The document discusses various methods for identifying and classifying prokaryotes, including microscopy, biochemical tests, serology, nucleic acid analysis, and detecting strain differences. Microscopy techniques include Gram staining, acid-fast staining, capsule staining, endospore staining, and fluorescent dyes. Biochemical tests involve catalase, sugar fermentation, and urease reactions. Identification is also possible through serology, polymerase chain reaction, rRNA sequencing, and analyzing fatty acids or genomic profiles. Strain differences can be detected using biochemical, serological, and genomic typing methods.

1. Chapter 10 Prokaryote Identification Microscopy Chapter 3 Other Phenotypic Characteristics Chapters 4, 17 Genomic Identification Strain Differences

2. Prokaryote Identification

3. Microscopy 1670s Antony van Leeuwenhoek

4. Microscopy

5. Microscopy: Stains

6. Gram Stain

7. Gram Stain: Clinical Specimen

9. Acid-Fast Stain Mycobacterium species Cell wall composition prevents dye uptake Red dye added heated over boiling water or concentrated dye added Rinse Decolorizing acid-alcohol wash Blue counterstain

10. Capsule Stain / Negative Stain Cryptococcus neoformans Capsule (glycocalyx) Gel-like layer for protection or attachment Distinct and gelatinous Stains poorly :

11. Endospore Stain Green dye added heated gently Rinse Red counterstain Bacillus and Clostridium species Endospores resistant to Gram stain

12. Fluorescent Dyes and Tags Living: green Dead: red Auramine Mycobacterium cell wall Metabolic activity alters fluorescent properties of a dye Fluorescent dyes bind to pathogen-specific compound Dye-conjugated antibodies: recognize pathogen-specific antigens S. pyogenes protein

13. Identification by Microscopy

14. Prokaryote Identification

15. Enrichment Culture

16. Streak Plate

17. Types of Growth Media

18. Complex vs. Defined Media

19. Selective & Differential Media Selective Media Inhibits growth of all but target organism Differential Media Target bacteria changes in a recognizable way MacConkey Agar Isolate/identify Gram negative rods from the intestine Selective: bile salts/crystal violet dye inhibit growth of all but Gram negative rods Differential: lactose fermenting bacteria produce pink colonies (pH indicator)

20. Differential Media: Blood Agar Differential media detects bacteria that lyse red blood cells (hemolysin production) Red blood cell lysis generates a zone of clearing (a) clear zone = beta hemolysis = S. pyogenes (b) greenish zone = alpha hemolysis = non-pathogenic Streptococcus

21. Prokaryote Identification

22. Biochemical Tests

23. Biochemical Tests Catalase test bubbles = catalase activity Sugar fermentation test Gas and acid production = fermentation activity Urease test Gas and ammonia (base) production = urease activity

24. Dichotomous Key

25. Biochemical Tests: Commercial Arrays API strip test add liquid culture to dehydrated media Enterotube Innoculation from rod drawn through liquid compartments Other formats: 96 well plates, Miniaturized systems

26. Prokaryote Identification Serology: Recognition of host antibody produced in response to bacterial antigen Direct recognition of bacterial antigen

27. Serology Seronegative No specific antibodies to a pathogen because no prior exposure Seropositive Production of specific antibodies to a pathogen Seroconversion Switch from seronegative to positive Serology In vitro study of antibody-antigen interactions Serum Fluid portion of the blood that remains after blood clots

28. Serology: Indirect ELISA ELISA Enzyme-Linked Immunosorbent Assay Bacterial antigen affixed to substrate Patient serum added if antibody vs. antigen present, it will stay in the well Peroxidase-conjugated anti-human IgG antibody added color reaction = antibody in serum recognizes antigen Testing for antibody production in response to antigen Often used as HIV test (anti-gp120, anti-p24 antibodies) “ Indirect” because second antibody required for signal

29. Western Blot Analysis To confirm positive HIV ELISA result HIV proteins run on SDS-PAGE gel protein separation based on size Separated proteins transferred to membrane Test serum added Enzyme-labeled secondary antibody added

30. Direct ELISA Known antibodies vs. antigen attached to substrate Patient sample added if bacterial antigen is present, it will stay in the well Peroxidase-conjugated antibody added Direct testing for presence of antigen “ Direct” because antibody directly conjugated to peroxidase

31. ELISA plates 96 well format for ELISA, other high-throughput protocols Antibody titer (concentration): serial dilutions of serum tested reciprocal of last dilution with positive reaction = titer (1:256 dilution = 256 titer)

32. Fatty Acid Analysis Bacteria differ in the type and quantity of membrane fatty acids Fatty acids removed and converted to methyl ester form (Fatty Acid Methyl Ester) Gas chromatography analysis

33. Prokaryote Identification

34. Nucleic Acid Hybridization

35. Polymerase Chain Reaction Nucleotide sequence must be known for primer design

36. Polymerase Chain Reaction

37. Polymerase Chain Reaction 30 cycles = billion-fold amplification

38. Prokaryote Identification: PCR Could also use DNA probe instead of gel Requires knowledge of a specific nucleotide sequence from the target pathogen

39. rRNA Sequencing Ribosomes protein + rRNA components S = Svedberg unit measure of sedimentation mRNA translation & protein synthesis important / conserved process Sequence rRNA directly Sequence DNA that codes for rRNA Usually 16S rRNA sequenced

40. Strain Differences E. coli strains K-12 EPEC EHEC STEC ETEC EIEC EAggEC Strains: related, but not identical, isolates of a species

41. Detecting Strain Differences

42. Biochemical Typing Some bacterial strains can be differentiated with biochemical tests Biovar/Biotype: strain with a characteristic biochemical pattern Vibrio cholerae : Pandemics 1-6 = Classical Biotype Pandemic 7 = El Tor Biotype

43. Serological Typing Strain differentiation based upon antigenic (protein/carbohydrate) differences Serovar/serotype: strain with characteristic antigen pattern V. cholerae O1 and O139 (LPS O region) E. coli O157:H7 1 st antigen = carbohydrate (LPS O region) 2 nd antigen = protein (flagella)

44. Genomic Typing RFLP: Restriction Fragment Length Polymorphism Strain differences based on subtle differences in DNA sequence

45. Genomic Typing PulseNet: national database of RFLPs from foodborne pathogens

46. Phage Typing Strain differences based on bacteriophage susceptibility patterns

47. Antibiograms Strain differences based on antibiotic susceptibility patterns

48. Summary Prokaryote Identification / Classification Microscopy Chapter 3 Other Phenotypic Characteristics Chapters 4, 17 Genomic Identification Strain Differences