APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1991, p. 1070-1074 Vol. 57, No.

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0099-2240/91/041070-05$02.00/0
Copyright 1991, American Society for Microbiology

Rapid Method for Direct Extraction of DNA from

Soil and Sediments
YU-LI TSAI AND BETTY H. OLSON*
Environmental Design and Analysis, Program in Social Ecology, University of California, Irvine, California 92717
Received 10 October 1990/Accepted 23 January 1991

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous
microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate
was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency
(>90%), the yields of DNA were high (38 and 12 ,ug/g [wet weight] from sediments and soil, respectively). This
method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an
Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required,
which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h)
period.

Isolation of bacterial DNA from natural environments has
become a useful tool with which to study the ecological
functions of certain characterized genes that encode impor-
tant metabolic pathways (25), allow tracking of genetically
engineered organisms (8, 10), and reveal bacterial DNA
diversity (21, 22) in microbial ecosystems. The application of
DNA extraction methods to environmental samples can
obviate the need for cell cultivation, since cell cultivation
has the disadvantage of obtaining only a very small propor-
tion of the total microbial community. DNA extraction
methods can also help researchers to understand the occur-
rence of particular bacterial genes in situ through applica-
tions of nucleic acid technology.
Several techniques have been described for direct detec-
tion and extraction of DNA from aquatic environments (3, 6,
13-15, 19), but there are only a few reports related to
extraction of DNA from soil and sediments (8, 12, 23). Two
current methods used to obtain microbial DNA from soils or
sediments are (i) bacterial cell extraction followed by cell
lysis and DNA recovery (8, 23) and (ii) direct extraction by
alkaline lysis (12). The direct extraction method produces a
better DNA yield than the cell extraction method (20).
However, both approaches share three disadvantages, i.e.,
they (i) are lengthy and laborious, (ii) require a relatively
large sample size (50 to 100 g), and (iii) produce low yields of
DNA.
In this study we have developed a rapid method for direct
extraction of DNA from soil and sediments by applying a
freeze-thaw approach. This method overcomes the draw-
backs of the previous techniques. The high yield from a
small sample size (1 g [wet weight]) means that the method
described in this report can be used efficiently to study in situ
gene occurrence and to detect the distribution of genetically
engineered microorganisms in the environment.
MATERIALS AND METHODS
Soil characterization. Subsurface soil samples from a man-
ufactured gas site in Southern California (SC) and sediment
samples from a settling pond in Oak Ridge, Tenn. (ORT),
were used for DNA extraction. The SC soils were contam-
*
Corresponding author.
1070
inated with polynuclear aromatic hydrocarbons (6.5 lug/g
[wet weight]) and mercury (0.07 ,ug/g), and the ORT sedi-
ments were contaminated with mercury (up to 80 ,ug/g) and
other heavy metals (Global Geochemistry Corp., Canoga
Park, Calif.; Oak Ridge National Laboratory, Oak Ridge,
Tenn.). The heterotrophic bacterial population in both sam-
ples was enumerated on plate count agar (Difco, Detroit,
Mich.). The indigenous mercury-resistant strains were re-
covered on plate count agar amended with 25 ,ug of Hg2+ as
HgCI2 per ml. Naphthalene degraders were selectively cul-
tivated on a minimal medium saturated with naphthalene
vapor (18). Sterile soil and sediment samples were produced
by autoclaving at 121C for 30 min. All soil and sediment
quantities are expressed as wet weight unless otherwise
indicated. The percentage of water was determined to be
51% in the ORT sediments and 10% in the SC soil (2).
Organisms. Two strains (V55 and VNM43) isolated from
SC soil were used as seed organisms. They were identified as
Pseudomonas luteola and Pseudomonas putida, respec-
tively, by the API Rapid NFT? kits (Analytab Products,
Plainview, N.Y.). In the remainder of this paper these
strains are referred to as P. luteola V55 and P. putida
VNM43. Both strains have the ability to degrade naphtha-
lene and are resistant to mercuric ions. They were grown to
late exponential phase in 50% plate count broth (Difco) with
25 pLg of Hg2+ per ml and were washed once with 10 mM
phosphate buffer (pH 7.0) and resuspended in phosphate
buffer before inoculation into the soils or sediments. The
inoculated soils and sediments were shaken at 100 rpm
(Orbit Shaker; Lab-Line Instrument, Inc., Melrose Park,
Ill.) at 23C for 30 min prior to DNA extraction.
Direct extraction of DNA. SC soil or ORT sediment sam-
ples (1 g) were mixed with 2 ml of 120 mM sodium phosphate
buffer (pH 8.0) by shaking at 150 rpm for 15 min. The slurry
was pelleted by centrifugation at 6,000 x g for 10 min. The
pellet was washed again with phosphate buffer, resuspended
in 2 ml of lysis solution (0.15 M NaCl, 0.1 M Na2EDTA [pH
8.0]) containing 15 mg of lysozyme/ml, and incubated in a
37C water bath for 2 h with agitation at 20- to 30-min
intervals, and then 2 ml of 0.1 M NaCl-0.5 M Tris-HCl (pH
8.0)-10% sodium dodecyl sulfate was added. Three cycles of
freezing in a -70C dry ice-ethanol bath and thawing in a
65C water bath were conducted to release DNA from the
VOL. 57, 1991 DIRECT DNA EXTRACTION FROM SOIL AND SEDIMENTS
microbial cells in the soil or sediments. The efficiency of
lysis was determined by direct cell counts with phase-
contrast microscopy and by acridine orange direct cell
counts (7) with fluorescence microscopy (model BH-2;
Olympus, Tokyo, Japan) and was found to be greater than
95% for P. putida VNM 43 and P. luteola V55 as well as a
Bacillus sp. After the freeze-thaw cycles, 2 ml of 0.1 M
Tris-HCl (pH 8.0)-saturated phenol (17) was added and the
sample was briefly vortexed to obtain an emulsion. The
mixture was centrifuged at 6,000 x g for 10 min. A 3-ml
sample of the top aqueous layer was collected and then
mixed with 1.5 ml of phenol and 1.5 ml of chloroform
mixture (chloroform/isoamyl alcohol ratio, 24:1). A 2.5-ml
portion of the resulting extract was further extracted with an
equal volume of chloroform mixture. Finally, nucleic acids
in the extracted aqueous phase (2 ml) were precipitated with
2 ml of cold isopropanol at -20C for 1 h or overnight. The
pellet of crude nucleic acids was obtained by centrifugation
at 10,000 x g for 10 min and then vacuum dried at 23C. The
nucleic acid pellets, which were brownish as a result of the
humic materials in both ORT and SC samples, were resus-
pended in 100 ,ul of TE buffer (20 mM Tris-HCl, 1 mM EDTA
[pH 8.0]). The RNA molecules in the crude extracts were
removed by incubation with heat-treated pancreatic RNase
A (final concentration, 0.2 jig/[ld) for 2 h at 37C. The
RNA-free DNA was then purified with an Elutip-d column
(Schleicher & Schuell, Keene, N.H.) attached to a Schlei-
cher & Schuell NA010/27 (0.45-,um [pore size] cellulose
acetate) prefilter. DNA was recovered from the column as
suggested by the manufacturer. The efficiency of recovery of
total DNA from sediment and/or soil crude extracts was
determined as the percentage of DNA extracted from a
known density of P. putida VNM43 in seeded sterile samples
compared with DNA recovered from the same density of
pure culture controls. Cell numbers used in all seeding
experiments resembled heterotrophic plate counts of the
sediments and soils under investigation. The yield of DNA
from the direct extraction was expressed in micrograms of
crude DNA obtained per gram of unseeded nonsterile sam-
ples.
Gel electrophoresis. Samples of extracted DNA were ana-
lyzed on a 0.7% agarose gel containing 1 p.g of ethidium
bromide per ml. To determine the quality of extracted DNA,
we analyzed undigested DNA extract and appropriate con-
trols including DNase-digested (0.5 pig/p.l) and RNase-di-
gested (0.2 p.g/p.l) extracts. The concentration of DNA in the
crude extract was determined spectrophotometrically at 260
nm. Restriction digestion of DNA was conducted with
EcoRI (2 U/p.l), BamHI (1 U/pul), and HindIll (1 U/pul) with
incubation for 5 h at 37C, followed by gel electrophoresis.
Hybridization. A 1-kb fragment of the merA gene encoding
mercuric reductase was labeled with [ot-32P]dCTP by random
priming (specific activity, 108 dpm/pg of DNA) and used as
a probe to detect the presence of mercury detoxification
genes in the environmental samples. The merA fragment of
plasmid pDU1358 was kindly provided by S. Silver. DNA
was transferred from the agarose gel to membranes by
vacuum blotting (11). The extracted DNA was also filtered
onto membranes through a slot blot apparatus (Minifold II;
Schleicher & Schuell). Nylon membranes (GeneScreen Plus;
NEN Products, Boston, Mass.) and nitrocellulose mem-
branes (BA85; pore size, 0.45 pum; Schleicher & Schuell)
were used as solid supports. Hybridization was carried out
at 42C in the presence of 50% formamide, followed by
high-stringency washes as previously described (24). The
Radioanalytic Imaging System (AMBIS Systems, San Di-
1071
A B
FIG. 1. Total DNA extracted from ORT sediments. (A) Ethid-
ium bromide-stained agarose gel; (B) autoradiogram of DNA hybrid-
ization signals with merA after Southern blot of the gel in panel A.
Aliquots (3 ,ul) of total extracts (100 ,ul) were loaded into each well.
Lanes: 1, HindlIl-cut lambda bacteriophage molecular size marker
(1 p.g); 2, unseeded; 3, unseeded and digested with RNase; 4,
unseeded and digested with DNase; 5, seeded; 6, seeded and
digested with RNase; 7, seeded and digested with DNase; 8, plasmid
size marker from E. coli V517.
ego, Calif.) was used to quantify the radioactivity signals of
hybridized DNA on the membranes.
RESULTS AND DISCUSSION
Figure 1A shows an ethidium bromide-stained agarose gel
used to visualize the DNA extracted from ORT sediments
after DNase and RNase treatments. The addition of 2.5 x
108 cells of P. luteola V55 to 1 g of nonsterile sediments
resulted in a larger quantity of DNA than in the unseeded
sample. The efficiency of recovery was calculated to be 92%
when sterile ORT sediment was seeded with a known cell
density of P. luteola V55. The sterile samples (autoclaved or
gamma irradiated) did not contain indigenous DNA after
extraction. The total DNA yield from nonsterile, unseeded
ORT sediments was determined to be 38 ,ug/g (Table 1). The
largest DNA was greater than 23 kb, and most DNA was in
the size range of 6.5 to 23.1 kb. The DNA-shearing effect of
the extraction procedure was less prominent than in the
method described by Ogram et al. (12), as evidenced by
smearing of smaller fragments. Because the DNA extracts
before RNase digestion contained RNA contaminants (Fig.
1A), introduction of RNase during the extraction procedure
was necessary. Impurities such as humic materials in the
extracts did not affect RNase or DNase digestion, but may
affect DNA hybridization efficiency. RNA was not observed
in unseeded samples, indicating that RNA degradation or
TABLE 1. Yields and efficiency of direct DNA
extraction method
Sample
Sample Heterotrophic
bacteria (CFU/g) Recovery(%)
efficiency' [wet (pLg/g
Yieldb
wt])
ORT sediments (8.0 + 0.7) x 107 92 3 38 5
SC soil (4.1 +0.1) x 106 95 4 12 4
aThe recovery efficiency was calculated from the total DNA obtained from
a known cell density of sterile seeded samples compared with DNA extraction
from bacterial pure cultures.
b
The yield was measured from nonsterile unseeded samples.
1072 TSAI AND OLSON APPL. ENVIRON. MICROBIOL.

FIG. 3. Autoradiogram of hybridization signals of extracted

FIG. 2. RNase treatment of nucleic acids extracted from SC soil. crude DNA with merA. (A) RNase-treated DNA from nonsterile
(A) Ethidium bromide-stained agarose gel; (B) DNA hybridization unseeded (slot 1) and sterile seeded (slots 3 to 8) SC soils. The sterile
signals with merA after Southern transfer. Aliquots (4 ,ul) of total SC soils were inoculated with (cells of P. putida VNM43 per g [wet
extracts (100 pLI) were loaded into each well. Lanes: 1, HindIII-cut weight]): no inoculum (slot 2), 1.1 x 108 (slot 3), 1.1 x 107 (slot 4),
lambda phage molecular size marker (1 ,ug); 2, sterile unseeded; 3, 1.1 x 106 (slot 5), 1.1 x 105 (slot 6), 1.1 X 104 (slot 7), and 1.1 X 103
sterile seeded; 4, nonsterile unseeded; 5, nonsterile seeded; 6 and 7, (slot 8). Slots 1, 3, 4, 5, and 6 each contained 1/10 and slots 7 and 8
pure culture of P. putida VNM43; 8, 10 ng of merA 1-kb fragments. each contained 1/2 of total DNA extracts. (B) RNase-treated DNA
from nonsterile unseeded ORT sediments (slots al to a4), seeded
ORT sediments (slots aS to a9), nonsterile seeded SC soils (slots bl
to b5), and pure culture of P. putida VNM43 (slots b6 to b9). The
nonsterile ORT sediments and SC soil were seeded with 2.5 x 108 p.
less RNA synthesis was occurring in the nonsterile, uninoc- luteola V55 and 7.0 x 108 P. putida VNM43 per g (wet weight),
ulated environmental samples. Southern blot analyses respectively. Slots: aS, 10 Rg; al, 5 jig; a2 and a6, 1 ,ug; a3 and a7,
showed that ORT sediments contained merA genes in the 0.1 ,ug; a4 and a8, 0.01 ,ug; a9, 0.001 ,ug; bl, 10 ,ug; b2 and b6, 1 ,ug;
indigenous microbial community (Fig. 1B). Small DNA b3 and b7, 0.1 ,ug; b4 and b8, 0.01 pug, b5 and b9, 0.001 jig of DNA.
fragments (less than 500 bp) resulting from DNase digestion
of the extracted DNA were homologous to partial merA
sequences (Fig. 1B, lane 7). The total heterotrophic bacterial support observations made by other investigators, who have
population in the ORT sediments was (8.0 + 0.7) x 107 reported that only a very small portion of the natural
CFU/g, with 17% of the population exhibiting phenotypic bacterial community is culturable (1, 4, 5, 16). Because the
mercury resistance. This determination supports the finding ORT sediment contains more water (51%) than the SC soil
of merA sequences in the ORT sediments. (10%), the yields based on dry weight would be 77 and 13 ,ug
Figure 2A illustrates the ethidium bromide-stained DNA of DNA per g, respectively.
extracted from unseeded nonsterile and sterile SC soils and Southern hybridization analyses did not indicate the oc-
from seeded (7.0 x 108 cells of P. putida VNM43 per g) currence of merA gene sequences in the nonsterile unseeded
nonsterile and sterile soils. No evidence of DNA was found
in the sterile soil, but DNA was recovered from the nonster-
ile soil (lanes 2 and 4, respectively). The heterotrophic
bacterial density in the nonsterile SC soil was (4.1 + 0.1) x
A B
106 CFU/g. Of these bacteria, 9% were phenotypically
mercury resistant and 15% were naphthalene degraders.
Most of the DNA fragments extracted from the seeded soils
were larger than 23.1 kb (Fig. 2A), indicating that the present
protocol does not cause severe DNA shearing. In addition,
the DNA eluted from agarose gels could easily be subjected
to restriction analyses for further subcloning. The efficiency
of extraction and the total recovery of DNA were 95% and
12 ,ug/g, respectively (Table 1). There was no linear corre-
lation between the total yield of DNA and the density of
heterotrophic bacteria based on dry weight when two dif-
ferent types of samples (sediment and soil) were used. This
discrepancy could be due to the presence of eukaryotic
organisms, chemolithotrophic bacteria, nonculturable bacte-
ria, or other microorganisms which were not enumerated
during the experiment.
On the average, each Escherichia coli cell contains 9.0 x FIG. 4. Restriction enzyme digestion patterns of DNA isolated
io-9 ,ug of DNA (9). Assuming that this holds true for from SC soil. (A) Ethidium bromide-stained agarose gel. (B) Auto-
average bacteria, the density of heterotrophic bacteria from
radiogram of merA hybridization signals from Southern blot. Lanes
1 to 3 and 4 to 6 each contained 1/10 of DNA extracts from P. putida
ORT sediments and SC soil would represent 0.72 and 0.037 VNM43 and seeded soil samples, respectively. Lanes: 1' Hindlll-
,ug of DNA per g, which would amount to 1.9 and 0.3% of cut lambda phage molecular size marker (1.25 p.g); 1 and 4, uncut; 2
total extracted DNA, respectively. These results further and 5, EcoRI cut; 3 and 6, Hindlll cut; 7, 20 ng of merA fragment.
VOL. 57, 1991 DIRECT DNA EXTRACTION FROM SOIL AND SEDIMENTS 1073

FIG. 5. Restriction enzyme digestion patterns of DNA extracted from ORT sediments. (A) Ethidium bromide-stained agarose gel. (B)
Autoradiogram of merA hybridization signals from Southern blot of gel in panel A. Lanes 2 to 4 and 5 to 7 each contained 1/30 of DNA extracts
from unseeded and seeded samples, respectively. Lanes 8 to 10 and 11 to 13 contained 1/20 of Elutip-d column-purified DNA from unseeded
and seeded samples, respectively. Lanes: 1, HindIll-cut lambda phage molecular weight marker with ladder of 23.1, 9.4, 6.5, 4.4, 2.3, and
2.0 kb; 2 and 8, unseeded and uncut; 3 and 9, unseeded and EcoRI cut; 4 and 10, unseeded and BamHI cut; 5 and 11, seeded and uncut; 6
and 12, seeded and EcoRI cut; 7 and 13, seeded and BamHI cut.

SC soil (Fig. 2B, lane 4), but the direct application of DNA
to membranes by the slot blot hybridization technique
showed that crude DNA extracted from the same soil did
contain merA genes (Fig. 3A, slot 1). The discrepancy
between these two observations was due to incomplete
transfer of DNA from the gel to the filters during Southern
blotting. Therefore, the existence of mercury-resistant bac-
terial strains such as P. putida VNM43 and P. luteola V55 in
the polynuclear aromatic hydrocarbon-contaminated SC
soils was further supported by the gene probe data. The
DNA extracts from seeded sterile (Fig. 2B, lane 3) and
nonsterile (lane 5) soils revealed smaller DNA fragments
(between 1 and 20 kb) containing homologous merA se-
quences. This shearing is probably due to the grinding effect
of the sand particles in SC soil during the extraction proce-
dure because pure-culture controls (lane 6 and 7) did not
exhibit this effect. The two soils differ in cation exchange
capacity and particle size distribution, with SC soils having
low cation exchange capacity (9 mEq/100 g) and high sand
concentration (18.4%), and ORT sediments having high
cation exchange capacity and low sand concentration (Uni-
versity of California, Riverside, Department of Soil and
Environmental Sciences; Oak Ridge National Laboratory).
Figure 3A shows the merA hybridization signals from the
crude DNA extracted from sterile SC soil seeded with
different quantities of P. putida VNM43. Signals were de-
tected in a 50% aliquot of total DNA extract when as few as
1.1 x 104 cells per g (wet weight) were inoculated into soil,
indicating that the sensitivity was 5.5 x 103 cells per g. A
comparative sensitivity of 4.3 x 104 cells per g (dry weight)
was reported for the cell extraction method (8). Figure 3B
illustrates hybridization signals of putative merA genes ex-
tracted from nonsterile unseeded and seeded ORT sediments
and nonsterile seeded SC soils. As the extracted DNA was
normalized at 1 ,ug, DNA from seeded samples (Fig. 3B,
slots a6 and b2) demonstrated higher merA hybridization
signals (7,049 and 24,477 cpm) than that from unseeded
samples (800 cpm; slot a2). As would be expected, the signal
for the SC soils inoculated with a higher cell density (slot b2)
had a higher-intensity signal of merA sequences than did
ORT sediments inoculated with a lower cell density (slot a6).
The pure-culture DNA control showed the highest signal
1074 TSAI AND OLSON
(28,648 cpm) in 1 ,ug of DNA extract. The radioactivity
counts for hybridized DNA were lower from soil and sedi-
ment samples than from pure cultures, indicating that DNA
had absorbed into the soil particles or that more nonhomol-
ogous merA sequences were present in the former samples.
The detection limit of homologous merA DNA was 10 ng of
total extracted DNA from both P. putida VNM43 pure
culture and P. putida VNM43-seeded SC soils (Fig. 3B). The
merA hybridization signals were found in 1 ,ug of total DNA
extracted from nonsterile unseeded ORT sediments. In
comparison, Ogram et al. (12) required 1.2 ,ug of total DNA
to detect nif nitrogen fixation genes from sediments other
than those used in the present study. In the present study
there is no indication that humic materials in the crude
extract affect DNA-DNA hybridization (Fig. 3).
Figure 4 shows the restriction patterns of DNA extracts
from a pure bacterial culture and from a seeded SC soil. The
unpurified DNA extract was clearly digested by Hindlll but
only slowly digested by EcoRI. The application of restriction
enzyme analyses to directly extracted DNA may allow more
precise studies of in situ gene rearrangements or amplifica-
tion. The impurities in ORT sediments did affect the restric-
tion enzyme digestion (Fig. 5). EcoRI and BamHI did not
digest DNA extracts from either unseeded or seeded sam-
ples within the 5-h incubation period. The DNA yield
suffered a 40% loss during the Elutip-d column purification
step, and the purified DNA (A260/A280 = 1.8) was cut by
EcoRI but not by BamHI. Therefore, for studying in situ
gene transfer the purification step might be needed depend-
ing on the soil type, but it is not necessary for determination
of gene occurrence.
In conclusion, this rapid method for the direct extraction
of DNA needs only 1 g of soil or sediments and can detect
the presence of a target gene, such as merA, from a minimum
of 5,000 bacterial cells. It is simple and can be completed in
7 h. In addition, because of the small sample size (1 g), a
large number of soil or sediment samples can be analyzed at
one time. Finally, the high yield in combination with the high
quality of DNA will enable microbial ecologists to study
DNA from environmental samples in a more detailed fashion
by molecular biology techniques.
ACKNOWLEDGMENTS
This study was funded by grant 8000-25 provided by the Electric
Power Research Institute.
We are grateful to Marie Park for developing the autoradiograms.
We also thank 0. A. Ogunseitan, P. A. Rochelle, and C. C. Tebbe
for valuable discussions and suggestions and R. Turner for provision
and analysis of ORT sediments.
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