1. A Survey of the Microbial WorldChapter 10- Classification of Microorganisms Siti Sarah Jumali Room 14 Level 3 (ext 2123) www.slideshare.net/sarah_jumali

2. The prokaryotes kingdom Bacteria and Archaea Classification and identification Bergey’s manual and bacteria taxonomy Biochemical tests The prokaryotes kingdom

3. Evolution Similarities among organisms such as having plasma membrane, the use of ATP, and possessing DNA are the result of evolution-descent from common ancestor Darwin (1859) says natural selection was responsible for similarities and difference among organisms.

4. Evolution cont’d

5. Phylogenetic relationship Evolution can be deduced from phylogeny. Taxa (singular: taxon)- a group of one or more organisms Taxonomy- put organisms into categories (taxa) to show degrees of similarities between organisms Phylogeny or systematics- study of evolutionary history of organisms

6. The Domain of Life (The 3 Domains) Comparing cells through ribosomes Ribosomes are different in every cells Ribosomes are present in all cells Comparing the sequences in ribosomal RNA (rRNA) gives 3 distinct groups: Eukaryotes Prokaryotes-bacteria and archaea (2 different types or prokaryotes) Differ in membrane lipid structure, transfer RNA molecules (tRNA) and sensitivity to antibiotics

7. Eukarya Animals Fungi Plants Protists

8. Eukarya

9. Prokayotes and Archaea Prokaryotes pathogenic and non-pathogenic Found in soil and water Archaea includes prokaryotes that do not have peptidoglycan in their cell walls Live in extreme environments and carry out unusual metabolic processes Includes Methanogens ( strict anaerobes that produce CH4 from CO2 and H2 extreme halophiles hyperthermophiles

10. The Domain of Life (The three Domains) Origin of mitochondria Origin of choroplast DNA passed from ancestors are called conserved Nucleoplasm grows larger

11. Endosymbionts Endosymbiotic theory Eukaryotic cells evolved from prokaryotic cells Eukaryotic cells and prokaryotic cells live in one another (mitochondria and chloroplast)

12. The 3 Domains And..only bacteria is sensitive to antibiotics

16. Domain divided into phyla

17. Based on rRNA sequence similarities

19. Classification of Eukaryotes Animals Fungi Plants Protists

20. Eukaryotic Cells

22. Not part of either domains Not composed of cells Use anabolic machinery within host cell to multiply Viral genome can direct biosynthesis inside a cell Some can be incorporated into the host’s genome Virus is more closely related to its host than to other virus Viral species- morphology, genes, enzymes Obligatory intracellular parasites Classification of Viruses

23. Viruses Hypotheses on the origin of viruses Arose independently replicating strands of nucleic acids such as plasmids They developed from degenerative cells that through many generations they gradually lost the ability to survive independently but could survive when associated with another cell

24. Virus

25. Methods of Classifying and Identifying organisms Classification Identification is for practical purposes E.g to determine appropriate treatment of infection Can be identified microscopically Morphology Bergey’s manual of Determinative Bacteriology Based on criteria Cell wall composition, morphology, differential staining, oxygen requirements and; biochemical test

27. In clinical microbiology, Information returned will allow further treatment uses transport media (from swab) Not nutritive Transport media prolongs viability of fastidious organisms

29. Identification of microorganisms Morphological Characteristics Cocci, rod, spirilla Tells us little about phylogenetic relationship Differences in structures such as endospores and flagella Differential staining Gram stain and acid-fast stain Based on chemical composition of cell wall Not useful for identification of wall-less and archaea with unusual cell wall

30. Identification of microorganisms

31. Biochemical tests Ability to ferment certain type of carbohydrate Gram identification All members of the family Enterobacteriacaea are oxidase-negative Genera Escherichia, Enterobacter, Shigella, Citrobacter and Salmonella All of these ferment lactose to produce acid and gas except for Shigella and Salmonella Selective media Differential media Rapid identification

32. Biochemical tests

34. Biochemical tests

35. Rapid Identification Method Manufactured for medically important group such as enterics Perform biochemical tests simultaneously Identify bacteria within 4-24 hours Numerical identification- result is assigned with number eg positive = 1, negative = 0 Results are compared to a database of unknown organisms

36. Rapid Identification Method cont’d Rapid Identification of bacteria using Becton Dickinson

37. Serology Study of serum and immune responses Microorganisms are antigenic, they stimulate antibodies Eg inject a rabbit with killed typhoid bacteria and the rabbit will produce antibody agaisnt typhoid bacteria Solution of antibody-Antiserum Slide agglutination test- put sample on slide and add different known antiserum Bacteria will agglutinate when mixed with antibodies in response to species or strain of bacterium Positive test is observed in the presence of agglutination

38. Serology cont’d Serological testing Can differentiate not only among microbial species but also strains within species Also include ELISA and Western Blotting Strains with different antigens are called serotypes, serovars or biovars E.g Different antigens in the cell walls of various serotypes of streptococci stimulate different antibodies

39. Serology cont’d But because closely related bacteria also produce some similar antigens, serological testing can be used to screen bacteriological isolates for possible similarities ELISA (Enzyme linked Immunosorbent Assay) Used to detect AIDS Fast and can be read by computer scanner Known antibodies are placed in microplate wells, and bacteria are added into it Reaction produced allows identification of bacteria

40. Serology cont’d Western blot

41. Western blot

42. ELISA

43. ELISA cont’d

44. Other techniques for identification Phage typing Fatty acids profile Flow cytometry DNA base composition DNA fingerprinting Polymerase Chained Reaction (PCR) Nucleic Acid Hybridization

45. Other techniques for identification Phage typing Looks for similarities among bacteria Useful to trace the origin and the cause of outbreak determines which phage the bacterium is susceptible to Fatty acids profile Fatty Acids methyl esther (FAME) Separate cellular fatty acids and compare to fatty acids profile of known organisms

46. Flow cytometry Identify bacteria without culturing Detect difference in electrical conductivity between cells and the surrounding medium Provide information on the size, shape, density and Can detect fluorescent cells such as Pseudomonas or fluorescent tagged cells

47. Flow cytometer

48. DNA base composition Percentage of guanine and cytosine (G + C), indirectly tells the composition of (A + T) Similar organisms may have about the same GC percentage DNA fingerprinting RFLP is used to break DNA fragments (Restriction enzymes) Determine source of hospital-acquired infection Polymerase Chained Reaction (PCR) Increase amount of DNA Detects presence of microorganisms on gel electrophoresis Taq polymerase Other techniques for identification

49. Nucleic acid Hybridization Southern Blotting The use of probe DNA chips The use of probe Fluorescent dye Ribotyping and rRNA sequencing All cells contain RNA RNA underwent lots of changes over time Does not require cells to be cultured in the laboratory FISH (Fluorescent In Situ Hybridization) Fluorescent-dye labeled Can be used to detect bacteria in drinking water or in patient less than 24 hr

50. Nucleic acid Hybridization

51. FISH- Ikan

52. Putting Classification Methods Together Dichotomous keys Identification based on successive questions: Yes/ No, Positive/ Negative Cladograms Branches Using computer

53. Questions???