BIOL 2010 Lecture 10-12 Supplemental Slides (PDF)

BIOL 2010 Lecture 10 - Enterobacteriaceae Supplemental Slides from Lecture November 4, 2024 Eosin Methylene Blue (EMB) Agar Encourages growth of some gram-negative bacteria – fecal and nonfecal coliforms (GNB, non-spore forming, motile/non-motile bacteria that can ferment lactose with the pr...

BIOL 2010 Lecture 10 - Enterobacteriaceae Supplemental Slides from Lecture November 4, 2024 Eosin Methylene Blue (EMB) Agar Encourages growth of some gram-negative bacteria – fecal and nonfecal coliforms (GNB, non-spore forming, motile/non-motile bacteria that can ferment lactose with the production of acid and gas- used as an indicator for sanitary quality in water/food) Lactose Fermenters: Escherichia, Klebsiella, Enterobacter, Serratia (LLF) Acidify the media, reduce the pH, dye produces – Dark purple complex associated with a green metallic sheen Eosin Y/Methylene blue (pH indicators): at low pH; form dark purple precipitate and inhibit GPB E.coli = Blue black colonies; green metallic sheen Green metallic sheen indicates vigorous lactose and/or sucrose fermentation ability typical of fecal coliform LLF: produce less acid = brown-pink colonies Non-Lactose Fermenters: increase the pH by deamination of proteins colourless, light pink colonies Salmonella spp. = colourless, amber Hektoen Enteric Agar Designed for Salmonella/Shigella A: Non-pathogens: Lactose Fermenter, low pH (acidic) A B B: Pathogen (NLF): Blue/green with dark centers – Salmonella spp. Produce H2S C C: Pathogen (NLF): Blue/green – Shigella spp. do not produce H2S XLD – Xylose Lysine Deoxychocolate Selectively promotes and differentiates Salmonella and Shigella Inhibits other enteric pathogens based on xylose fermentation xylose with a phenol red indicator + sucrose + lactose (in excess) Lysine detects lysine decarboxylation Thiosulfate detects hydrogen sulfide A B  A: Non-pathogen (LF): Fermenters/those not producing lysine decarboxylase Yellow/Orange E.coli, Citrobacter spp. B. Pathogen (NLF) Red colony, black center C Salmonella spp.  C. Pathogen (NLF) colourless/red colony Shigella spp. BIOL 2010 Lecture 12 – Non-fermenting Bacilli (NFB) Supplemental Slides from Lecture November 4, 2024 Dark/Purple = E. coli (LF) A Pseudomonas aeruginosa – cultural characteristics Odour – fruity, grape-like Corn-tortilla-like odour Growth on MAC (Fig. A) Non-lactose fermenter B Pigment may be present Growth on BAP (typical colonies) –(Fig. B) Colonies are large with an irregular shape (serrated edges); confluent growth Metallic (blue/green) sheen; scaling appearance “alligator skin” morphology Gun-metal colour and spreading, flattened topology Beta Hemolytic Turquoise-blue diffusible pigment Pseudomonas aeruginosa producing pyocyanin Pseudomonas aeruginosa - ID Motile (single polar flagella) Oxidase - Positive Catalase - Positive Nitrate - Positive (usually beyond nitrite to nitrogen gas) left: nitrate to Nitrogen gas Right: reduction of nitrite to nitrogen gas (more gas produced from nitrite than nitrate) Arginine Dihydrolase (ADH) positive Citrate - Positive OF Test - Non-Fermenter (Oxidizer of Carbohydrates) TSI - Alk/Alk or K/K (2nd tube) Growth at 42C - Positive  Motile Non-motile Motility Motility B Medium: contains TTC (2,3,5- triphenyltetrazolium chloride) indicator motility difficult to observe in NFB as mostly tend to grow in the upper (most aerobic) portion of the tube Tetrazolium detects motility as organisms that reduce TTC appear red along the stab line as well as in the area the cells have migrated making it easier to ID Motile (A) = red stab line; red upper portion where cells have migrated to oxygen exposure Non-motile (B) = red along the inoculation line; no migration Tube 1 Tube 2 OF Test OF Media: Hugh-Leifson oxidative fermentative (OF) media Tube 1: overlaid with mineral oil Excludes exposure to atmospheric oxygen Tube 2: no oil, open to atmospheric oxygen Acid (yellow) seen on top portion of tube 2 Green seen on bottom (unable to oxidize in absence of oxygen) The organism is capable of oxidizing glucose but incapable of fermenting glucose  Tube Tube 2 1 TSI Tube 1: K/A - Alkaline slant/Acid butt Fermentation of glucose, no fermentation of lactose = NLF Enterobacteriaceae Tube 2: K/K – Alkaline slant/Alkaline butt NFB- unable to utilize lactose, glucose, sucrose in TSI