3. A.Identification of prokaryotes using phenotypic
1. Microscopic Analysis
• An important step is to determine:
• size, shape and staining characteristics of a
• Microscopic examination sometime gives
information enough to make a presumptive
• Trichomonas (protozoa) in vaginal secretion
• Round worms eggs in stool can be
identified based on their shape and size
under the microscope.
4. Identification of prokaryotes using
•Gram stain is a differential method.
•Gram stain of a specimen by itself - generally not
sensitive and specific enough to diagnose the cause
of most infection,
•but very useful tool in narrowing the possible
identities of an organism.
• In certain cases, it gives enough information to
start appropriate antimicrobial therapy while waiting
more accurate identification.
5. Identification of prokaryotes using
•Certain microorganisms have unique characteristics
that can be detected with special staining procedure
– Fungus Cryptococcus neoformans (capsule
– Mycobacterium tuberculosis (acid-fast stain).
7. Metabolic differences
1. Culture characteristics
•can give initial clues for the identification of
– Colonies of Streptococci are generally
fairly small relative to many other bacteria.
– Pseudomonas aeruginosa often produces
a soluble green greenish pigment which
discolors the growth media and has a
distinct fruity odor.
8. Metabolic differences
Use of selective and differential media
•Blood agar media: differential media.
– Beta-hemolytic colonies are characteristics of
•MacConkey agar: both selective and differential media.
– It inhibits the growth of most Gram positive bacteria
and Gram negative cocci.
– It has bile salts which inhibits the growth of most
nonintestinal organisms thus usually it is used to
select intestinal gram negative bacteria.
9. MacConkey agar:
– It also differentiates lactose fermenting bacteria
from nonlactose fermenter
e.g. E. coli a lactose fermenter, forms
characteristic pink colonies on MacConkey
10. Biochemical tests
•Culture characteristics can narrow the number of
possible identities of bacteria.
•but biochemical tests are generally necessary for a
more conclusive diagnosis.
• nearly all bacteria which grow in the presence of
oxygen are catalase positive.
• Catalase positive bacteria break down hydrogen
peroxide to release oxygen gas which cause
• Important exception are lactic acid bacteria which
11. •Thus if a throat culture has beta-hemolytic
colonies on blood agar but are catalase positive,
then Streptococcus pyogenes is ruled out.
12. Biochemical tests
•Most biochemical tests rely on a pH
indicator or chemical reaction that results
in color change when a compound is
• Fermentation of sugar results in
acid production, which lowers the pH,
resulting in a color change from pink
to yellow and gas production.
• No color change (central tube)
indicate that sugar is not used.
• A medium designed to detect urease
enzyme that degrades urea to produce
carbon dioxide and ammonia, utilizes a
different pH indicator that turns bright
pink in alkaline conditions.
14. Biochemical tests
• The basic strategy for identifying
bacteria based on biochemical test
relies on the use of a dichotomous key,
which is a flow chart of tests that give
either a positive or negative result.
• The biochemical tests are usually
initiated simultaneously to speed
15. Biochemical tests
•In certain cases, biochemical test can be done
without culturing the organism e.g.
-Breath test which assays for the presence of
urease is done to detect Helicobacter pylori
•Commercial modifications of traditional biochemical
•e.g. API test strip, enterotube and Biolog
microtiter plate methods.
•In some cases, proteins and polysaccharides present on the
surface of the bacterium are considered as identifying markers.
•The most useful of these are the molecules that make up
surface structures including the cell wall, glycocalyx, flagella
•Antibodies directed against surface proteins and
polysaccharides are frequently used to identify various
•Methods which use antibodies for the detection of antigens are
•Some serological tests such as used to identify Streptococcus
pyogenes are quite specific, simple and rapid.
17. Fatty acid analysis (FAME)
•Bacteria differ in the type and relative quantity of fatty acids
that make up their membranes. Thus cellular fatty acid
compositions can be used as an identification marker.
•The bacterial cells are grown under standardized conditions
and then chemically treated with sodium hydroxide and
methanol to release fatty acids and to convert those acids to
their more volatile methyl ester form (FAME stands for fatty
acid methyl ester).
•FAME are analyzed by gas chromatography.
•By comparing the pattern of peaks, or chromatogram, to
those of known species, an isolate can be identified.
20. •Genotypic characteristics are used in the identification of
microorganisms particularly which are difficult to cultivate.
•Nucleic acid probes: are used to detect specific nucleotide
sequences that characterize a particular species of
•Fluorscence in situ hybridization (FISH) is increasingly being
used to identify intact microorganisms in environmental and
•By using the rRNA specific probes, either specific species or
groups of related organisms can be identified
22. Polymerase chain reaction (PCR)
• PCR can be used to amplify specific nucleotide sequences of
microorganisms from samples such as body fluids, soil, food,
• This technique can be used to detect microorganisms that are
present in extremely low numbers as well as those can not be
grown in culture.
• In order to use PCR to detect microorganism of interest, a
sample should be first treated to release and denature DNA.
• All ingredients needed for PCR along with specific primers
known and designed for particular microbe are then added.
23. • After ~30 cycles of PCR, sufficiently amplified DNA fragment
is visualized as discrete band on an ethidium bromide stained
• Alternatively, a DNA probe can be used to detect the amplified
24. Sequencing ribosomal RNA genes
•Ribosomal RNA genes (DNA sequences) are highly
conserved, and can be used to identify organisms.
•This method is particularly useful for identification of those
prokaryotes which are difficult or currently impossible to grow
•Three different rRNAs: 5S, 16S, and 23S.
•Some regions of 16S rRNA are virtually same in all
prokaryotes whereas others have quite variable sequence and
this variable region is used to identify an organism.
•In certain cases, 16S rDNA is used to identify uncultivable
27. Characterizing strain differences
• In some situations it is useful to distinguish among different
stains of bacteria especially when only certain strains cause
• Example: only certain strains of E. coli cause intestinal disease as
only a few strains have the virulence factors such as toxin production
• Detecting strains differences is also helpful in tracing source
of an outbreak.
• The following methods are used for charactering various
– Biochemical typing
– Serological typing
– Genomic typing
– Phage typing
28. Biochemical and serological typing
1. Biochemical typing
•Biochemical tests are mainly used to identify various
species of bacteria but they can also be used to distinguish
•A strain that has characteristic biochemical pattern is
called a biovar or biotype.
29. 2. Serotyping
•Serological procedures used to differentiate strains
[serovars, serotypes] of microorganisms that differ in the
antigenic composition of a structure or product.
•Important in identifying a pathogen out of the group,
primarily using cell wall antigens.
Examples: Lancefield system to identify streptococci.
–Serotypes are identified by a letter A-O and is based on
specific antibody agglutination reactions with cell wall
carbohydrate [Polysaccharide O}.
30. 2. Serotyping
–Further subdividing of Group A Strep based on specific
M protein antigens can also be performed.
•Other examples of serotyping:
– Identifying specific antigen-antibody reactions involving Flagella
–Capusular [K] antigens, and
–Cell wall [O] antigens.
31. 2. Serological typing
•Proteins and carbohydrates that vary among strains can
be used to differentiate strains.
•Example: E. coli vary in the antigenic structure of
certain parts of the LPS portion of the cell wall, the O
antigen. The composition of the flagella, the H antigen,
can also vary.
•The strain designation of E. coli O157:H7 refers to the
structure of LPS and its flagella.
•A strain that varies serologically from other strains is
sometime called a serovar or a serotype.
32. 3. Genomic typing
•Molecular methods can be used to detect genomic variations
that characterize certain strains.
•In some cases these differences include genes that encode
for toxins or other proteins related to disease.
•Example: E. coli O157:H7. The toxin gene can be detected
using a probe that consists of a specific nucleotide
sequence unique to that gene.
•Subtle differences in DNA sequences can be used to
distinguish among strains that are phenotypically identical.
This helps in tracing epidemics of foodborne illnesses.
33. 3. Genomic typing
•One method of genomic typing is to compare the pattern of
fragment sizes produced when the same restriction enzyme is
used to digest DNA from each organism.
•When the lengths of restriction fragments vary among
organisms, it is termed ‘restriction fragment length
34. 3. Genomic typing
Common methods used to look for RFLPs :
1. Pulse-field gel electrophoresis:
•The bands can be visualized by staining gel with ethidium
•Uses a restriction enzyme that cuts genomic DNA into
many small fragments.
- As bacteria usually have several different rRNA
genes, the probe hybridizes to several different
restriction fragments, the pattern of which varies
- Southern blot hybridization is then done using a probe
that hybridizes to only those fragments that have
sequences encoding ribosomal RNA.
37. Phage typing
•Strains of a given species sometimes differ in their
susceptibility to various types of bacteriophages.
•The susceptibility of an organism to a particular type of phage
can be readily demonstrated in the laboratory.
•The patterns of clearing around the bacteriophage spot
indicate the susceptibility of the test organism to different
•Different patterns are compared to determine strain
•Bacteriophage typing is largely replaced by molecular
41. •Antibiotics susceptibility patterns or antibiograms, are also
used to distinguish among different strains.
•Again this method has largely been replaced by molecular
•Different strains will have different patterns of clearing
around antibiotics disks.