1. Classification of Microorganisms10
Classification of Microorganisms

3. The Study of Phylogenetic Relationships
Learning Objectives 10-1 Define taxonomy, taxon, and phylogeny Discuss the limitations of a two-kingdom classification system Identify the contributions of Linnaeus, Whittaker, and Woese Discuss the advantages of the three-domain system List the characteristics of the Bacteria, Archaea, and Eukarya domains.

4. The Study of Phylogenetic Relationships
Taxonomy is the science of classifying organisms
Shows degree of similarity among organisms
Systematics, or phylogeny, is the study of the evolutionary history of organisms

5. The Study of Phylogenetic Relationships
1735: Linnaeus—kingdoms Plantae and Animalia
1800s: Bacteria and fungi put in kingdom Plantae (Nägeli); Kingdom Protista proposed for bacteria, protozoa, algae, and fungi (Haeckel)
1937: Prokaryote introduced to distinguish cells without a nucleus
1968: Murray—Kingdom Prokaryotae
1969: Whittaker—five-kingdom system

6. Check Your Understanding  Of what value is taxonomy and systematics
Check Your Understanding  Of what value is taxonomy and systematics? 10-1  Why shouldn't bacteria be placed in the plant kingdom? 10-2, 10-3

7. The Three Domains
Developed by Woese in 1978; based on sequences of nucleotides in rRNA
Eukarya
Animals, plants, fungi
Bacteria
Archaea
Methanogens
Extreme halophiles
Hyperthermophiles

8. Figure 10.1 Three-Domain System.

9. Table 10.1 Some Characteristics of Archaea, Bacteria, and Eukarya

10. Table 10.2 Prokaryotic Cells and Eukaryotic Organelles Compared

11. The Three Domains
Eukaryotes originated from infoldings of prokaryotic plasma membranes
Endosymbiotic bacteria developed into organelles

12. Figure 10.2 A model of the origin of eukaryotes.
Early cell
Bacteria
Chloroplast
Archaea
Mitochondrion
DNA
Eukarya

13. Figure 10.3 Cyanophora paradoxa.
Bacterium
Eukaryotic host cell

14. A Phylogenetic Tree Grouping organisms according to common properties
Fossils
Genomes
Groups of organisms evolved from a common ancestor
Each species retains some characteristics of its ancestor

15. Figure 10.4a Fossilized prokaryotes.

16. Figure 10.4b Fossilized prokaryotes.

17. Figure 10.4c Fossilized prokaryotes.

18. Check Your Understanding  What evidence supports classifying organisms into three domains? 10-4  Compare archaea and bacteria; bacteria and eukarya; and archaea and eukarya. 10-5

19. Classification of Organisms
Learning Objectives 10-6 Explain why scientific names are used List the major taxa Differentiate culture, clone, and strain List the major characteristics used to differentiate the three kingdoms of multicellular Eukarya Define protist Differentiate eukaryotic, prokaryotic, and viral species.

20. Scientific Nomenclature
Common names vary with languages and geography
Binomial nomenclature is used worldwide to consistently and accurately name organisms
Genus
Specific epithet (species)

21. Table 1.1 Making Scientific Names Familiar

22. The Taxonomic Hierarchy
A series of subdivisions developed by Linnaeus to classify plants and animals
Eukaryotic species: a group of closely related organisms that breed among themselves

23. Figure 10.5 The taxonomic hierarchy.

24. Check Your Understanding  Using Escherichia coli and Entamoeba coli as examples, explain why the genus name must always be written out on first use. Why is binomial nomenclature preferable to common names? 10-6  Find the gram-positive bacteria Staphylococcus in Appendix F. To which bacteria is this genus more closely related: Bacillus or Streptococcus? 10-7

25. Classification of Prokaryotes
Prokaryotic species: a population of cells with similar characteristics
Culture: bacteria grown in laboratory media
Clone: population of cells derived from a single parent cell
Strain: genetically different cells within a clone

26. Figure 10.6 Phylogenetic relationships of prokaryotes.

27. Classification of Eukaryotes
Protista: a catchall kingdom for a variety of organisms; autotrophic and heterotrophic
Grouped into clades based on rRNA
Fungi: chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores or hyphal fragments
Plantae: multicellular; cellulose cell walls; undergo photosynthesis
Animalia: multicellular; no cell walls; chemoheterotrophic

28. Classification of Viruses
Not a part of any domain; not composed of cells; require a host cell
Viral species: population of viruses with similar characteristics that occupies a particular ecological niche

29. Check Your Understanding  Use the terms species, culture, clone, and strain in one sentence to describe growing methicillin-resistant Staphylococcus aureus (MRSA)  You discover a new multicellular, nucleated, heterotrophic, organism with cell walls. To what kingdom does it belong? 10-9  Write your own definition of protist  Why doesn't the definition of a viral species work for a bacteria? 10-11

30. Methods of Classifying and Identifying Microorganisms
Learning Objectives Compare and contrast classification and identification Explain the purpose of Bergey's Manual Describe how staining and biochemical tests are used to identify bacteria Differentiate Western blotting from Southern blotting.

31. Methods of Classifying and Identifying Microorganisms
Learning Objectives Explain how serological tests and phage typing can be used to identify an unknown bacterium Describe how a newly discovered microbe can be classified by DNA base composition, DNA fingerprinting, and PCR Describe how microorganisms can be identified by nucleic acid hybridization, Southern blotting, DNA chips, ribotyping, and FISH Differentiate a dichotomous key from a cladogram.

32. Methods of Classifying and Identifying 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

33. Methods of Classifying and Identifying Microorganisms
Bergey's Manual of Determinative Bacteriology provides identification schemes for identifying bacteria and archaea
Approved Lists of Bacterial Names lists species of known classification

34. Methods of Classifying and Identifying Microorganisms
In clinical microbiology, lab requisition forms are used to note types of specimens collected and tests to be conducted
Transport media is used to collect and transport pathogens to a laboratory

35. Figure 10.7 A clinical microbiology lab report form.
Filled out by one person
Filled out by different person

36. Methods of Classifying and Identifying Microorganisms
Morphological characteristics: useful for identifying eukaryotes; tell little about phylogenetic relationships
Differential staining: Gram staining, acid-fast staining; not useful for bacteria without cell walls
Biochemical tests: determine presence of bacterial enzymes

37. Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.

38. Applications of Microbiology 10.1

39. Biochemical Tests
Rapid identification methods perform several biochemical tests simultaneously
Results of each test are assigned a number

40. Figure One type of rapid identification method for bacteria: EnteroPluri test from BD Diagnostics.
One tube containing media for 15 biochemical tests
is inoculated with an unknown enteric bacterium.
After incubation, the tube is observed for results.
Phenylalanine
Glucose
Ornithine
Adonitol
Arabinose
Lysine
Indole
Lactose
Sorbitol
Dulcitol
Urease
Citrate
Gas
H2S
V–P
The value for each positive test is circled, and
the numbers from each group of tests are
added to give the code number.
Comparing the resultant code number with a
computerized listing shows that the organism in
the tube is Citrobacter freundii.
Code Number
Microorganism
Atypical Test Results
62352
Citrobacter freundii
Citrate
62353
Citrobacter freundii
None

41. Serology The science that studies serum and immune responses in serum
Microorganisms are antigenic—they stimulate the body to form antibodies in the serum
In an antiserum, a solution of antibodies is tested against an unknown bacterium

42. Serology
In the slide agglutination test, bacteria agglutinate when mixed with antibodies produced in response to the bacteria
Serological testing can differentiate between species and strains within species

43. Figure 10.10 A slide agglutination test.

44. Serology Enzyme-linked immunosorbent assay (ELISA) Western blotting
Known antibodies and an unknown type of bacterium are added to a well; a reaction identifies the bacteria
Western blotting
Identifies antibodies in a patient's serum; confirms HIV infection

45. Figure An ELISA test.

46. Figure 18.14a The ELISA method.

47. Figure 10.12 The Western blot.
If Lyme disease is suspected in a patient:
Electrophoresis is used to separate Borrelia
burgdorferi proteins. 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
Smaller
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.
Salt solution
Sponge
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 antihuman
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.

48. Phage Typing
Test for determining which phages a bacterium is susceptible to
On a plate, clearings called plaques appear where phages infect and lyse bacterial cells

49. Figure 10.13 Phage typing of a strain of Salmonella enterica.

50. Fatty Acid Profiles
FAME: Fatty acid methyl esters provide profiles that are constant for a particular species

51. Flow Cytometry
Uses differences in electrical conductivity between species or fluorescence

52. Figure 18.12 The fluorescence-activated cell sorter (FACS).
A mixture of cells is
treated to label cells
that have certain
antigens with
fluorescent-antibody
markers.
Cell mixture leaves
nozzle in droplets.
Fluorescently
labeled cells
Laser beam strikes
each droplet.
Laser beam
Detector of
scattered light
Laser
Fluorescence detector
identifies fluorescent
cells by fluorescent
light emitted by cell.
Electrode
Fluorescence
detector
Electrode gives
positive charge to
identified cells.
Electrically
charged
metal plates
As cells drop between
electrically charged
plates, the cells with
a positive charge
move closer to the
negative plate.
The separated cells
fall into different
collection tubes.
Collection
tubes

53. DNA Base Composition DNA base composition Guanine + cytosine %
Two organisms that are closely related have similar amounts of various bases

54. DNA Fingerprinting DNA fingerprint
Electrophoresis of restriction enzyme digests of an organism's DNA
Comparing fragments from different organisms provides information on genetic similarities and differences

55. Figure 10.14 DNA fingerprints.

56. Nucleic Acid Amplification Tests (NAATs)
Use of PCR to amplify DNA of an unknown microorganism that cannot be cultured

57. Nucleic Acid Hybridization
Nucleic acid hybridization measures the ability of DNA strands from one organism to hybridize with DNA strands of another organism
Greater degree of hybridization, greater degree of relatedness

58. Figure 10.15 DNA-DNA hybridization.

59. Nucleic Acid Hybridization
Southern blotting uses nucleic acid hybridization to identify unknown microorganisms using DNA probes

60. Figure 10.16 A DNA probe used to identify bacteria.
Plasmid
Salmonella
DNA
fragment
Unknown bacteria
are collected
on a filter.
A Salmonella DNA
fragment is cloned in
E. coli.
The cells are lysed,
and the DNA
is released.
Cloned DNA fragments are marked
with fluorescent dye and separated
into single strands, forming
DNA probes.
The DNA is separated into
single strands.
DNA probes are added
to the DNA from the
unknown bacteria.
Fluorescent probe
Salmonella DNA
DNA probes hybridize with
Salmonella DNA from sample.
Then excess probe is washed
off. Fluorescence indicates
presence of Salmonella.
DNA from
other bacteria

61. DNA Chips
A DNA chip (also known as a microarray) contains DNA probes and detects pathogens by hybridization between the probe and DNA in the sample
Detected by fluorescence

62. Figure 10.17a-b DNA chip.

63. Figure 10.17c-d DNA chip.

64. DNA Chips Ribotyping Fluorescent in situ hybridization (FISH)
rRNA sequencing
Fluorescent in situ hybridization (FISH)
Fluorescent DNA or RNA probes stain the microorganisms being targeted
Determines the identity, abundance, and relative activity of microorganisms in an environment

65. Figure 10.18 FISH, or fluorescent in situ hybridization.

66. Putting Classification Methods Together
Dichotomous keys
Identification keys based on successive questions
Cladograms
Maps that show evolutionary relationships among organisms; based on rRNA sequences

67. Figure 10.19 Building a cladogram.

68. Dichotomous Keys: Overview
PLAY
Animation: Dichotomous Keys: Overview

69. Dichotomous Keys: Sample with Flowchart
PLAY
Animation: Dichotomous Keys: Sample with Flowchart

70. Dichotomous Keys: Practice
PLAY
Animation: Dichotomous Keys: Practice

71. Check Your Understanding  What is in Bergey's Manual
Check Your Understanding  What is in Bergey's Manual?  Design a rapid test for a Staphylococcus aureus. (Hint: See Figure 6.10, page 162.)  What is tested in Western blotting and Southern blotting? 10-15

72. Check Your Understanding  What is identified by phage typing
Check Your Understanding  What is identified by phage typing?  Why does PCR identify a microbe?  Which techniques involve nucleic acid hybridization?  Is a cladogram used for identification or classification? 10-12, 10-19