ORGANISM IS GRAM-POSITIVE: GO TO SECTION I

ORGANISM IS GRAM-NEGATIVE: GO TO SECTION II

I. ALL GRAM POSITIVE ORGANISMS

A. All Gram-Positive Organisms Studied

1. Organism is a coccus: Go to Section B.

2. Organism is a bacillus: Go to Section E.

B. All Gram-Positive Cocci

1. Catalase positive; some arranged in grape-like clusters and others in regular arrangements of 2,4,6 or 8: Go to Section C.

2. Catalase negative; most characteristic arrangement is in chains although chain length can be as small as 2; some members are not true cocci appearing as cocco-bacilli: Go to Section D.

C. Micrococceaceae

1. Random arrangement in grape-like clusters; nutrient agar colonies with flat generally white in color with even margins; luxurious growth on 6.5% salt media; characteristically commensals or pathogens of vertebrates including man:

Go to Section C.3.

2. Regular arrangement in groups of 2,4,6, or 8; moderate to light growth on 6.5% salt media; nutrient agar colonies moderate to small domed, round, entire colonies; most are gray but some can be highly pigmented when cultured at room temperature; characteristically saprophytic organisms: Go to Section C.4

3. Staphylococcus

a. Ferments mannitol: Organism: Staphylococcus aureus

b. Does not ferment mannitol: Organism: Staphylococcus epidermidis

4. Micrococcus

a. Urease positive: Ferments maltose and mannitol; gelatinase positive. Arrangement usually in groups of 4; Organism: Micrococcus ureae

b. Urease Negative: Does not ferment mannitol; gelatinase negative. Arrangement most often observed is groups of 4, 6 & 8 from colonies on nutrient media.

Organism: Micrococcus luteus

D. Streptococceaceae

1. Blood hemolysis

a. b-hemolytic: Go to D.2

b. a-hemolytic: Go to D.3

c. g-hemolytic: Go to D.4

2. b-hemolytic Streptococci

a. Ferments mannitol: Organism: Streptococcus pyogenes

b. Does not ferment mannitol: Organism: Streptococcus agalactiae

3. a-hemolytic Streptococci

a. Almost always observed in Gram stains as diplococci; ferments inulin & raffinose (Ask instructor for results of this test if media is not available):

Organism: Streptococcus pneumoniae

b. Appears in short to medium numbered chains; does not ferment inulin & raffinose (Ask instructor for results of this test if media is not available).

Organism: Streptococcus salivarius

4. g-hemolytic streptococci

a. Salt tolerant; ferments mannitol: Organism: Enterococcus faecalis

b. Salt intolerant; does not ferment mannitol:

Organism: Streptococcus mutans

E. All Gram-Positive Bacilli Studied

1. Cellular Morphology

a. Cells regular shaped looking like rectangles: Go To Section E.2

b. Cells irregular in shape: Go To Section E.5

2. Catalase Test

a. Catalase and amylase positive: Go To Section E.3

b. Catalase and amylase negative: Go to Section E.4

3. Bacillus: All members form endospores that are best observed in Gram stains of older

cultures.

a. Ferments mannitol: Organism: Bacillus subtilis

b. Does not ferment mannitol: Organism: Bacillus thuringiensis

4. Lactobacillus

a. Ferments raffinose: Organism: Lactobacillus casei

b. Does not ferment raffinose: Organism: Lactobacillus acidophilus

5. Cellular Morphology

a. Cells observed in a variety of polymorphic forms: X, Y and V shaped very common.

Go To Section E.6

b. Cells observed as long slender pointed rods in chains resembling long strands like fungal mycelia. Go To Section E.7

6. Corynebacterium

a. Urease positive Organism: Corynebacterium

pseudodiphtheriticum

b. Urease negative Organism: Corynebacterium xerosis

7. Mycobacterium

a. Ferments mannose and rhamnose. (Ask instructor for results of this test if media is not available.) Organism: Mycobacterium phlei

b. Does not ferment mannose and rhamnose. (Ask instructor for results of this test if media is not available) Organism: Mycobacterium smegmatis

II. ALL GRAM NEGATIVE ORGANISMS

A. Organism is a coccus: go to Section C.

B. Organism is a bacillus or coccobacillus: Go to Section D.

C. Neisseria

1. Lactose fermentation

a. Does not ferment lactose: Organism: Neisseria gonorrhoeae

b. Ferments lactose: Go To C.2

2. Sucrose fermentation

a. Does not ferment sucrose Organism: Neisseria meningitidis

b. Ferments sucrose Organism: Neisseria sicca

D. All Gram-negative bacilli

1. Ferments Lactose: Go to Section E

2. Does not ferment Lactose: Go to Section F

E. All Lactose Fermenting bacilli

1. Indole test:

a. Indole positive; citrate negative Organism: Escherichia coli

b. Indole negative: Go to Section E.2

2. Hydrogen Sulfide Production

a. H₂S produced; citrate positive Organism: Citrobacter freundii

b. H₂S not produced: Go to Section E.3

3. Urease Test

a. Urease positive Organism: Klebsiella pneumoniae

b. Urease negative Organism: Enterobacter aerogenes

F. Lactose Nonfermenters

1. Produce hydrogen sulfide: Go to Section G

2. Do not produce hydrogen sulfide: Go to Section J

G. Hydrogen Sulfide Producers

1. Urease positive: Go to Section H.

2. Urease negative: Go to Section I.

H. Proteus

1. Indole positive Organism: Proteus vulgaris

2. Indole negative Organism: Proteus mirabilis

I. Salmonella

1. Citrate negative: Organism: Salmonella typhi

2. Citrate positive: Organism: Salmonella typhimurium

J. Hydrogen Sulfide Negative Organisms

1. Oxidase negative; ferments glucose: Go to Section K.

2. Oxidase positive; does not ferment glucose or other carbohydrates. Go to Section N.

K. Oxidase Negative Glucose Fermenting Organisms

1. Citrate positive: Go to Section L.

2. Citrate negative: Go to Section M.

L. Serratia Organism: Serratia marcescens

M. Shigella

1. Indole positive: Organism: Shigella flexneri

2. Indole negative: Organism: Shigella sonnei

N. Oxidase Positive Nonfermenting Organisms

1. Gelatinase positive: Organism: Pseudomonas aeruginosa

2. Gelatinase negative: Organism: Alcaligenes faecalis