Journal of Experimental Microbiology and Immunology (JEMI) Vol.

6:52-58
Copyright December 2004, M&I UBC
An Evaluation of the Relationship between the Most Probable Number
(MPN) Assay and 16S rRNA Hybridization Technique in Characterizing
and Quantifying Nitrosomonas Species in Sludge Wastewater
CAROL NG
Department of Microbiology and Immunology, UBC

Nitrosomonas species are the main organisms involved in the oxidation of ammonium to
nitrite in nature. The traditional method for enumerating Nitrosomonas species is via the
Most Probable Number (MPN) Technique. However, this is a slow and labourious method
due to the slow growth rate of nitrifiers. Also, the method does not distinguish different
species of nitrifiers. Oligonucleotide probes are now used as a method for identifying
particular species of nitrifying bacteria. However, the correlation between these methods is
unclear. Sludge wastewater sample from a bioreactor was used to study the potential
correlation between the MPN technique and the oligonucleotide probe technique. In order
to investigate this relationship, multiple replicate dilutions of the sludge wastewater were
made and each dilution was grown and processed using the MPN technique. After a period
of growth, a record was made of the presence or absence of ammonia oxidation. RNA was
extracted from these growth samples were probed with 16S rRNA specific probes Nso 190
and Nsm 156. It was found that the original sludge sample had 1.4 x 10
6
viable cells/ 100mL
with 95% confidence lower limit of 6.1 x 10
5
and an upper limit of 2.8 x 10
6
. However, the
poor integrity of the isolated RNA prevented any identification of Nitrosomonas species in
the MPN sample.
_________________________________________________________________

The biogeochemical cycles describe the cyclic
nature of the five essential nutrients of life. One of
which is nitrogen. Nitrogen is an essential component
in amino acids, which are the building blocks of
proteins, and in nucleic acids, which form the genetic
material of the cell. Nitrogen gas makes up about 78%
of the earths atmosphere, but the triple bonds in this
molecule makes the gas inert. Nitrogen fixation in
bacteria converts N
2
to forms of fixed nitrogen
compounds that are more available to the biosphere. To
complete the cycle, the denitrification process returns
nitrogen gas (N
2
) back to the atmosphere. In nature,
nitrogen is found in a variety of valence states from -3
in NH3 to +5 in NO3- (3, 8). The energy released by
changes in the redox potential of the element is
harnessed by organisms to maintain life (7).
Nitrification is the process by which ammonium is
oxidized to nitrate. This process is carried out by two
distinct groups of bacteria: the ammonium oxidizing
bacteria (AOB), which oxidize ammonium to nitrite,
and the nitrite oxidizing bacteria (NOB), which oxidize
nitrite to nitrate (11). Nitrosomonas europaea, an
obligate lithoautotrophic ammonia-oxidizing
bacterium, is the most studied of the ammonia-
oxidizing bacteria that are participants in the
biogeochemical nitrogen cycle. These bacteria also are
important players in the treatment of industrial and
sewage waste in the first step of oxidizing ammonia to
nitrate. N.europaea is also capable of degrading a
variety of halogenated organic compounds (6). The
ability of nitrifying organisms to degrade some
pollutants may make these organisms attractive for
controlled bioremediation in nitrifying soils and waters.
N. europaea is a gram negative chemolithotroph
that oxidizes ammonia to nitrite and can be found in
places such as soil, sewage, freshwater, and building
surfaces in polluted areas where the air contains high
levels of nitrogen compounds (3). N. europaea grows at
an optimum pH of 6.0-9.0, temperature range of 20C
to 30C and under aerobic metabolism conditions. In
addition to the previously mentioned conditions, a
special selective medium that is inorganic and includes
ammonia as an electron donor, bicarbonate as the sole
carbon source for the isolation of Nitrosomonas.
Because of the large amounts of ammonia this
bacterium needs to consume for energy to divide, cell
division can take up to several days. Due to their slow
growth rates and undefined growth requirements,
lithotrophic nitrifiers are difficult to isolate (2).
The standard method to enumerate nitrifying
bacteria is by the most probable number technique
(MPN) which indicates numbers of viable nitrifying
bacteria. The MPN assay is performed by setting up
multiple replicate dilutions of samples and recording
the presence or absence of nitrifying activity.
Unfortunately, the MPN assay is inefficient method for
the enumeration of nitrifying bacteria due to their slow
growth rate and moreover, nitrifiers are small
organisms and growth cannot be determined using
optical density measurements. With the development of
52
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC
molecular techniques, researchers have applied
methods such as PCR and probe hybridization in the
identification of nitrifying bacteria. In a RNA blot,
Ribonuclease (RNA) from nitrifying bacteria is
immobilized on a nylon membrane by slot blotting.
Then the blot is hybridized with nucleotide probes with
specific affinity for 16S rRNA of isolated species of
nitrifiers. The detection of probes bound to the
membrane would indicate the presence of a particular
strain of nitrifying bacteria in the original sample.
However, previous studies have detected a consistent
discrepancy between the nitrification activity in
bioreactors and the binding of 16sRNA probes of
known nitrifiers (1, 11). For example, the level of
ammonium converted to nitrite measured remains high
when the amount of organisms detected by the Nso190
probe (specific to all -proteobacteria ammonia-
oxidizers) is decreased by 50% (12, 13). This
observation could be caused by mismatches between
the RNA probe and the RNA sequences due to
nucleotide sequence differences. If the nucleotide
sequence does not completely match the probe, the
probe-RNA hybrid will dissociate at a lower
temperature. Other causes for mismatches could be the
presence of other nitrifying organisms, or other species
of Nitrosomonas.
This study attempts to investigate the relationship
between the MPN assay and the results of the probe
assays. In addition, this study will also determine if the
MPN enriches for a diversity of AOB. MPN assay was
performed on a sludge sample was obtained from a
bioreactor to determine the number of Nitrosomonas
cells in the initial sludge sample. The samples from the
MPN assay was then assayed by DNA probes specific
to the 16sRNA of Ammonia-oxidizing -
Proteobacteria and species of Nitrosomonas to
determine the identity of AOB enriched by the MPN
assay (1).
MATERIALS AND METHODS

MPN growth tubes and media. The Most Probable Number
Assay for determining the number of Nitrosomonas cells in a sludge
sample was set up as follows. A sludge sample (Methanol Bio-P
Sludge- October 7, 2004) was obtained from UBC Department of
Civil Engineering. Sludge sample was centrifuged at 4200g for 10
minutes at 4C. The MPN assay was conducted in 50mL test tubes
with an inverted Durham tube to trap any gas produced as a result of
fermentation. The fermentation tubes filled with Nitrosomonas
europaea medium (ATCC medium 2265. Details can be found at
http://www.atcc.org/mediapdfs/2265.pdf). Seven series of
fermentation tubes were set up, ranging from 10
-1
to 10
-7
.
Quadruplicate fermentation tubes were set up for series 10
-1
and 10
-2
.
Series 10
-3
, 10
-4
, 10
-5
, 10
-6
, and 10
-7
were set up in replicates of ten.
Final volume of media in fermentation tube was 30mL. The sludge
pellet was inoculated into series 10
-1
and subsequent serial dilutions
were made. The fermentation tubes were incubated in the dark at
30C for 5 weeks.
Nitrogen assay. The nitrate reduction test was used to monitor
the growth of Nitrosomonas. At week 3 and 5, the fermentation tubes
were examined for pH change, the presence of gas and the production
nitrite ions in the medium. A pH change would be indicated by a
change in media color as the pH indicator Phenol red (14) was added
to the media. The presence of nitrogen gas would be indicated by gas
bubbles trapped in the Durham tube. The production of nitrite ions
were detected by the addition of sulfanilic acid and N, N-dimethyl-1-
naphthylamine to culture (7).
RNA extraction protocol. The Nitrosomonas cells from the
MPN tubes were harvested by vacuum filtration through a 0.22um
(Millipore GVWP). Cells were washed off from the membrane and
resuspended in 1ml of fresh media. The cells were then microfuged at
14000 rpm for 15 minutes.
The cell pellets from the MPN tubes and a frozen pellet from a
pure Nitrosomas culture were suspended in 1 mL of Trizol reagent
(Invitrogen) was added to disrupt the cells. The Trizol reagent is a
monophasic solution of phenol and guanidine isothiocyanate and it
disrupts cells while inactivating RNase activity. For every milliliter
of Trizol, 0.2mL of chloroform was added to the cell pellet for phase
separation, and 0.1 mm diameter glass beads (SIGMA) were added to
the vial until it was full. The cells were lysed by mechanically
disrupting the cells using a Beadbeater (Minibeadbeater, Biospec
Products) at 4800rpm for a total of 3 minutes, cooling the mixture on
ice after every minute. The mechanical disruption was then followed
by a 10 minute cooling at room temperature, and centrifugation at
14000 rpm for 15 minutes.
After centrifugation, two layers formed: a clear aqueous top
layer, and a pink organic lower layer. The aqueous layer, which
contains the RNA, was removed. A second extraction was performed
on the remaining organic layer by adding again Trizol reagent and
chloroform and repeating the beating and centrifugation procedures
as mentioned above. The clear aqueous top layer was removed and
combined with the aqueous layer from the first extraction and
precipitated with isopropanol. The mixture was incubated at room
temperature for 10 minutes, and the RNA was pelleted by
centrifugation. The RNA pellet was washed with 75% ethanol, and
then resuspended and dissolved in 100L of diethylpyrocarbonate
(DEPC) treated water to minimize the amount of contaminating
ribonuclease. RNA was then quantified by measuring the absorbance
at 260nm and 280nm (A260 and A280) using the relationship
[RNA]=0.063(A260)-0.036(A280). The isolated RNA was then
stored at -80C until it was used.
The purity and integrity of the RNA samples were verified by gel
electrophoresis. The apparatus was DEPC treated to ensure that it
was ribonuclease (RNase) free.
Determination of the Quality of RNA using Gel
Electrophoresis. The integrity of the extracted RNA was
characterized by gel electrophoresis in 1X TAE buffer (40 mM Tris
pH 8.0, 1.14 ml of glacial acetic acid, 1 mM EDTA) with 1.2%
agarose gel at 100V for 1 hour. The gels were stained with ethidium
bromide and run at 70 V for about 1 hour. The gels were visualized
and photographed under a UV transilluminator. Note that due to the
lack of RNA from the MPN cultures, only the quality of the RNA
extracted pure Nitrosomonas sample was assessed by gel
electrophoresis.
Slot Blotting. RNA samples were immobilized onto BioRad
transblot nitrocellulose membrane (6.45m) as per the Alkaline RNA
Denaturation and Fixation protocol in the Bio-Dot SF Microfiltration
Apparatus Instruction Manual. The RNA samples were denatured
with 5 volumes of ice cold 10mM NaOH, 1mM EDTA immediately
before blotting onto membrane. The samples are then applied onto a
pre-wet membrane. Then sample wells were washed with cold 10mM
NaOH, 1mM EDTA. Vacuum was applied to pull liquid through the
membrane after each application. After blotting, the membrane was
air-dried and then baked at 80C for 30 min before tube
hybridization.
Primer Design. Oligonucleotide probes were synthesized and
purified at the Nucleic Acid Protein Service Unit (NAPS) at
University of British Columbia using Applied Biosystems automated
synthesizers. The probes were biotin-labeled for detection with
streptavidin-peroxidase (POD) and the chemiluminescent substrate
luminol.
53
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC
TABLE 1. Names, target positions, sequences, and specificities of the probes used in this study.
Hybridization. BM Chemiluninescence Blotting Kit
(Biotin/Streptavidin) from Roche Applied Science was used. The
processing of the membrane and detection of nucleic acid probes was
performed as per manufacturers protocol. Due to the limited RNA
samples, the membrane was divided into two parts. Membrane 1 was
first probed with Nso 190 and membrane 2 probed with Nsm 156.
After initial hybridization, the probes were stripped off and probed
with the other oligonucleotide probe. Prior to hybridization, the nylon
membranes with the immobilized RNA were placed in hybridization
tubes and pre-warmed in hybridization buffer (5x SSC, 2% blocking
reagent, 0.1% N-laurylsarcosine, 0.02% SDS, 50% formamide) at
40C for 3 hours. Probes were then added to the hybridization tubes
at 800 ng/ml. The nylon membranes and the probes were left
overnight to hybridize at 40C. After hybridization, the membranes
were washed at twice in 2x washing solution (2x SSC, 0.1% SDS) for
5 minutes per wash at room temperature. Followed then by two
washes in 0.1x washing solution (0.1x SSC, 0.1% SDS) for 15
minutes per wash at 65C for Nso190 probe and 46C for Nsm 156
probe. After washing, the membranes were blocked with 1%
blocking solution for 30 minutes at room temperature followed by
incubation of the membranes in Streptavidin-POD diluted in 1%
blocking solution at 10 mU/ml for 30 minutes. The membranes are
then washed with a solution of maelic acid and 0.3% Tween 20 for 15
minutes and subsequently with just maelic acid.
Chemiluminescence detection. Detection reagent was prepared
by combining substrate solution A and starting solution B included in
the chemiluminescence kit in a ratio of 100:1. After draining off
excess maleic acid, the detection reagent was poured onto the
membranes allowing the horseradish peroxidase (POD) to catalyze
the oxidation of luminol in the presence of hydrogen peroxide. This
reaction forms an activated intermediate product which emits light
energy in its conversion back to ground state. The POD was allowed
to react with the bound probes for 1 minute and exposed to
autoradiography film for intervals of 5, 10, and 30 minutes.
Stripping and reprobing. The bound probes to the membrane
were stripped by incubating the membrane twice with 50%
dimethylformamide, 1% SDS, 50 mM Tris-HCl at 68C for 30
minutes. The membranes were then rinsed with 2x SSC and reprobed
with the alternate probe in the same manner as described above.

RESULTS

Most Probable Number Assay. Bacterial density
can be estimated by the Poisson formula or from the
table using the number of positive tubes in multiple
dilutions. MPN tables are based on the assumption of a
Poisson distribution (random dispersion). The most
probable number for bacteria density in the original
sludge sample is calculated by using Table 2. Using
results from 10
-5
, 10
-6
, and 10
-7
, the MPN of viable cells
in the original sludge sample is 1.4 x 10
6
cells/ 100mL
with 95% confidence lower limit of 6.1 x 10
5
and an
upper limit of 2.8 x 10
6
.

TABLE 2. Summary of results of the MPN assay.



RNA preparation and assessment of RNA
integrity. The RNA preparation from frozen
Nitrosomonas pellet and sludge sample yielded two
bands corresponding to the 23S and 16S rRNA via
detection by gel electrophoresis (data not shown). The
overall integrity of the RNA was determined to be
adequate by the marginally acceptable ratio of
A
260
/A
280
spectrophotometric methods but the presence
of bands showed that the degree of RNA degradation
was small. However, the RNA extractions from MPN
assay samples were determined to be low yield and low
quality via spectrophotometric methods and gel
electrophoresis was not able to make out any distinct
16S rRNA bands (Table 3).
Hybridization autoradiogram. As indicated in
Table 1, Nso 190 probe should be able to identify all -
proteobacteria and Nsm 156 probe is specific to a
couple species of Nitrosomonas. By using differential
probing by Nso 190 and Nsm 156, this study hoped to
identify Nitrosomonas and distinguish them from other
AOB that may be enriched by the MPN method. The
autoradiograms were exposed to the chemiluminescent
probes that were in turn bound to the immobilized
RNA on the nylon membranes. Therefore, the
intensities of the bands not only allude to the presence
of the 16S rRNA of that the particular probe is specific
to, but also to the amount of RNA that is present on the
nylon membrane. A standard of Nitrosomonas RNA
titrating from 5g to 0.5g so that any bands resulting
from the MPN assay samples can be used to extrapolate
54
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC
TABLE 3. Summary of RNA integrity analysis via spectrophotometry.


TABLE 4. Schematic detailing layouts of the slot blot membrane. The amount and identity of the RNA samples are indicated. The
membrane was cut into two with part (a) becoming membrane 1 and part (b) becoming membrane 2.

the amount of RNA resulting from that sample. The
probe Nso 190 bound very little to the Nitrosomonas
RNA on membrane 1 with only a very faint band at the
5g as seen in figure 2. Compared to the probing of
membrane 2 with Nsm 156, in which the
autoradiogram produced a distinct band even at 0.5g
of Nitrosomonas RNA (Fig. 1).
Figure 1 shows detection of Nitrosomonas 16S
rRNA in the RNA extracted from sludge wastewater
sample using Nsm 156 probe on membrane 2. All 3
concentrations (5g, 4g, and 3g) were detected and
the intensity of the bands decreased with decreased
amounts of RNA immobilized on the nylon membrane.


After stripping and reprobing with the alternate probe,
similar results were seen. Nsm 156 was able to detect a
gradient of Nitrosomonas RNA on membrane 1 (Fig. 4)
whereas Nso 190 did not reveal any distinct bands to
the Nitrosomonas RNA on membrane 1 (data not
shown).

55
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC


FIG. 1 Autoradiogram of hybridization of Nsm 156 onto
membrane 2 (10 minute exposure). Refer to Table 4 for identity of
samples.



FIG. 2 Autoradiogram of hybridization of Nso 190 onto
membrane 1 (10 minute exposure). Refer to figure 1 for identity of
samples. The streak in the middle was a shadow produced by the
wrinkles in the plastic wrap in the exposure process.






FIG. 3 Autoradiogram of hybridization of Nsm 156 onto
membrane 1 (30 minute exposure). Refer to figure 1 for identity of
samples. The large black area that obscures the top right corner of the
autoradiogram was caused by inadequate sealing of the film cassette
during film exposure. Therefore light leaked into the cassette and
bleached out that area of the autoradiograph film.

DISCUSSION

MPN assay enumerates the amount of viable
organisms in a sample. According to the MPN assay,
there are 1.4 x 10
6
viable growth units (GU) in the
original sludge sample. Although MPN cannot
distinguish between the different organisms that may
be present in the sludge wastewater (9), certain
parameters of the MPN assay allude to the identity of
the organism that was enriched in the MPN assay. First,
the media used in the fermentation tubes only consisted
of inorganic materials such as ammonia so that only
AOB are able to grow. Also other parameters such as
temperature and lack of light were optimal for AOB
such as Nitrosomonas to flourish (2). Second, the
nitrate reductase test of the supernatant detected nitrite.
Since there was no nitrite in the original media, the
nitrate must be the product of the nitrification. It would
be possible to have nitrite oxidizing bacteria (NOB)
since such organisms oxidize nitrite to nitrate in their
metabolic growth, so NOB can flourish using the
products of AOB metabolism. However, the nitrate
reductase test detected no nitrate in any of the sample
supernatants which suggests that NOB are present in
the samples. Also, the absence of nitrogen gas
production further signifies the lack of NOB in the
sludge. Therefore, the MPN assay can conclude that
56
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC
there are AOB present in the original sludge
wastewater sample and all the growth units can be
attributed only to the presence of AOB in the sludge
wastewater sample. Even though Nitrosomonas
europaea is the primary AOB found in wastewater,
other species of Nitrosomonas could be found in
wastewater such as Nitrosomonas halophila,
Nitrosomonas communis, Nitrosomonas

ureae,
Nitrosomonas marina, Nitrosomonas aestuarii,
Nitrosomonas

oligotropha, Nitrosomonas cryotolerans,
and Nitrosomonas nitrosa (11, 12). Therefore, the 16s
rRNA assay was performed to identify the particular
Nitrosomonas species present in the sludge wastewater.
From the autoradiogram, it is evident that the two
probes Nso 190 and Nsm 156 have a very different
binding affinity to the Nitrosomonas RNA in the blot
analysis. The binding of oligonucleotide probes is
dependent on many factors such as temperature, NaCl
concentration and formamide concentration of the wash
buffer (7). One possible explanation for the binding of
Nsm 156 to the pure Nitrosomonas RNA sample but
the Nso 190 probes greatly reduced affinity to the
RNA is that the washing temperature of the Nso 190
probe was set too high. The washing temperature used
in this study was 68C but according to Wong (15), the
Nso 190 probe dissociation temperature ranges from
44.1C to 51.9C (14), which is much lower than the
melting temperature described by Mobarry et al at 68C
(11). The more than 15C difference in washing
temperature might be enough to cause total dissociation
of bound probes off the nylon membrane. Nsm 156 has
an optimum NaCl concentration of 56 mM (5).
Compared to Nso 190, the optimum NaCl
concentration is 20 mM (4). This detail is important
because higher NaCl concentration will favour the
nonspecific binding and may be misleading. The
concentration of NaCl in 0.1x washing solution is
15mM. The NaCl concentration in the washing solution
is much lower than optimum for Nsm 156 which may
have allowed the probe to bind more efficiently than
Nso 190 because the comparatively lower NaCl
concentration might have allowed for more
mismatching. Formamide concentration should not
have played a part in probe binding because no
formamide was used in the wash buffer. However, it
can be seen from the autoradiogram that the Nso 190
probe is sensitive to Nitrosomonas RNA at 5g while
the Nsm156 is sensitive to amounts of RNA less than
0.5g.
If there were slight mismatching of the nucleotides
to the RNA coupled to too high of a washing
temperature, significant amounts of probes that were
supposed to be bound to the RNA would be washed off
(14). Although the sequence of Nso 190 is specific to
16s rRNA of -proteobacteria which include
Nitrosomonas, there are still many species of
Nitrosomonas that are not yet isolated or sequenced.
The particular strain of Nitrosomonas present on the
nylon membrane may have sequences corresponding to
the Nsm 156 probe but not the Nso 190 oligonucleotide
probe. Although it should be noted that this hypothesis
is highly unlikely since 16s rRNA is highly conserved.
The probe Nso 190 is designed to detect all
ammonia oxidizers of the -subclass of proteobacteria.
However, according to Purkhold et al. (12) Nso 190
probe does not react with species of Nitrosomonas such
as N. communis, N nitrosa, N. oligotropha, and N.
ureae. The probe Nsm 156 targets species of
Nitrosomonas such as N. europaea, N. eutropha, N.
C56 and Nitrosococcus mobilis (11). A possible
explanation of the differential sensitivity to the
Nitrosomonas RNA is that the species of organism is
one that Nsm 156 is specific to, but not Nso 190 such
as Nitrosococcus mobilis, which is a AOB in the -
subdivision of the Proteobacteria class. Since Nso 190
is specific to -proteobacteria, it would not be able to
detect N. mobilis RNA while Nsm 156 can.
A study by Behrens et al (4) investigated the
hybridization of rRNA-targeted oligonucleotides. They
hypothesized that if probes were targeted to the surface
of the ribosomal subunit, then the increase accessibility
of the probe to RNA binding site would increase
sensitivity of probe assays. However, the results
showed little correlation between probe hybridization
efficiency and the proximity of the probe target region
to the surface of the three-dimensional model of the
30S ribosomal subunit. Moreover, according the
predicted secondary structures of E. coli 16s rRNA
model, both Nso 190 and Nsm 156 probes are located
at exposed portions of the secondary structure of the
ribosomal subunit. Therefore, the secondary structure
of the RNA is not believed to have any pronounced
effect on the binding of the oligonucleotide probes.
None of the RNA samples from MPN assay were
detected by either Nso 190 or Nsm 156 as seen in
figures 1, 2, and 3. The integrity of the RNA extracted
from the MPN assay was very poor as assessed by
spectrophotometric method of ratio A
260
/A
280
. Due to
the small amounts of RNA extracted, RNA integrity
analysis by gel electrophoresis was not performed in
order to converse RNA for slot blotting. It is strongly
hypothesized that the RNA degradation by RNase was
very great so that the probes would not be able to bind
efficiently to the RNA samples immobilized on the
nylon membrane. Therefore, even if Nitrosomonas
RNA was extracted from the MPN assay, the RNase
degradation would have prevented the 16s rRNA
probes from identifying the presence of Nitrosomonas
RNA.



57
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:52-58
Copyright December 2004, M&I UBC

FUTURE EXPERIMENTS

Since the present study did not yield any conclusive
results as to whether the MPN assay enriched for
Nitrosomonas species, the present study should be
repeated. The lack of good integrity RNA extracted
from the MPN assay is the main barrier in this study.
Therefore, it is suggested that this study be repeated
using extra anti-RNase precautions to prevent RNase
degradation. To obtain higher yield from RNA
extraction, the MPN assay can be performed in larger
vessels to obtain more cells. Future experiments can
also include the use of other 16s rRNA probes such as
Nsc 128 to detect -proteobacteria (11) such as
Nitrosococcus mobilis that are also AOB that may be
enriched by the Nitrosomonas media in the MPN assay.
Other aspects of the enrichment of MPN assays can
be studied such as the temporal enrichment of the MPN
assay. Perhaps different nitrifiers are enriched at
different times during an MPN assay. Also, by varying
the growth conditions of the MPN assay, one can study
the effect of growth condition on the expression of
rRNA in nitrifying bacteria.


ACKNOWLEDGEMENTS

I thank Dr. William Ramey and Jennifer Sibley for
their guidance with the experimental design and
technical support. I also thank Ronan Yu for his
counseling throughout this study and Raphael Fugere
for providing sludge samples.



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