1. Dichotomy key

2. 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

3. 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

4. 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

5. Part 8- Gram negative facultative rods and coccobacilli

6. 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

7. 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

8. 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

9. 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

10. 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

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