. Ann. Rev. Microbiol,. 37:143-187.
Schmidhuber, S., Ludwig, W. and Schleifer, K.H. 1988.
Construction of a DNA probe for the specific identification of
Streptococcus oralis. J. Cl|n» Microbiol. 26:1042-1044.
Schmidt, E.Xi. 1974. Quantitative autecological study of
microorganisms in soil by immunofluorescence. Soil Science
118:141-149.
Schmidt, F.R.J., Nucken, E.J., and Henshke, R.B. 1989. Structure
and function of hotspots providing signals for site-directed
specific recombination in Tn21 transposons. Molecular
Microbiology 3:1545-1555.
Sharrock, W.J. and Rabinowitz, J.C. 1979. Fractionation of
ribosomal particles from Bacillus subtilis. Methods in
Enzvmoloav 59:371-382.
Shaw, J.J. and Kado, C.I. 1986. Development of a vibrio
bioluminescence gene-set to monitor phytopathogenic bacteria
during the ongoing disease process in a non-disruptive manner.
Bio/Technoloav 4:560-564.
Simonet, P., P. Nonnand, A. Moiroud and R. Bardin. 1990.
Identification of Frankia strains in nodules by hybridization of
polymerase chain reaction products with strain-specific
oligonucleotide probes. Arch. Mierobiol. 153:235-240.
88
-------
Smit, E. and van Elsas, J.D. 1991. Methods for studying genetic
transfer in soil. In: (eds. J.D. van Elsas and E.M.H.
Wellington) Genetic interactions between microorganisms in the
microenviromnent. , Manchester University Press (in press).
Smith, C.L. and Cantor, C.R. 1987. Preparation and manipulation
of large dna molecules advances and applications. Trends
Biochem. Sei. 12:284-287. ,.
Smith, L.M., Sanders, J.Z., Kaiser, R.J., Hughes, P., Dodd, C.,
Conell, C.R., Heiner, C., Kent, S.B.H. and Hood, L.E. 1986.
Fluorescence detection in automated DNA sequence analysis.
Nature 321:674-679.
Sommerfelt, H., Svennerholm, A.-M., Kalland, K.H., Hankanes, B.-
I. and Bjorvatin, B. 1988a. Comparative study of colony
hybridization with synthetic oligonucleotide probes and ELISA for
identification of enterotoxigenic Escherichia coli. J. clin. .
Microbiol. 26:530-534.
Sommerfelt, H., Kalland, K.H., Raj, P., Moseley, S.L., Bhan, M.K.
and Bjorvatin, B. 1988b. Cloned polynucleotide and synthetic
oligonucleotide probes used in colony hybridization are equally
efficient in the identification of enterotoxigenic Eseherichia
coli. J. Clin. Microbiol. 26:2275-2278.
Somerville, C.C., Knight, I.T., straube, W.L. and Colwell, R.R.
1989. Simple rapid method for direct isolation of nucleic acids
from aquatic environments. APP!. Environ. Microbiol. 55:548-554.
Southern, E.M. 1975. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol.
98:503-510.
Stahl, D.A., Flesher, B., Mansfield, H.R. and Montgomery, L.
1988. Use of phylogenetically based hybridization probes for
studies of ruminal microbial ecology. Appl. Environ. Microbiol.
54:1079-1084.
Stambach, M.N., S. Falkow and L.S. Tompkins. 1989. Species-
specific detection of Leaionella pneumophila in water by DNA
amplification and hybridization. J. clin. Microbiol. 27:1257-
1261.
Steffan, A., and Atlas, R. 1988. DNA amplification to enhance
detection of .genetically engineered bacteria in environmental
samples. APP!. Environ. Microbiol. 54:2185-2191.
Steffan, R.J., Breen, A., Atlas, R.M., and Sayler, 6.S. 1989.
Application of gene probe methods for monitoring specific
microbial populations in freshwater ecosystems. Can. J.
Microbiol. 35:681-685.
89
-------
Steffan, R.3., Goksoyr, J, Bej, A.K., and Atlas, R.M. 1988.
Recovery of DNA from soils and sediments. Aopl. Environ.
Mierobiol. $4:2908-2915.
Steffan, R.J., Breen, A., Atlas, R.M., and Sayler, G.S. I989a.
Monitoring genetically engineered microorganisms in freshwater
microcosms. J. Ind. Mierobiol. 4:441-446.
Steffan, R.J., Breen, A., Atlas, R.M., and Sayler, G.S. 1989b.
Application of gene probe methods for monitoring specific
microbial populations in freshwater ecosystems. Can. J.
Mierobiol. 35:681-685.
Steffan,.R. and Atlas, R. 1990. Solution hybridization assay
for detecting genetically engineered microorganisms in
environmental samples. Biotechnioues. 8:316-318.
Stotzky, G., Devanas, M.A., and Zeph, L.R. 1990. Methods for
studying bacterial gene transfer in soil by conjugation and
transduction. Adv. Apol. Microbiol. 35:57-170.
Syvanen, A.C., Laaksonen, M. and Soderlund, H. 1986. Time-
resolved fluorometry: a sensitive methods to quantify DNA-
hybrids. Nucleic Acids Res. 14:1017-1028.
Talbot, H., Yamamoto, D., Smith, M., and Seidler, R. 1980.
Antibiotic resistance and its transfer among clinical and
nonclinical Klebsiella strains in botanical environments. ABpl,,.
andEnviron,Microbiol. 39:97-104.
Tannoclc, G.W. 1989. Biotin-labeled plasmid DNA probes for
detection of epithelium-associated strains of lactobacilli.
ADD!. Environ. Mierobiol. 55:461-464.
Taylor, M.A., Wise, K.S. and Mclntosh, M.A. 1985. Selective
detection of Mycoplasma hvorhinis using cloned genomic DNA
fragments. Infgffti IfflMn- 47:827-830.
Tchen, P., Fuchs, R.P.P., Sage, £., and Leng, M. 1984.
Chemically modified nucleic acids as immuno detectable probes in
hybridization experiments. Proe. Natl. Acad. Sei. PSA 81:3466-
3470.
Tenover, F.C. 1988. Diagnostic DNA probes for infectious
diseases. Clin. Microbiol. Rev. 1:82-101.
Thorpe, G.H.G., Kricka, L.J., Gillespie, E., Moseley, S*, Amess,
R., Baggett, N. and Whitehead, T.P. 1985. Enhancement of the
horseradish peroxidase-catalyzed chemiluminescent oxidation of
cyclic diacyl hydrazides by 6-hydroxybenzothiazoles. Anal.
Biochem. 145:96-100.
90
-------
Tjernberg, I., Lindh, E. and Ursing, J. 1989. A quantitative
bacterial dot method for DNA-DNA hybridization and its
correlation to the hydroxyapetite method. Curr. Microbiol.
18:77-81.
Top, E., Mergeay, M., Springael, D., and Verstraete, W. 1990.
Gene escape model: Tmasfer of heavy metal resistance genes from
Escherichia eoli to Alcaliaenes eutrophus on agar plates and in
soil samples. ADD!. Environ. Microbiol. 56:2471-2479.
Torsvik, V.L. 1980. isolation of bacterial DNA from soil. Soil
Biol. Biochem. 12:15-21.
Torsvik, V.L., and Goksoyr, J. 1978. Determination of bacterial
DNA in soil. Soil Biol. Bioehem. 10:7-12.
Torsvik, V.L., Goksoyr, J., and Daae, F.L. 1991. High diversity
in DNA of soil bacteria. APP!. Environ. Microbiol. 56:782-787.
Torsvik, V.L., Salte, K. and Serheim, R. 1990a. Comparison of
phenotypic diversity and DNA heterogeneity in a population of
soil bacteria. ADD!. Environ. Microbiol. 56:776-781.
Torsvik, V.L., Goksoyr, J., and Daae, F.L., 1990b. High
diversity of DNA in soil bacteria. ADD!. Environ. Microbiol.
56:782-787.
Totten, P.A., Holmes, K.K., Handsfield, H.H., Knapp, J.S.,
Ferine, P.L. and Falkow, S. 1983. DNA hybridization technique
for the detection of Neisseria aonorrhoeae in men with
urethritis. J. Infect. Pis. 148:462-471.
Trevors, J.T., and van Elsas, J.D. 1989. A review of selected
methods in environmental genetics. Can. J. Microbiol. 35:895- ..
902.
Trevors, J.T. and Oddie, K.M. 1986. R-plasmid transfer in soil
and water. Can. J. Microbiol. 32:610-613.
Trevors, J.T. and Starodub, M.E. 1987. R-plasmid transfer in
non-sterile agricultural soil. Systematics and ApplT Microbiol.
9:312-315.
Urdea, M.S., Running, J.A., Horn, T., dyne, J., Ku, L. and
Warner, B.D. 1987. A novel method for the rapid detection of
specific nucleotide sequences in crude biological samples without
blotting or radioactivity: application to the analysis of
hepatitis B virus in human serum. Gene 61:253-264.
-------
van Elsas, J.D., Govaert, J.M., and van Veen, J.A. 1987.
Transfer of plasmid pFT30 between Bacilli in soil as influenced
by bacterial population dynamics and soil conditions. Soil Biol.
Biochem. 19:639-647.
van Elsas, J.D., Trevors, J.T., and Starodub, M.E. 1988.
Bacterial conjugation between pseudomonads in the rhizosphere of
wheat. FEMS Microbiol. Ecology 531299-306.
van Elsas, J.D., Nickel, M., and Van Overbeeck, L.S. 1989.
Detection of plasmid RP4 transfer in soil and rhizosphere, and
the occurrence of homology to RP4 in soil bacteria. Curr.
Microbiol. 19:375-381.
van Elsas, J.D., L.S. van Overbeek and R. Fouchier. 1991. A
specific marker, pet, for studying the fate of introduced
bacteria and their DNA in soil using a combination of detection
techniques. Plant and soil (in press).
Verdlov, E.D., Monastyrskaya, 6.S., Guskowa, L.I., Levitan, T.L.,
Sheichenko, V.I. and Budowsky, E.I. 1974. Modification of
cytidine residues with a bisulfite-o-methyl hydroxylamine
mixture. Biochim. Biophvs. Acta 340:153-165.
Verreault, H., Lafond, M., Asselin, A., Banville, G. and
Bellemare, G. 1988. Characterization of two DNA clones specific
for identification of Corynebacterium sepedonicum. Can. J.
Microbiol. 34:993-997. ,
Vincent, C., Then, P., Cohen-Solal, M. and Kourilsky, P. 1982.
Nucleic Acids. Res. 10:6787-6796.
Viscidi, R.P., Connelly, C.J. and Yolken, R.H. 1986. J. Cliri.
Microbiol. 20:1083-1088.
Vogeli, G., and Kaytes, P. 1987. Amplification, storage, and
replication of libraries, pp.407-415. In; Methods in
Enzymology. Volume 152. Guide to Molecular Cloning Techniques.
(Berger, S., Ximmel, A., eds.), Academic Press, New York, NY.
Wahl, G., and Berger, S. 1987. Screening colonies or plaques
with radioactive nucleic acid probes. In: Methods in
Enzymology. Volume 152. Guide to Molecular Cloning Techniques.
(Berger, S. and Kimmel, A., eds.), Academic Press, New York, NY.
Wahl, G., Berger, S., and Kimmel, A. 1987. Molecular
hybridization of immobilized nucleic acids: Theoretical concepts
and practical considerations, pp. 399-407. In: (Berger, S. and
Kimmel, A., eds.) Methods in Enzymology. Volume 152. .Guide to
Molecular Cloning Techniques, Academic Press, New York, NY.
92
-------
Wallace, R.B., Johnson, M.J., Hirose, T., Miyaka, T., Kawashima,
E.H. and Itakura, K. 1981. The use of synthetic
oligonucleotides as hybridization probes. 2. Hybridization of
oligonucleotides of nixed sequence to rabbit beta-globin DNA.
Nucleic Acids Res. 9:879-894.
Wallace R., and Miyada, C. 1987. Oligonucleotide probes for the
screening of recombinant DNA libraries, pp. 432-443. In: (eds.}
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, New York.
Walter, M., Porteous, A., and Seidler, R. 1987. Measuring
genetic stability in bacteria of potential use in genetic
engineering. ADD!. Environ. Microbiol. 53:105-109.
Walter, M.V., Olsen, R.H., Prince, V., Seidler, R.J., and Lyon,
F. 1988. Use of catechol dioxygenase for the direct and rapid
identification of recombinant microbes taken from environmental
samples. In; Proc. V. Intl. Sym. on Rapid Methods and
Automation in Microbiology and Immunology, Springer-Verlag.
Wang, Z., Crawford, D.L., Pometto, A.L. Ill, and F. Rafii. 1989.
Survival and effects of wild-type, mutant, and recombinant
streetomvces in a soil ecosystem. Can. J. Microbiol. 35:535-543.
Wang, Z. and Crawford, D.L. 1990. Effects of genetically
engineered microorganisms in the environment: Lignin peroxidase-
expressing recombinant Streptomyces can effect the rate of carbon
mineralization in an agricultural soil, (in preparation and
abstracted in Abstr. FirstIntl. Conf. on the Releaseof
Genetically—engineered Microorganisms. p. 13)*
Ward, D.M., We Her, R., and Bateson, M.M. 1990. 16S rRNA
sequences reveal numerous uncultured microorganisms in a natural
community. Nature 345:63-65.
Waller, R. and Ward, D.M. 1989. Selective recovery of rRNA
sequences from natural microbial communities in the form of cDNA.
Appl. Environ. Mierobiol. 55:1818-1822.
Wellington, E.M.H., Saunders, V.A., Cresswell, N., and Wipat, A.
1988. Plasmid transfer between streptomycetes in soil. pp. 300-
305. In: Biology of Actinomycetes. '88, eds. Y. Okami, T. Beppu
and H. Ogaware, Japan Scientific Societies Press, Tokyo.
Wellington, E.M.H., Cresswell, N., and Saunders, V.A. 1990.
Growth and survival of streptomycete inoculants and the extent of
plasmid transfer in sterile and non-sterile soil. Appl. Environ.
Microbiol. 54:1413-1419.
93
-------
Weinberg, S.R. and Stotzky, G. 1972. Conjugation and
recombination of Eschcrichia coli in soil. Soil Biol. Biochem.
4:171-180.
Wetherall, 8.L., McDonald, P.J. & Johnson, A.M. 1988. Detection
of Campvlobacter pylori DNA by hybridization with non-radioactive
probes in comparison with a phosphorus32 labelled probe. J. Med.
Microbiol. 26:257-264.
Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-
271.
Wood, P.K., Morris, J.G. Jr., Small, P.L.C., Sethabutr, o.,
Toledo, R.F., Trabulsi, L. and Kaper, J.B. 1986. Comparison of
DNA probes and the Sereny test for identification of invasive
Shioella and Escherichia eoli strains. J. Clin. Microbiol.
24:498-500.
Yang, H.-L. 1985. R-plasmid identification using biotinylated
DNA probe, p. 161-164. In: L. Lieve (ed.), Microbiology-1985.
American Society for Microbiology, Washington, DC.
Yates, J.R., Lobos, J.H. and Holmes, D.S. 1986. The use of
genetic probes to detect microorganisms in biomining operations.
J. Indtist. Mierobiel. 1:129-136.
Yogev, D., Halachmi, D., Kenny, 6.E. and Razin, S. 1988a.
Distinction of species and strains of Mvcoplasmas mollicutes by
genomic DNA fingerprints with a ribosomal RNA gene probe. J.
Clin. Microbiol. 26:1198-1201.
Yogev, D., Sela, S., Bercovier, H. and Razin, S. 1988b.
Elongation factor (EF-Tu) gene probe detects polymorphism in
Mvcoplasma strains. FEMS Microbiol, Letters 50:145-149.
Zechman, J.,- and Casida, L.E., Jr. 1982. Death of Pseudomonas
aeruqinosq in soil. can. J. Microbiol. 28:788-794.
Zeph, L.R., Onaga, M.A., and Stotzky, G. 1988. Transduction of
Escherichia eeli by bacteriophage PI in soil. Appl. Environ.
Microbiol. 54:1731-1737.
Zeph, L.R., X. Lin, and G. Stotzky. 1990. Comparison of three
nonradioactive and a radioactive DNA probe for the detection of
target DNA by DNA hybridization. Curr. Mierobiol. 22:79-84.
-------
TABLE 1. DETECTION OF GENE TRANSFER IN STERILE SOIL: RECIPIENT SELECTION AND DONOR COUNTERSELECTION.
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Weinberg &
Stotzky, (1972)
Graham &
Istock, (1978
& 1979)
Trevors &
Oddie, (1986)
van Elsas et
(1987)
Krasovsky &
Stotzky, (1987)
Intraspeciflc crosses
between E. coli
auxotrophic recipient
& prototrophic donor.
Intraspecific crosses
between B. Subtilis in
autoclaved potting
soil.
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
transfer.
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
Sm.
-------
TABLE 1. (Continued)
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Rafii &
Crawford,
(1988)
Lorenz et al
(1988)
Wellington
al.. (1988)
Zeph et al..
(1988)
Inter- & Intraspecific
crosses between
streptomycetes.
Autoclaved soil,
moisture 60% MHC.
Competent Bf subtilis
(auxotrophic, TrpC2)
attached to sterile
sand grains.
Conjugal piasmid
transfer & piasmid
mobilization.
Transformation of naked
DNA Isolated from
B. subtilis 168
prototroph.
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)
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Richaume et
al.. (1989)
Herron &
Wellington,
(1990)
Top et alt.
(1990)
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%
chitin.
Intergeneric crosses
between E. coli donor
and A. eutroohus.
autoclaved soils,
moisture 75% field
capacity.
Conjugal plasmid
transfer (CpRK2073:Tn5)
Actinophage infection,
formation of lysogens.
Conjugal plasmid
transfer, and plasmid
mobilization.
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
gene.
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)
-------
TABLE 2. DETECTION OP GENE TRANSFER IN NON-STERILE SOIL:
COUNTERSELECTION.
RECIPIENT SELECTION AND DONOR
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Schilf &
Klingmuller,
(1983)
van Elsas et
(1987)
Krasovsky &
Stotzky, (1987)
Trevors &
Starodub,
(1987)
Zeph et al
(1988)
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
transfer.
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
transfer.
Selection of transconjugants
by selection of plasmid encoded
antibiotic resistance genes.
No counterselection, no transfer
detected.
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)
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Germida &
Khachatourians,
(1988)
Wellington
(1990)
Co-inoculation of Transduction by phage
transducing Pi phage Pi.
lysates and auxotrophic
E. coli K12-GK401, soil
amended.
As in Table 1.
As in Table 1.
Herron &
Wellington,
(1990)
As in Table 1.
Soil unattended.
As in Table 1.
Top et al,
(1990)
As in Table 1.
Soil unamended &
amended.
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)
REFERENCE.
EXPERIMENTAL
CONDITIONS.
MODE OF GENETIC
TRANSFER.
METHOD FOR SELECTION OF
EXCIPIENTS AND COUNTER-
SELECTION OF DONORS.
Henscke &
Schmidt, (1990)
Smit & van
Elsas, (1990)
E. coli SM10 plasmid
donor, study of
transfer to indigenous
microflora.
Conjugal plasmid
mobilization.
P. fluorescens plasmid
donor (R2F RP4;;pat
Rpr), study of transfer
to indigenous microflora.
Conjugal RP4 plasmid
transfer.
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).
PLASMID PLASMID TYPE
pBC16 conjugative
pBC16
PBC16
RP4 conjugative
RP4
8a conjugative
8a
R68.45 conjugative
R68.45
R$8.45
DONOR/RECIPIENT
fi. subtilus
fi. subtilus/
fi. 1 icheni f ormis
fi. subtilus/
B. meaaterium
CONDITIONS
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 _ «_ _ _ . *.
RATE
10'2-10*5D
10*SD
10'*D
10'5D
l.OD
10'3D
10**D
10-*D
10'5D
10"2D
REF
Koehler and Thorne (1987)
Davidson and Summers
(1983)
Walter et a_l. (1987)
O'Morchoe et aJL. (1988)
*
R68.45
lake water
plus filter
field: non-
sterile lake
water
1
-------
TABLE 3. (Continued)
PLASMID PLASMID TYPE DONOR/RECIPIENT
CONDITIONS
RATE
REF
PMQ1 conjugative
PMQ1
PMQ1
£. aeruqinosa/
£. aeruqinosa
PMQ1
pBR325 nobilizable £. coli/E. coli
pBR325
pFT30
pFT30
conjugative Bacillus cereus/
B. subtilis
lab: broth 10'1R
with sterile
stone & filter
lab: sterile 10'2R
river water,
field: unencl. 1(T*R
nonsterile
river water,
sterile stone
& filter
Bale et al. (1987)
field: unencl.
nonsterile
water, nonsterile
stone , & filter
lab: broth
& mobilizer
plasnid
lab: raw,sterile
wastewater &
mobilizer plasmid
lab: filter
lab: sterile 10"7D
soil
10'7R
10'5D
10'7D
McPherson and Gealt (1986)
Van Elsas (1987)
-------
TABLE 3. (Continued)
PLASMID PLASMID TYPE
pFT30
*
pFT30
R plasmids conjugative
pRTrSa conjugative
DONOR/RECIPIENT CONDITIONS RATE
lab: nonsterile OD
soil
lab: nonsterile 10"7D
soil & bentonite
Klebaiella/ lab: nonsterile 10'6D
Klebaiella radish rhizosphere
Rhizobium trifolii/ lab: in situ ?
B. leauminsarura
REF
Talbot et al. (1980)
Hooykaas et al.
(1981)
-------
TABLE 4. DMA extraction from the environment
Reference
Environment
Detection
Notes
Abbot et al.
(19S8)
Attwood
et al. (1988)
Bej et al.
(1990)
Deflaun
et al. (1986)
Deflaun &
Paul (1989)
FredricJcson
et al. (1988)
Fuhrman
et al. (1988)
Hay et al.
(1990)
Holben et al.
(1988)
Kniaht et al.
(1990)
Kuritza et al,
(1986)
HIV/HTLV in
Human T-cells
Rumen
Water
Water
Fresh water/
marine
soil
Water
Cerebrospinal
fluid
Soil
Water
Faeces
50ng DNA
(2X107 cfu)
IBacteroidesl
1-10 fg
DNA or 1-5
cfu/lOOml
E. coll
volume
dependent
Cells isolated,
DNA extracted,
FCR amplified.
Indirect lysis,
hybridisation.
Indirect lysis,
PCR, hybridisa
tion.
Indirect lysis
Hoechst 33258.
167 fg Herpes Indirect lysis,
DNA in E. coll hybridisation.
plasmid
10-100 cfu/g
(Rhizobium/
E. colil
25-50% of
total
bacterial
65
fTreponema)
Enrichment, in
direct lysis,
hybridisation,
MPN.
Indirect lysis,
agarose gel
electrophoresis,
Hoechst 33258.
Direct lysis,
PCR, hybridisation.
0.2 pg DNA/104 Indirect lysis,
cfu/g hybridisation.
(Bradyrhizobium)
7-15 ng Indirect lysis,
DNA/2.6 x 103 hybridisation.
cfu/ml
(Salmonella1
(Baeteroidesl
Indirect lysis,
hybridisation,
(radioactive
count).
-------
TABLE 4.
(Continued)
Reference
Environment
Detection
Notes
Kuritza &
Salyers (1985)
Nannipieri
et al. (1986
Ogram et al.
(1987)
Paul &
Carlson (1984)
Paul & Myers
(1982)
Preston et al.
(1990)
Somerville
et al. (1989)
Steffan &
Atlas (1988)
Steffan et al.
(1988)
Torsvik &
Goksoyr (1977)
Torsvik (1980)
Torsvik et al.
(1990)
Faeces
Soil
Sediment
Fresh/marine
water
Water
Waste water
Marine
Sediment
Soil/sediment
2% of popula-
tion
(Bacteroides)
Soil
Soil
Soil
40-91% total
95-100% total
DNA
104 pfu
(Poliovirus)
1-3 cfu/ml
100 cfu/ioog
(Pseudo monas1
Indirect lysis,
hybridisation
(radioactive
count)
Indirect lysis,
diphenylamine
assay.
Direct lysis,
hybridisation.
Indirect lysis,
Hoechst 33258.
Indirect lysis,
Hoechst 33258.
Indirect lysis,
hybridisation.
Indirect lysis,
agarose gel
elect.
Indirect lysis,
PCR, hybridisation.
Direct lysis/
Indirect lysis,
agarose gel,
electrophoresis,
absorbance.
Indirect lysis,
DABA.
Indirect lysis,
absorbance.
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
Identification
Method
Label
Reference
Ac inet obacter spp.
Bacteroides spp.
Bacteroides spp.
Campylobacter
pylori
Campy 1 obacter spp.
Chlarovdia tracho-
matis
Enterobacter
aaalomerans
Mobiluncus spp.
MvcobacteriuM
leprae
whole-cell dot
blot
whole-cell dot
blot
whole-cell dot
blot
dot blot
dot blot
dot blot
whole-cell dot
blot
whole-cell dot
blot
dot blot
125!
32p
32p
32p
Acetyl-
amino-
fluorene
32p
125!
32p
32p
Tjernberg et al.,
1989
Roberts et al.,
1987
Horotomi et al.,
1988
Wetherall et al.,
1988
Chevrier et al.,
Hyypia et al.,
1985
Tjernberg et al.,
1989
Roberts et al.,
1984
Athwal et al.,
1984
-------
TABLE 6: Examples of DNA probes derived from 16 or 23S rRNA targets
Speci-
ficity
Type of Probe
Detection
Reference
Universal
probe
Kingdom
Supragenus
Genus
Species
Subspecies
Oligonucleotide
(16S)
Cloned fragment
(23S)
Oligonucleotide
(16S)
01igonucleotide
(16S)
Cloned fragment
Of 23S rRNA
Oligonucleotide
(16S)
Oligonucleotide
(16S)
Cloned fragment
(23S)
Cloned fragment
(16S)
Cloned fragment
(23S)
Oligonucleotide
(1€S)
Oligonucleotide
(not specified)
Cloned rRNA gene
fragment
Oligonucleotide
(16S)
Oligonucleotide
(16S)
Oligonucleotide
(16S)
Oligonucleotide
(16S)
Oligonucleotide
(16S)
.Oligonucleotide
(16 and 23S)
Oligonucleotide
(not specified)
Oligonucleotide
(16S)
Oligonucleotide
(16S)
all organisms
all organisms
Giovannoni et al.,
1988
Regensburger
et al., 1988
eucaryotes
eubacteria
eubacteria
eubacteria
archaebacteria
Micrococcus -
Arthrobacter
Mvcoplasma
Pseudomonas
Proteus
Legjonella
Giovannoni et al.,
1988
Giovannoni et al.,
1988
Regensburger
et al., 1988
Chen et al., 1989
DeLong et al., 1989
Regensburger
et al., 1988
Gobel and
Stanbridge, 1984
Festl et al. ,1986
Haun and Gobel,
1987
Enns, 1988
Leqionella pneu-
mophila
Mvcoplasma pneu-
moniae
Proteus species
Grimont et al.,
1985
Gobel et al.,
1987
Haun and Gobel,
1987
Species associated Chubaetal., 1988
with human perio-
dontitis
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
niae
Mvcobacteriuro avium Drake et al.,
complex 1987
Vibrio ancmillarum Rehnstam et al.,
1988
Bacteroides
sueeinoaenes
strains
Stahl et al.,
1988
-------
TABLE 7: Examples of DNA probes derived from randomly cloned DNA
fragments
Speci-
ficity
Origin
Identification
Reference
Genus
Species
Sub-
species
Genomic DNA
plasmid
Genomic DNA
Genomic DNA
Genomic DNA
Oligonucleotide
(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
Cryptic
plasmids
Genomic DNA
Salmonella spp.
Spiroplasma spp.
Bacteroj.des thetaio-
taomicron
Bacteroides vulcraris
Bacteroides fraoilis
Campvlobacter jeiuni
Chlamydia trachomatis
CorvnebacteriuBi sepe—
donicun
culosia
MvcobacteriuM leprae
Mvcoplaama hvorhinis
Mycoplasma pneumonias
Mvcoplasma aallisep-
Mvcoplasma-like .
organism
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.,
1983
Kuritza and
Salyers, 1986
Kuritza et al.,
1986
Bryan et al,, 1986
Palva, 1985
Verreault et al.,
1988
Eisenach et al.,
1988
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.,
1986
Schmidhuber et al.,
1988
Yates et al., 1986
Attwood et al.,
1988
Tannock, 1989
Yates et al., 1986
-------
TABLE 8: Examples of DMA probes from genes
Speci-
ficity
Strains of
different
species
Gene coding for
Identification
Reference
Delayed hyper- Listeria strains
sensitivity factor
-Lactamases
0-Lactamases
Different anti-
biotic resistance
genes
Elongation factor
Tu
Pili and outer
membrane proteins
Hemolysin
Virulence-asso-
ciated plasmid
£. coli and
Pseudomonas aeru-
ainosa
various gram-nega-
tive eubacteria
various eubacte-
ria
Mycoplasma spp.
Neisseria spp.
Vibrio parahaemo-
lyticus. V.
hollisae
Yersinia spp.
Notermans et al.,
1989
Cooksey et al.,
1985
Huovinen et al.,
1988
Halbert, 1988
Yogev et al.,
1988
Aho et al., 1987
Nishibuchi et al.,
1985; 1986
Gemski et al.,
1987; Rabins-Browne
et al., 1989
Species
Surface protein
Hemolysin
Capsular antigen
Deoxyribonu-
clease
Autolysin
Anaolasma mar-
Listeria mono-
cytoaenes
Salmonella tvpfai
Staphylococcus
aureyp
Streptococcus
Eriks et al., 1989
Datta et al., 1988
Rubin et al., 1985
Liebl et al., 1987
Pozzi et al., 1989
-------
TABLE 8 (continued)
Speci-
ficity
Gene coding for
Identification
Reference
Strains Delta-endotoxin
Gentamicin
resistance
Heat-labile and
heat-stable
toxins
Invasion process
Shiga-like toxi
Adhesin-factor
Capsular anti-
gens
Toluene degrada-
tion
Exfoliative
toxin
Virulence
(plasmid)
Mercury
resistence
Bacillus thuringensis
strains
£• coli strains
enterotoxigenic
£• coli strains
£• coli and
Shioella spp.
enterohemorrhagic
£• coM strains
enteropathogenic
£• SSSUL strains
encapsulated
£• eoli strains
Pseudomonas
putida strains
Staphylococcus
aureus strains
Yersinia entero-
colitiea
whole-community
genome (water)
Prefontaine et al.,
1987
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.,
1988
Sayler et al,,
1985
Rifal et al., 1989
Jagow and Hill,
1986
Barkay et al.,
1989
-------
TABUS 9: Non-radioactive reporter groups for indirect detection of
hybrids
Reporter group
Biotin
N-2-Acetylamino-
fluorene
Sulfone
4-dinitrophenyl
Digoxygenin
Detection
Labelled avidin,
Labelled antibody
Labelled antibody
Labelled antibody
Labelled antibody
Labelled antibody
Reference
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.
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
£•
PI Cm cts;tTnSOl
Streptomyces sp.2
Pseudomonas sp.3
?. ceoacia
conjugative
plasmid, pIJ303
chromosomal
insertion of
vector sequences
from suicde
vector,pRL425,
with
dot blot on DNA
extracted from
clonies isolated
on selective media
using biotinylated
probe to repA
colony
hybridization
with selective
media using a
nick translated
biotinylated
probe made from
the whole plasmid
Southern
hybridization on
DNA isloated from
colonies
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
verify
transduction
detect
mobilization of
conjugative
plasmid in
Streptomvces sp.
recepients
verify the
presence of the
plasmid and
determine
modifications in
new host by RFLP.
use restrction
length
polymorphisms to
detect plasmid
sequence deletion
as a result, of a
double cross-over
recombination
event
-------
Table 10. (Continued)
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
P. cepacia4
Pseudomonas sp.
notll chromosomal
insert
P. nutida5
leaumisarium
Tnfi chromsomal
insertion
Colony blot
analysis with
probe made to 32P-
labeled pRL425
vector
slot blots on
total DNA using a
32P-labeled
single- stranded
DNA probe
soil microcosm
Dot blots on DNA
from isolated
colonies using
nick translated
32P-labeled vector
containing Tn£ as
probe
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
cells
follow the
population
dynamics,
movement, and
leaching of
introduced
organism
determine if Tn£
insert is stable
in soil
determine
recovery of and
follow growth of
organisms over
time
-------
Table 10. (Continued)
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
£. fluorescens6
conjugative,
plasmid RP4
pBR322 modified
with Drosphila
gene
E. coli8
Rhizobium fredi,
pRK2073::Tn5
colony
hybridization
with selective
media using a
nick translated
32P-labeled probe
made from whole
plasmid
colony
hybridization
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
heterlogous
plasmid
containing Tnj>
non-sterile soil
detect plasmid
transfer to
homologous
recepient
sterile and non-
sterile soil
sterile soil
determine
stability of
plasmid and
foreign DNA
transfer of
conjugative
plasmid from £.
coli and fi.
fredii
-------
Table 10. (Continued)
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
E .coli9
£• putida10
Alcaligenes AS11
P.cepacia
non-conj uga t1ve
plasmid
containing herpes
simplex thymidine
kinase (tkl gene
plasnid RK2 and
biodegredative
plasmid TOL
encoding the xvl
operon colony.
biodegredation
plasnid, pSSSO
allows the
degradation of 4-
chlorobiphenyl
genes involved in
2-4-5-trichloro-
phenoxyacetic
acid degredation
colony
hybridization
with selective
media using a
nick translated
32p-labeled 850 bp
tk fragment as a
probe
hybridization
with non-
selective media
using nick-
tanslated 32P-
labeled probes to
RK2 and xvlR gene
on TOL plasmid
colony
hybridization
with selective or
non-selective
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
treatment
facility
groundwater
aqifer material:
uncontaminated
and
experimentally
contaminated
non-contaminated
and contaminated
freshwater
microcosm
demonstrate
mobilization of
non-conj ugative
plasmid in a
tripatental
mating with
recepient cells
demonstrate
stability and
maintenance of
introduced
biodegredative
strain and
plasmids
monitor growth
and perslstance
of organisms
-------
Table 10. (Continued)
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
£• coll12
£. putida13
hybrid plastnid
between pBR325
and pEML159 from
Alcallaienes sp.
encoding mercury
resistance and
2,4-dichloro-
phenoxyacetic
acid degredatlon
non-conj ugatlve
IncQ broad-host
range plasmid In
which xvlE gene
has been Inserted.
located
chromosomally and
on plasmlds In £.
cepacia or to
pSSSO for
Alcaliaenes
colony
hybridization
with selective
and non-selective
media using a
nick translated
biotinylated
probe to whole
plasmid
Dot blots of DNA
isolated from
filtered cells
lysed in situ on
nitrocellulose
membranes using a
0.6 kb xylE
fragment randomly
primed and
labeled with
32P-dCTP
filtered and
unfiltered lake
water
survival and
monitoring study
filtered and
unfiltered lake
water
survival and
monitoring study
-------
Table 10. (Continued)
ORGANISM
TARGET DNA
DETECTION METHOD
SAMPLE MATRIX
PURPOSE
Azospirillum
lipoferum-14
Clavibactei
cvnodontis^
chromosomal
insert
chromosomal
insertion using
insertion vector
containing
^acillus
thurinaensia
delta-endotoxin
gene and
Streptococca1 tet
gene
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
oligo-nucleotide
(30 base pair)
probe to nptll
end-labeled with
32P-ATP.
Hybridization
blots on colonies
grown in
selective and
non-selective
media,
transferred to
nitrocellulose
and lysed ip situ;
probed with
isolated delta-
entotoxin and tet
genes labeled by
random priming with
digoxygenin-dUTP
corn seeds coated
with &. lipoferum
germinated in
microcosm
corn plants
planted in the
field for a
small-scale
release
determine levels
in rhizosphere,
endorhizo-sphere,
and xylem
exudates;
determine extent
of leaching into
soils
study gene loss
and segregation
rates of inserts
in corn;
determine rate of
survival in plant
debris post-
harvest; monitor
dissemination
into trap plants
-------
Table 10. (Continued)
ORGANISM TARGET DMA DETECTION METHOD SAMPLE MATRIX PURPOSE
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.
ORGANISM
Pseudoroonas
tmtida*
Azospirilum
lipoferum*
Pseudomonas
cepacia3
Bradvrhizpbium
j aponicum*
E. coli5
£• outida
TARGET
SEQUENCE
TOL plasmid
notll
species-
specific
repeat 1.3KB
sequence
nptll
xylE
PROBE
radiolabeled
TOL plasmid
radiolabeled
heterologous
plasmid
containing
nptll sequence
radiolabeled
fragment
containing 1
KB of RS1100-I
radiolabeled
single-
stranded DNA
radiolabeled
822 base-pair
DETECTION
METHOD
colony
hybridization
colony
hybridization .
dot blot
dot blot
dot blot
SAMPLE MATRIX SENSITIVITY
(DETECTION
LIMIT)
sediment 1 colony in
106
soil 103colonies/
gram of soil
sediment 103 cells/
gram of
sediment
soil 4 X 104/9rara
of soil or
0.02 picograms
target
DNA/microgram
total DNA
lake water 104colonies/ml
£• cepacia6
RS1100-I
fragment
T7-generated
radiolabeled
transcription
product of
RS1100-I
solution
hybridization
sediment
102-103
cells/gram of
sediment
-------
TABLE 11. (Continued)
ORGANISM
TARGET
SEQUENCE
PROBE
DETECTION
METHOD
SAMPLE MATRIX
SENSITIVITY
(DETECTION
LIMIT)
B.
notll
Rhizobiun Tn£
leouminosarum®
radiolabeled
single-
stranded DMA
radiolabeled
nick
translated
plasmid DNA
containing Tn£
sequences
Southern blot
soil
MPN-DNA
hybridization
soil
0.1 picogram
target
DNA/microgram
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
Environment
Soil
Soil
Soil
Alnus
nodules
Infected
wheat
Sediment
Aquatic
Aquatic
(lake, sew.)
Aquatic
* Detection
amplified;
of target
fraotn./fr.
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)
optimization
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
amplification
£. ceoacia 1.0 kb 15-20 Primer selection
important
Coliform LacZ/ 1?
bacteria LamB
£• coli 0.2 kb multiple
(plasm.)
Leaionella 0.65 kb 1
pneumophila mip fr.
Detection
limit
103-104
-103
"103(cfu);
~200(IU)
?
?
-l/0.3pg
<103
1-10
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;
References
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;
-------
This Page Intentionally Blank
-------
This Page Intentionally Blank
-------
This Page Intentionally Blank
------- |