Environmental Microbiology of Aquatic and Waste Systems

4.1 Taxonomy of Microorganisms in Aquatic Environments 55

serve as excellent antigens. The test includes

production of antibodies in an animal host and test­

ing of the antiserum by either the agglutination or

precipitation test. In the agglutination test, a drop of

the culture of a particular bacterium is mixed on a

slide with the anti­serum of an individual infected

by it and examined under a microscope. If clump­

ing occurs, the test bacterium is considered to be

the same or closely related to the bacterium used as

the antigen.

Nucleic Acid Methods
The methods for the characterization and identification
of bacteria which have been discussed so far are based
on phenotypic properties, i.e., the outward manifesta­
tion of the innate (genetic) attributes of the organism.
The properties to be discussed in this section are those
of the nucleic acids of the organisms. They alone how­
ever do not define the organism and must be taken along
with the phenotypic properties. They are very useful in
refining the description of an organism. They are par­
ticularly useful in identifying strains within a species.
(a) G + C ratio
The G + C ratio is the percentage of guanine + cyto­
sine in an organism’s DNA. Several methods exist
for determining this ratio. One method is to deter­
mine the Tm or temperature of melting of the DNA.
At room temperature, DNA is double stranded.
However, as its temperature is raised gradually,
the two strands separate and the rapidity of separa­
tion with increasing temperature depends on the
amount of G and C in the organism’s DNA. G and
C are linked by triple bonds and are therefore less
likely to separate than A and T bonds, which have
double bonds. The higher the G + C content, the

higher the temperature at which the DNA separates

completely.

DNA begins to separate at 70–75°C and separates

completely at about 90°C, when it is said to have

melted. When cooled slowly, it begins to anneal

(i.e., to reform itself into double strands). In anneal­

ing, the strands do not return to their previous

“ partners” but will anneal with any strand with com­

plimentary bases no matter the source, including

those coming from the same organism, other orga­

nisms, or even those synthesized in the laboratory.

This phenomenon of annealing with complimentary

strands from any source is important in other proce­

dures such as in the identification of unknowns, the

Polymerase Chain Reaction (PCR), etc.

When the Tm method is used to determine the

G + C composition, the temperature of the double

(ds) DNA is raised slowly and subjected to spectro­

photometric reading at 260 nm. The graph of the

spectrophotometric readings is plotted against

the change in temperature (see Fig. 4.8). The Tm is

the midpoint of the resulting graph. Two organisms

with similar phenotypic properties and the same

G + C ratio are likely to belong to the species.

(b) DNA–DNA hybridization

This technique measures the degree of genetic

similarity between pools of DNA sequences. It is

usually used to determine the genetic distance

between two species.

It was seen above that when melted DNA is

allowed to cool slowly, the single­stranded DNA

will anneal with any single­stranded DNA no

matter its source, as long the bases are complimen­

tary. To determine how closely related an unknown

organism is with a known one, DNA from the two

Fig. 4.8 Determination of
temperature of melting
(Tm) of DNA