1. Distinguishing Strains Individual bacterial species and strains may be distinguished by: RFLP or rep-PCR analysis Protein profiling Immunological tests Phage typing Biochemical tests This allows ID for treatment as well as for tracing disease outbreaks For example, if it can be shown that two pathogens are identical (clonal) then it may be inferred that they came from the same place/person

2. RFLP & Rep-PCR

3. Protein Profile

4. Immunological Tests Slide agglutination  antiserum and slides (will be doing in lab) Enzyme-Linked Immunosorbent Assay (ELISA)  antibodies and microplates (will discuss in detail last lecture) Western Blotting: antigens are separated by a gel-electrophoresis and detected using radioactive or fluoresence tagged antibodies Along with ELISA serves as basis for assay for HIV seroconversion

5. Monoclonal Antibodies (Mabs) Note that though clonal, Monoclonal Antibodies are (or at least used to be not) produced by processes that have nothing to do with Gene Cloning

6. Phage Typing

7. Biochemical Tests (1/2)

8. Biochemical Tests (2/2)

9. Type Strain In classifying an organism, it is helpful to have some standard to compare it to Such standards for a given strain are termed type strains Often the type strain is the first example of a species or strain Type strains are kept preserved by the American Type Culture Collection (ATCC) (yes, I know, this is an exact quote from the lecture notes—what can I say, it was late….)

10. Bergey’s Manual

11. Direct Sequencing of Environmental Samples DNA PCR DNA 1. Collect water or soil sample containing bacteria and archaea. 2. Lyse cells and purify DNA. 3. Use universal primers to amplify rRNA genes in sample by PCR. 4. Purify rRNA genes on a gel, Then separate them by inserting individual rDNAs into plasmids. 5. Insert plasmids into E.coli cells. The cells grow rapidly to produce millions of copies of each rDNA. 6. Purify rDNA from plasmids. …AGGCTTACCGTAAC... 7. Sequence rDNA; compare to known sequences.

12. Learning the Binomials 1.Look for Default Categories: Facultatively Anaerobic Gram- Negative Rods (Escherichia, Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, Proteus, & Yersinia = Family Enterobacteriaceae; plus Gardnerella, Haemophilus, Pasteurella, & Vibrio) 2.Learn the categories: Spirochetes; Aerobic Motile helical Gram- Negative; Gram-Negative Aerobic Rods and Cocci; Anaerobic Gram-Negative Rods; Rickettias; Chlamydiaes; Mycoplasmas; Gram-Positive Cocci; Endospore-Forming Rods and Coccis, etc. 3.Start by distinguishing by Gram-staining characteristics, shape, and oxygen requirements 4.Associate disease with genera 5.Associate genera with specific epithets 6.Learn only that stuff not in brackets 7.Don’t forget to use proper binomial-nomenclature conventions 8.Start learning binomials for the previous midterm! 

13. Chlamydia Life Cycles

14. Link to Next Presentation

15. Acknowledgements http://www.ulm.edu/~sdavis/b122/lectures/14.ppt http://www.sw.vccs.edu/msht2/biology/Lecture%20in%20Power%20Point/Chapter%2022.ppt http://www.botany.hawaii.edu/BOT201/Algae/BOT201%20CYANOPHYTE%20LECTURE%20DELIVERED.PPT http://zadaw.com/presentations/arboviruses.ppt www.bishops.ntc.nf.ca/wells/redas/webpages/ powerpoint/Kingdoms_Taxonomy.ppt http://www.colby.edu/biology/BI163/Bacteriappt/bacteriaarchaea.ppt http://www.laredo.edu/science/rviswanath/BIOL1410/Chapter12.ppt http://microbiology.okstate.edu/courses/micro2124/babus/Chapter10_files/Chapter10.ppt

16. DNA Hybridization Group A Streptococcus Direct Test Nucleic acid hybridization tests are based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes (7). The GEN-PROBE DNA Probe assay uses a single-stranded DNA probe with a chemiluminescent label which is complementary to the ribosomal RNA of the target organism. After the ribosomal RNA is released, the labeled DNA probe combines with the target organism’s ribosomal RNA to form a stable DNA:RNA hybrid. The Selection Reagent differentiates non-hybridized from hybridized probe. The labeled DNA:RNA hybrids are measured in a GEN-PROBE luminometer (LEADER® I, 50, 400, 450, or 450i). A positive result is a luminometer reading greater than or equal to the cut-off. A value below this cut-off is a negative result. http://www.gen- probe.com/PI/3890 PI.pdf http://www.gen- probe.com/PI/3890 PI.pdf

17. Immunological Tests Slide agglutination  antiserum and slides (will be doing in lab) Enzyme-Linked Immunosorbent Assay (ELISA)  antibodies and microplates (will discuss in detail last lecture) Western Blotting: antigens are separated by a gel-electrophoresis and detected using radioactive or fluoresence tagged antibodies Along with ELISA serves as basis for assay for HIV seroconversion Group A Streptococcus Direct Test Phadebact® Streptococcus Test is a co- agglutination test. Antibodies which are specific against streptococcal groups A, B, C and G are bound to Protein A on the surface of non-viable staphylococci. When a sample containing streptococci belonging to one of these four groups is mixed with the reagent, specific antigens on the surface of the streptococci bind to the corresponding specific antibodies. A co-agglutination lattice is formed, which is visible to the naked eye. Test time: 15 min or less. http://www.boule.se/diagnostics/10558312.htm http://www.boule.se/diagnostics/10558312.htm