Dichotomy key

1. Dichotomy key

2. Gram negative Not obligate Intracellular parasite Obligate Intracellular parasite Part 18 Curved rods Part 6 Straight rods or cocci rods cocci Anaerobic Part 9 Aerobic Part 7 Facultative Part 8 Aerobic or Facultative Part 7 & 8 Anaerobic Part 11

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

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

5. Part 8- Gram negative facultative rods and coccobacilli

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

7. Part 8B- Gram negative facultative rods and coccobacilli Lactose not fermented Within 48 h Growth well on common media Proteus Growth very slow or non existent on common media Yersinia 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

8. Glucose not fermented Micrococcus 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 Manitol fermented S. aureus Novobiocin sensitive S. epidermidis Manitol not fermented Novobiocin resistant S. saprophyticus colonies yellow M. luteus colonies not yellow M. roseus Part 14- Gram positive cocci 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

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

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

11. 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.

12. Malachite green staining of Clostridium botulinum highlights spore formation