Classification and Identification of

Organisms

Classification and Identification ofMicroorganisms• 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 theyferment lactose?

Can they usecitric acid as their

sole carbon source?

Can they usecitric acid as their

sole carbon source?

Can theyfermentsucrose?

Do theyproduceacetoin?

Escherichia spp. E. coli O157 Citrobacter Enterobacter

Shigella:produces lysinedecarboxylase

Salmonella:generally

produces H2S

No Yes

No YesNo Yes

No Yes No Yes

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.

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.

Glu

cose

Gas

Lys

ine

Orn

ithin

e

H2S

Indo

le

Ado

nito

l

Lac

tose

Arab

inos

e

Sor

bito

l

V–P

Dul

cito

l P

heny

lala

nine

Ure

ase

Citr

ate

2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1

1 2 0 0 7

ID Value

21006

21007

21020 Salmonella choleraesuis

Organism Atypical TestResults

ConfirmatoryTest

Proteus mirabilisProteus mirabilis Ornithine–

Ornithine–

Lysine–

Sucrose

Comparing the resultant ID value with a computerized listing shows that the organism in the tube is Proteus mirabilis.

Bergey’s Manual of Determinative BacteriologyProvides identification schemes for identifying bacteria and archaea

Morphology, differential staining, biochemical tests

Bergey’s Manual of Systematic BacteriologyProvides phylogenetic information on bacteria and archaea

Based on rRNA sequencing

Book References

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

Positive test

Figure 10.10 A slide agglutination 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.

Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ).

Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ).

(a) A positive direct ELISA to detectantigens

4 4

(b) A positive indirect ELISA to detectantibodies

The Western Blot

Lysedbacteria

Polyacrylamidegel

Proteins

Sponge

Paper towels

Salt solution

Gel

Nitrocellulosefilter

Smaller

Larger

If Lyme disease is suspected in a patient: Electrophoresis is used to separate Borrelia burgdorferi proteins in theserum. Proteins move at different rates based on their charge and size when the gel is exposed to an electric current.

The bands are transferred to a nitrocellulose filter byblotting. Each band consists of many molecules of aparticular protein (antigen). The bands are not visible atthis point.

The proteins (antigens) are positioned on the filterexactly as they were on the gel. The filter is thenwashed with patient’s serum followed by anti-humanantibodies tagged with an enzyme. The patientantibodies that combine with their specific antigen arevisible (shown here in red) when the enzyme’ssubstrate is added.

The test is read. If the tagged antibodies stick to thefilter, evidence of the presence of the microorganism inquestion—in this case, B. burgdorferi—has been foundin the patient’s serum.

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)

Collectiontubes

Laser beam strikeseach droplet.

Fluorescencedetector

LaserLaser beam

Fluorescentlylabeled cells

The separated cellsfall into differentcollection tubes.

As cells drop betweenelectrically chargedplates, the cells witha positive chargemove closer to thenegative plate.

Electrode givespositive charge toidentified cells.

Fluorescence detectoridentifies fluorescentcells by fluorescentlight emitted by cell.

Electricallychargedmetal plates

Cell mixture leavesnozzle in droplets.

A mixture of cells istreated to label cellsthat have certainantigens withfluorescent-antibodymarkers.

Detector ofscattered light

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2

3

4

5

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6

7

Electrode

FLOW CYTOMETRY

Genetic Identification

• rRNA sequencing• NCBI Blast

• Polymerase Chain Reaction (PCR)• PCR Animation

• DNA Fingerprinting• Electrophoresis of restriction enzyme digests of Nucleic Acids

DNA Fingerprints

1 2 3 4 5 6 7

*Also known as DNA Footprints

Organism A DNA

DNA-DNA Hybridization

Heat to separate strands.

Organism B DNA

Determine degreeof hybridization.

Cool to allow renaturationof double-stranded DNA.

Combine singlestrands of DNA.

Complete hybridization:organisms identical

Partial hybridization:organisms related

No hybridization:organisms unrelated

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2

3

4

Plasmid

A DNA probe used to identify bacteria

SalmonellaDNAfragment

A Salmonella DNAfragment is cloned in E. coli.

Cloned DNA fragments are marked with fluorescent dye and separated into single strands, forming DNA probes.

Unknown bacteriaare collectedon a filter.

The cells are lysed,and the DNAis released.

The DNA is separated intosingle strands.

DNA probes are addedto the DNA from theunknown bacteria.

Fluorescent probe

Salmonella DNA

DNA fromother bacteria

DNA probes hybridize with Salmonella DNA from sample. Then excess probe is washed off. Fluorescence indicates presence of Salmonella.

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2

6

7

3

4

5

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