Dichotomy key unknown Gram Gram Gram negative Obligate

Dichotomy key unknown Gram - Gram+

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

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

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

Part 8 Gram negative facultative rods and coccobacilli Lactose fermented within 48 h Part 8 A Lactose not fermented within 48 h Part 8 B

Part 8 AGram negative facultative rods and coccobacilli Lactose fermented Within 48 h Indole present Indole not present Citrate not utilized Eschrichia coli H 2 S produced C. freundii Red methyl Positive Methyl red Negative Citrobacter Enterobacter H 2 S not produced C. intermedius Gas not produced by fermentation of glycerol E. cloacae Urease Negative and Motile E. aerogenes

Part 8 BGram negative facultative rods and coccobacilli Lactose not fermented Within 48 h Indole present H 2 S Positive Urease Positive Proteus vulgaris Indole not present H 2 S Negative Urease Positive Proteus mirabilis Urease Positive Proteus rettgeri Urease Negative Edwardsiella tarda Mannitol not fermented Morganella morganii Has Motility H 2 S Positive P. rettgeri Urease Negative Does not have Motility H 2 S Negative Serratia marcescens Shigella sonnei

Part 14 Gram positive cocci Cells in irregular clusters Tetrads or packs of 8. Catalase positive Glucose not fermented Micrococcus Glucose fermented Staphylococcus Manitol fermented S. aureus Manitol not fermented Novobiocin sensitive S. epidermidis Cells in pairs or chains Catalase negative Streptococcus colonies yellow M. luteus Novobiocin resistant S. saprophyticus colonies not yellow M. roseus Grow well at 450 c and In 6. 5% Na. Cl broth E. faecalis Does not grow well at 450 c and in 6. 5% Na. Cl broth S. lactis

Part 15 Endospores- forming rods and cocci Cells sperical, In sarcinal packs Sporosarcinal urea Cells are rod shaped Bacillus Starch hydrolysis VP Positive Citrase Produced B. subtilis Starch not hydrolysis VP Negative B. megaterium Citrase Produced B. cereus Catalase produced B. barinus Catalase not produced B. larvae

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

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