BIO 206SPRING SEMESTER 2019INSTRUCTOR: DR. RITA MOYES01 March 20194. Chemical Analysis● Analyzing the types of specific structural substances that the microorganismcontains○ Example: bacterial■ Peptidoglycan■ Outer membrane■ D amino acids○ Further differentiate bacteria:■ Gram stain● Gm +/Gm-■ Where the tetrapeptides connect● Gm- bacteria= peptide bond (direct bond)● Gm+ bacteria= peptide bridge (bridge of amino acids)■ Observe the 3rd amino acid in tetrapeptide● Gm+ bacteria= Lysine● Gm- bacteria= diaminopimelic acid5. Phage Typing● Uses bacteriophages○ Very specific- must interact with complementary receptor on host bacterialcell○ No receptor → virus can't bind and infect○ Can differentiate strains of the same bacteria species1○ Different strains of E. coli → different plaque patterns○ Same strains → same plaque pattern● Experiment:○ Draw a grid on the bottom if the plate and make a template of the squares○ Make a confluent layer of test bacteria on the agar plate○ Put drops of known phages-one per square inch■ According to your template○ Incubate and look for lysis of the bacterial cell■ Plaques (clearings)■ Incubate 24-48 hours● Observation of which phages lysed the bacteria helps determine bacteriarelatedness○ Bob- food poisoning from Burger Shack-isolated Staphylococcus aureus■ Is it burger shacks fault?● No, they are different strains → different phage formation● Burger Shack is not the source6. Genetic and Molecular Analysis● Microbial genomes can be compared directly, and taxonomic similarity can beestimated in many ways○ Look at the DNA● Nucleic acid base composition○ DNA is made up of subunits called Nucleotides○ One of the simplest techniques:■ Determine the DNA base composition○ 4 nucleotides are used to make DNA: ■ DNA contains two purines (adenine and guanine)■ Two pyrimidines (cytosine and thymine)○ Double stranded DNA: A pairs with T and G pairs with C○ The G+C:A+T ratio or G+C% in DNA can be determined and used as indicatorof relatedness○ G + C% in prokaryotes range from 25-85%■ % within species is constant2○ G + C: involves 3 hydrogen bonds○ A + T: involves 2 hydrogen bonds■ The number of hydrogen bonds determines the melting point● Apply heat● More heat needed to break triple bond and double bond■ Higher the G + C% = higher temperature = higher melting point● Experiment○ Heat double stranded DNA until the strands separate → single stranded DNA○ Note DNA melting point- more heat is required to break triple bonds so thereis a higher melting point= more G + C%○ Results:■ Bacteria 1 45 G + C%■ Bacteria 2 29● Not related■ Bacteria 1 50■ Bacteria 2 50● Could be related (or the same) but you have to consider thesequence of the nucleotides○ Must compare G + C% along with other traits○ 50 G + C%■ T,C,T,C■ G,G,A,A■ A,T,G,C● Different order means different organism● Must use other tests to determine relatedness● DNA Sequencing○ The most detailed information comes from determining the actual orderand types of nucleotides bases that code the DNA○ This is an automated process that is based on the synthesis and analysis ofcDNA → Complementary DNA■ DNA→ 2 single stranded DNA→ template→ fed into sequencingmachine● Contains all 4 nucleotides that are labeled (i.e. different colors)3○ Compare the sequence to a known bacteria sequence in computer genebank○ After testing the organism is ready to be placed within the taxonomic