Dichotomy key

Gram negative Anaerobic Part 11 Obligate Intracellular parasite Not obligate Aerobic or Facultative Part 7 & 8 rods Curved rods Part 6 Straight rods or cocci Aerobic Part 7 Facultative Part 8 Part 9 cocci Gram negative

Gram positive Cocci Rods or filaments Straight rods Part 16 Endospores produced Part 15 Irregular rods Filaments Part 17 Gram positive Endospores not produced Produced Part 14

Part 7 - Gram negative aerobic rods and cocci aerobic rods / cocci Fluorescent pigments not on common media Alcaligens No obvious pigments by agar grown colonies Yellow cream become Brown with aging Litmus milk –alkaline A. faecalis Litmus milk – unchanged A. aquamarinus Colonies –yellow slimy, glistening A. paradoxus Colonies cream Initially then brown A. eutrophus Fluorescent pigments on common media Pseudomonas Reduce nitrate Gelatin hydrolyzed Does not reduce nitrate Gelatin not hydrolyzed Pseudomonas aeruginosa stutzeri putida

Part 8- Gram negative facultative rods and coccobacilli

Part 8A- Gram negative facultative rods and coccobacilli Lactose fermented Within 48 h Citrate not utilized Eschrichia coli Citrate utilized Red methyl Positive Citrobacter Methyl red Negative Enterobacter Gas not produced by fermentation of glycerol E. cloacae Gas produced by Fermentation of glycerol E. aerogenes H2S produced Indole negative C. freundii H2S not produced Indole variable C. intermedius

Part 8B- Gram negative facultative rods and coccobacilli Lactose not fermented Within 48 h Growth very slow or non existent on common media Yersinia Growth well on common media Proteus Urease positive Urease Negative P. inconstans Gelatin hydrolyzed Gelatin not hydrolyzed Maltose fermented P.vulgaris Maltose not fermented P.mirabilis Mannitol fermented P.rettgeri Mannitol not fermented M. morganii

Part 14- Gram positive cocci Cells in irregular clusters Tetrads or packs of 8. Catalase positive Cells in pairs or chains Catalase negative Streptococcus Glucose fermented Staphylococcus Glucose not fermented Micrococcus colonies yellow M. luteus colonies not yellow M. roseus Manitol fermented S. aureus Manitol not fermented Novobiocin sensitive S. epidermidis Novobiocin resistant S. saprophyticus Grow well at 450c and In 6.5% NaCl broth E. faecalis Does not grow well at 450c and in 6.5% NaCl broth S. lactis

Part 15- Endospores- forming rods and cocci Cells are rod shaped Bacillus Cells sperical, In sarcinal packs Sporosarcinal urea Spors ellipsoid and centrally located Butanediol produced B. cereus Butanediol not produced B. megaterium

Part 16- Gram positive, asporogenous, rod shaped bacteria No gas production from glucose fermentation L. casei Gram positive, asporogenous, rod shaped bacteria Lactobacillus Gas production from L. brevis

Spore stain-malachite green Prepare two smears from a colony of the spore-formers. Cut a piece of blotting paper the size of the smears, but small enough so it doesn't hang over the edges of the slide. Place the slide over the boiling streamer and lay the paper on top of the smears. Flood the paper with the malachite green stain (CARCINOGEN) and time for 3 min. Keep the PAPER WET with the stain if it begins to dry. After 3 min. remove the slide and lift off the paper with your loop and discard. Gently wash the slide with water. Flood the slide with the counter-stain safranin to stain the vegetative cells. After one minute, wash off the safranin, blot the slide dry and examine under the microscope.

Malachite green staining of Clostridium botulinum highlights spore formation