Download Lab Aseptic Techniques and Classification

Classification and
Identification of Organisms
Classification and Identification of
Microorganisms
• Classification: placing organisms in groups of related species
• Lists of characteristics of known organisms
• Identification: matching characteristics of an “unknown” organism to
lists of known organisms
• Clinical lab identification
• Microorganisms are identified for practical purposes such as determining
treatment for infection
Clinical Identification Methods
• Morphological characteristics: useful for identifying eukaryotes but
can be used for prokaryotes
• Shapes of bacterium; colony characteristics
• Differential staining: Simple staining, Gram staining, and acid-fast
staining
• Based on cell membrane differences
• Biochemical tests: determines presence of bacterial enzymes
• Catalases, peroxidases, agglutination tests, fermentation tests, etc.
Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.
Can they
ferment lactose?
No
Yes
Can they use
citric acid as their
sole carbon source?
Can they use
citric acid as their
sole carbon source?
No
Shigella:
produces lysine
decarboxylase
Yes
No
Yes
Can they
ferment
sucrose?
Salmonella:
generally
produces H2S
No
Escherichia spp.
Do they
produce
acetoin?
Yes
E. coli O157
No
Citrobacter
Yes
Enterobacter
Figure 10.9 One type of rapid identification method for bacteria: Enterotube II from Becton Dickinson.
One tube containing media for 15 biochemical tests
is inoculated with an unknown enteric bacterium.
Citrate
Urease
2
Dulcitol
Phenylalanine
Sorbitol
4 +
V–P
Arabinose
Lactose
Adonitol
H2S
Indole
Ornithine
Lysine
Glucose
Gas
After incubation, the tube is observed for results.
The value for each positive test is circled, and the
numbers from each group of tests are added to give
the ID value.
2+1 4 + 2 + 1
2
Comparing the resultant ID value with a
computerized listing shows that the organism in the
tube is Proteus mirabilis.
4 + 2 + 1
1
0
+
1
4 + 2 + 1
0
7
ID Value
Organism
Atypical Test
Results
Confirmatory
Test
21006
Proteus mirabilis
Ornithine–
Sucrose
21007
Proteus mirabilis
Ornithine–
21020
Salmonella choleraesuis
Lysine–
Book References
Bergey’s Manual of Determinative
Bacteriology
Provides identification schemes for
identifying bacteria and archaea
Morphology, differential
staining, biochemical tests
Bergey’s Manual of Systematic
Based on rRNA sequencing
Bacteriology
Provides phylogenetic information on
bacteria and archaea
Serology
• Serology is the science that studies serum and immune responses
that are evident in serum (does not contain blood cell or clotting
factors)
• Combine known anti-serum plus unknown bacterium
• Rabbit immune system injected with pathogen produces antibodies against
that pathogen
• Strains of bacteria with different antigens are called serotypes,
serovars, biovars
• Slide Agglutination Test
Figure 10.10 A slide agglutination test.
Positive test
Negative test
ELISA
• Enzyme-Linked Immunosorbent Assay
• Direct or Indirect
• Direct ELISA looks for the presence of bacterium in the serum
• Indirect ELISA looks for the presence of antibodies in the serum
• Both use antibodies linked to enzyme
• Enzyme contains substrate that produces color
Figure 18.14.4 The ELISA method.
4
Enzyme's substrate ( ) is
added, and reaction produces
a product that causes a
visible color change ( ).
(a) A positive direct ELISA to detect
antigens
4
Enzyme's substrate ( ) is
added, and reaction produces
a product that causes a
visible color change ( ).
(b) A positive indirect ELISA to detect
antibodies
If Lyme disease is suspected in a patient: Electrophoresis is used
to separate Borrelia burgdorferi proteins in the
serum. Proteins move at different rates based on their charge and
size when the gel is exposed to an electric current.
Lysed
bacteria
Polyacrylamide
gel
Proteins
Larger
Paper towels
The bands are transferred to a nitrocellulose filter by
blotting. Each band consists of many molecules of a
particular protein (antigen). The bands are not visible at
this point.
Smaller
Sponge
Salt solution
Gel
Nitrocellulose
filter
The proteins (antigens) are positioned on the filter
exactly as they were on the gel. The filter is then
washed with patient’s serum followed by anti-human
antibodies tagged with an enzyme. The patient
antibodies that combine with their specific antigen are
visible (shown here in red) when the enzyme’s
substrate is added.
The test is read. If the tagged antibodies stick to the
filter, evidence of the presence of the microorganism in
question—in this case, B. burgdorferi—has been found
in the patient’s serum.
The Western Blot
Phage Typing of a strain of Salmonella enterica
Bacteriophages are viruses that infect bacteria
Flow Cytometry
• Uses differences in electrical conductivity between species
• Fluorescence of some species
• Cells selectively stained with antibody plus fluorescent dye
• Also can be used in FACS analysis (Fluorescent Antibody Cell Sorter)
Fluorescence-activated cell sorter (FACS)
Fluorescently
labeled cells
1
A mixture of cells is
treated to label cells
that have certain
antigens with
fluorescent-antibody
markers.
2
Cell mixture leaves
nozzle in droplets.
3
Laser beam strikes
each droplet.
Laser beam
Detector of
scattered light
Laser
Electrode
4
Fluorescence detector
identifies fluorescent
cells by fluorescent
light emitted by cell.
5
Electrode gives
positive charge to
identified cells.
6
As cells drop between
electrically charged
plates, the cells with
a positive charge
move closer to the
negative plate.
7
The separated cells
fall into different
collection tubes.
Fluorescence
detector
Electrically
charged
metal plates
Collection
tubes
6
FLOW CYTOMETRY
Genetic Identification
• rRNA sequencing
• NCBI Blast
• Polymerase Chain Reaction (PCR)
• PCR Animation
• DNA Fingerprinting
• Electrophoresis of restriction enzyme digests of Nucleic Acids
1
DNA Fingerprints
*Also known as DNA Footprints
2
3
4
5
6
7
DNA-DNA Hybridization
Organism A DNA
Organism B DNA
1
Heat to separate strands.
2
3
4
Combine single
strands of DNA.
Cool to allow renaturation
of double-stranded DNA.
Determine degree
of hybridization.
Complete hybridization:
organisms identical
Partial hybridization:
organisms related
No hybridization:
organisms unrelated
A DNA probe used to identify bacteria
Plasmid
Salmonella
DNA
fragment
1
A Salmonella DNA
fragment is cloned in
E. coli.
3 Unknown bacteria
are collected
on a filter.
4
2
Cloned DNA fragments are marked with
fluorescent dye and separated into single
strands, forming DNA probes.
6
7
DNA probes are added
to the DNA from the
unknown bacteria.
DNA probes hybridize with
Salmonella DNA from sample. Then
excess probe is washed off.
Fluorescence indicates presence of
Salmonella.
The cells are lysed,
and the DNA
is released.
5
The DNA is separated into
single strands.
Fluorescent probe
Salmonella DNA
DNA from
other bacteria
DNA chip (DNA Microarray)
(a) A DNA chip can be manufactured to
contain hundreds of thousands of synthetic
single-stranded DNA sequences. Assume
that each DNA sequence was unique to a
different gene.
(b) Unknown DNA from a sample is separated into single strands, enzymatically cut,
and labeled with a fluorescent dye.
DNA chip
(c) The unknown DNA is inserted into the chip and
allowed to hybridize with the DNA on the chip.
(d) The tagged DNA will bind only to the
complementary DNA on the chip. The bound DNA
will be detected by its fluorescent dye and analyzed
by a computer. In this Salmonella antimicrobial
resistance gene microarray, S. typhimurium-specific
antibiotic resistance gene probes are green, S.
typhi-specific resistance gene probes are red, and
antibiotic-resistance genes found in both serovars
appear yellow/orange.
Microarray Analysis
• Cory L. Blackwell Dissertation
• Pg. 61
FISH
• Fluorescent in situ hybridization
• Used to identify specific sequences in DNA/Chromosomes
• Add DNA probe for S. aureus
• In Situ Hybridization
FISH, or fluorescent in situ hybridization