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
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
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
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.
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
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