Student Perspectives on the Use of Biolog GenIII Plates in Undergraduate Research and a General Microbiology Course
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1 Student Perspectives on the Use of Biolog GenIII Plates in Undergraduate Research and a General Microbiology Course Jordan Krebs & Jeff Newman Lycoming College Williamsport, PA
2 Talk Contents The use of Biolog GenIII in: General microbiology class Research Novel Species work Correlation to Genomics
![sensitivity, ph, [NaCl], Temperature, Oxygen](/docs-images/94/122302156/images/3-4.jpg "requirements - Kirby-Bauer Disk Diffusion assay (10)")
3 The LycoMicro Unknown Microbe Lab Week 1 - Aseptic technique/inoculation - pipetting sterile media - selection of knowns and unknowns - streak plates, inoculation of liquid - preparation of frozen permanents Week 2 Staining & Microscopy - Gram stain - Endospore stain - Wet mount Week 3 Antibiotic sensitivity, ph, [NaCl], Temperature, Oxygen requirements - Kirby-Bauer Disk Diffusion assay (10) - oxidase, catalase, Gas-Pak Jar
4 The LycoMicro Unknown Microbe Lab Week 4 Carbohydrate & Nitrogen Metabolism - MRVP, citrate, phenol red, TSI - urease, nitrate reduction, SIM Week 5 Exoenzymes, Differential/Selective media - caseinase, lipase, amylase, DNase, - EMB, HEA, MSA, PhenylEthanol, - Bile Esculin, Brilliant Green, EG Min. Week 6 - PCR of 16S rdna, gel, send for sequencing. - Bergey s Manual Hypothesis 27f 330f 16S rrna gene ~ 1500 bp 1492r
5 The LycoMicro Unknown Microbe Lab Week 7 MIDI FAME Analysis - BUG+Blood (Hemolysis) Biolog GenIII Plates Week 8 Analyze DNA - Construct Phylogenetic Tree w/mega, - Literature Research (IJSEM) 0.02 Pantoea anthophila JJM Escherichia coli Acinetobacter johnsonii Pseudomonas aeruginosa Neisseria gonorrhoeae Aquaspirillum sinuosum Helicobacter pylori Bdellovibrio bacteriovorus Blastopirellula marina Cytophaga hutchinsonii Sphingobacterium anhuiense Chryseobacterium indologenes Prochlorococcus marinus Geovibrio ferrireducens Lactococcus lactis Streptococcus pyogenes Exiguobacterium undae Bacillus subtilis Staphylococcus aureus Oerskovia jenensis Arthrobacter aurescens Streptomyces coelicolor Corynebacterium callunae Nitrospira moscoviensis Aquifex pyrophilus Thermomicrobium roseum Chloroflexus aurantiacus
6 Biolog GenIII plates A Acidic ph Sugars Hexose-PO 4 s Amino s Hexose s Carboxylic s, esters, and fatty s B C D E F G H BiOLOG NaCl Lactic Reducing power GN vs. GP 71 carbon sources 23 chemical sensitivity assays A A B B C D E F BiOLOG C D E F BiOLOG G G H H isolate under study database
7 Compared to: Gram Negative Kirby-Bauer Gelatinase API Amylase 20NE 20E/ Positive Biolog GenIII Plate A B negative control D-raffinose gal-α1-6- glu fru Dextrin amylose glu- 1-4, 1-6 poly -Dlactose gal glu ( ) D-maltose glu glu D- melibiose gal-α1-6- glu D- trehalose glu glu -methyl-dglucoside D- cellobiose glu glu D-salicin aspirin- 1- glu gentiobiose glu- 1-6-glu Sucrose glu fru D-turanose glu fru N-acetyl-Dglucosamine N-acetyl- - D-mannosamine N-acetyl-Dgalactosamine Stachyose gal(α1 6) gal(α1 6) glc(α1 2β) fru N-acetyl neuraminic positive control ph 6 ph 5 1% NaCl 4% NaCl 8% NaCl C -Dglucose D-mannose D-fructose D- galactose 3-methyl glucose D-fucose L-fucose L- rhamnose inosine 1% Nalactate fusidic D-serine D D-sorbitol D-mannitol D-arabitol glycerol myoinositol D-glucose- 6-PO4 D-fructose- 6-PO4 D-aspartic D-serine Troleandomycin rifamycin SV minocycline E gelatin L-alanine L-arginine L-aspartic L-glutamic L-histidine glycyl-lproline L-pyroglutamic L-serine lincomycin guanidine HCl niaproof 4 F pectin D-galacturonic L-galacturonic lactone D-gluconic D- glucuronic Glucuronamide mucic quinic D-saccharic vancomycin tetrazolium violet tetrazolium blue G p-hydroxyphenylacetic sodium methyl pyruvate D-lactic methyl ester L-lactic citric -ketoglutaric D-malic L-malic bromosuccinic nalidixic LiCl K-tellurite H tween-40 -aminobutyric -hydroxybutyric -hydroxy- D,L-butyric -ketobutyric acetoacetic propionic acetic formic aztreonam Na butyrate Na bromate
8 Biolog GenIII plates Used for identification of environmental unknowns Used for confirmation of phenotypes, comparison to literature, comparison to genomes. Amylase dextrin Gelatinase gelatin well more sensitive Kirby-Bauer Nalidixic Acid, Rifamycin API 20E gelatin, lactose, glucose, arabinose, mannitol, melibiose, citrate, arabinose, sucrose, rhamnose, b-methyl-glucoside API 20NE gelatin, lactose, glucose, arabinose, mannose, mannitol, maltose, malic, N-acetyl-glucosamine, gentiobiose, citrate, b-methyl-glucoside API ZYM lactose, melibiose, b-methyl-glucoside, fucose, maltose, cellobiose Biolog GN & GP many
9 Output Included in Lab Report
10 Biolog GenIII plates Used for identification of environmental unknowns 10% correct species, (but >25% novel species) 80-90% correct genus Used for confirmation of phenotypes, comparison to literature, comparison to genomes. Amylase dextrin Gelatinase gelatin well more sensitive Kirby-Bauer Nalidixic Acid, Rifamycin API 20E gelatin, lactose, glucose, arabinose, mannitol, melibiose, citrate, arabinose, sucrose, rhamnose, b-methyl-glucoside API 20NE gelatin, lactose, glucose, arabinose, mannose, mannitol, maltose, malic, N-acetyl-glucosamine, gentiobiose, citrate, b-methyl-glucoside API ZYM lactose, melibiose, b-methyl-glucoside, fucose, maltose, cellobiose Biolog GN & GP many
11 Green = Correct species Yellow = correct genus Biolog GENIII results: Used for identification of environmental unknowns : 10% correct species, (but >25% novel species) 80-90% correct genus
12 Class Phylogenetic Clustering
13 Class Phylogenetic Clustering
14 Research Biolog GenIII plates used to compare novel species candidates to closest relatives.
15 Compare GenIII Results to Rapid Annotation with Subsystem Technology (RAST) Subsystem category expansion show individual subsystems, which can be compared to utilization and resistance patterns.
16 Carbohydrate Utilization RAST Subsystems Subsystem Feature Count Carbohydrates 196 Central carbohydrate metabolism 87 Methylglyoxal Metabolism 7 Pyruvate metabolism II: acetyl CoA, acetogenesis from pyruvate 10 Pyruvate Alanine Serine Interconversions 4 Glycolysis and Gluconeogenesis 14 Entner Doudoroff Pathway 11 Dehydrogenase complexes 13 TCA Cycle 15 Pentose phosphate pathway 8 A Pyruvate metabolism I: anaplerotic reactions, PEP 5 Aminosugars 0 Di and oligosaccharides 6 Maltose and Maltodextrin Utilization 6 Glycoside hydrolases 0 One carbon Metabolism 37 Serine glyoxylate cycle 31 Correlation? B C D E F BiOLOG One carbon metabolism by tetrahydropterines 6 Organic s 0 Fermentation 25 Butanol Biosynthesis 11 G H Acetyl CoA fermentation to Butyrate 14 CO 2 fixation 0 Sugar alcohols 0 Carbohydrates no subcategory 0 Polysaccharides 15 Glycogen metabolism 7 Cellulosome 8 Monosaccharides 28 Mannose Metabolism 6 D ribose utilization 2 Deoxyribose and Deoxynucleoside Catabolism 6 D Galacturonate and D Glucuronate Utilization 14
17 Biolog GenIII Plates Identification Metabolic profiles of related species Genotype vs. Phenotype studies
18 Biolog GenIII plates in the Undergraduate Laboratory FUN get to use multi-channel pipettor Less expensive and provides more info than API tests ~$10 includes GenIII plate, BUG+Blood plate, inoculating fluid, tips, reservoir Web-based database search tool in planning stages ASMCUE A B C D E F BiOLOG G H
19 Questions? From left to right: Back: Dr. Newman, Andrew Gale ( 15), Dillon Snyder ( 14), Tom Sontag ( 14), Logan Mariano ( 13), Me, Clark Thompson ( 13) Front: Jessica Hoffman ( 15), and Jessica Lehman ( 13)
Student Perspectives on the Use of Biolog GenIII Plates in Undergraduate Research and a General Microbiology Course
Student Perspectives on the Use of Biolog GenIII Plates in Undergraduate Research and a General Microbiology Course Jordan Krebs & Jeff Newman Lycoming College Williamsport, PA Talk Contents The use of
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