Identification flow charts

Bergeys Manual of Determinative Bacteriology

All of the unknowns will fall into the following groups in Bergey's Manual of Determinative
Bacteriology (The pink book on the shelf in the laboratory).

GROUP 4
Description: Gram Negative, Aerobic/Microaerophilic rods and cocci
Key differences are: pigments/fluorescent, motility, growth requirements, denitrification,
morphology, and oxidase, read Genera descriptions
Examples: Acinetobacter, Pseudomonas, Beijerinckia, Acetobacter

GROUP 5
Description: Facultatively Anaerobic Gram negative rods
Key differences are: growth factors, morph., gram rxn., oxidase rxn., read Genera descriptions
Examples: Family Enterobacteriaceae and Vibrionaceae

GROUP 17
Description: Gram-Positive Cocci
Key differences are: oxygen requirements, morph., growth requirements (45C and supplements),
read Genera descriptions
Examples: Micrococcus, Staphylococcus, Streptococcus, Enterococcus, Lactococcus, Aerococcus

GROUP 18
Description: Endospore-Forming Gram positive rods and cocci
Key differences are: oxygen requirements, motility, morph, catalase
Examples: Bacillus, Clostridium

GROUP 19
Description: Regular, Nonsporlating Gram positive rods
Key differences are: morph., oxygen require, catalase
Examples: Lactobacillus, Listeria

GROUP 20
Description: Irregular, Nonsporlating Gram-positive rods
Key differences are: catalase, motility, morph., read Genera descriptions
Examples: Actinomyces, Corynebacterium, Arthrobacter, Propionibacterium

GROUP 21
Description: Weakly Gram-Positive Nonsporlating Acid Fast Slender Rods
Key differences are: acid fast, growth
Examples: Mycobacterium

Identification flow charts

Differentiation via Gram stains and cell morphology.

Gram Stain & Morphological

Flowchart
Some Examples

Gram Positive
Cocci

Bacilli
Filamentous

Round in clusters
& tetrads:
Staphylococcus
Micrococcus
Peptococcus

Oval shape in
Chains:

Streptococcus
Peptostreptococcus
Enterococcus

Club-shaped and/or in
palisades:

Corynebacterium
Listeria
Erysipelothrix
Mycobacterium (Acid Fast)
Propionobacterium

Spore-bearing,
large, uniform:
Bacillus
Clostridium

Extensive
branching:

Actinomyces
Arachnia
Nocardia (partially
acid-fast)

Branching rudimentry or absent:

Erysipelothrix
Lactobacillus
Eubacterium

Streptomyces

Gram Negative
Cocci

Neisseria
Veillonella

Bacilli

Medium-sized
cocco-bacillary:
Acinetobacter
Moraxella

Tiny-coccobacillary:
Brucella
Bordetella
Bacteroides

Pleomorphic
coccobacillary
& filamentous:

Haemophilus
Bacteroides
Pasteurella
Francisella
Actinobacillus
Eikenella
Cardiobacterium
Flavobacterium

Exaggerated
pointed ends:

Fusobacterium
nucleatum

Coiled &
sphero-plastic:
Streptobacillus
Fusobacterium

Curved,
comma-shaped:
Vibrio
Campylobacter
Spirillium

Uniformly
Bacillary:

Enterobacteriaceae
Pseudomonas
Aeromonas
Alcaligenes
Chromobacterium

Identification flow charts

Gram Positive Rods ID Flowchart

Gram Positive Rods

Bacillus spp.
Clostrididium spp.
Corynebacterium spp.
Lactobacillus spp.
Mycobacterium spp.

Mycobacterium smegmatis
Spore Forming
+

Corynebacterium spp.
Lactobacillus spp.
Mycobacterium spp.

Bacillus spp.
Clostridium spp

Acid Fast

Strict Anaerobes

Corynebacterium spp
Lactobacillus spp.
-

+
Clostridium spp.

You sure you have this?

Go to Bergey's.

Bacillus spp.

See separate flowchart.

Catalase
+

Corynebacterium spp.

Lactobacillus spp.

Starch Hydrolysis
Glucose Ferm. Activity
+
Corynebacterium kutsceri

Acid

Acid & Gas

Corynebacterium xerosis

Lactobacillus fermenti

Lactobacillus casei
Lactobacillus delbrueckii

Mannitol
+
Lactobacillus casei

Lactobacillus delbrueckii

Identification flow charts

Bacillus spp. ID Flowchart

Bacillus spp.
B. subtilis
B. cereus
B. megaterium
B. stearothermophilus
B. polymyxa
B. mycoides
B. macerans
B. thuringiensis
B. macquariensis
B. licheniformis
B. lentus
B. alvei
B. anthracis
B. alcalophilus
B. coagulans
B. brevis
B. circulans
B. firmus
B. pantothenticus

Starch Hydrolysis
(amylase)

+
(add creatine as catalyst
w/ reagents)

+
B. subtilis
B. cereus
B. polymyxa
B. mycoides
B. thuringiensis
B. licheniformis
B. alvei
B. anthracis
B. coagulans

(makes insecticidal
protein)

B. mycoides

(round spore, rest

are oval)

B. popilliae
B. pasteurii
B. lentimorbus
B. azotoformans
B. badius
B. insolitus
B. larvae
B. laterosporus

VP

B. megaterium
B. stearothermophilus
B. macerans
B. pantothenticus
B. macquariensis
B. lentus
B. alcalophilus
B. badius
B. brevis
B. circulans

B. popilliae

Catalase

(spore is not
round, rest are)

(colony has a
rhizoidal appearance)

B. azotoformans

Swollen Cell

Swollen Cell

B. stearothermophilus

B. megaterium

(growth at 55C-rest neg.)

(citrate pos.)

B. macerans
B. pantothenticus
B. macquariensis
B. lentus

B. badius

(citrate neg.)

B. sphaericus
B. pasteurii
B. laterosporus

(oval spore,
rest are round)

B. alcalophilus
B. brevis
B. circulans

(Motile - It is this
one )

B. larvae
(gelatinase pos.)
B. popilliae
(gelatinase neg.)

(containing spore)

(catalase neg.-rest pos.)

B. cereus

B. insolitus

(acid from glucose)

B. marinus

(acid from glucose)

Nitrate Reduction

B. pasteurii

B. sphaericus

Citrate
6.5% NaCl Growth

+
B. coagulans
B. licheniformis
B. subtilis

B. insolitus
B. laterosporus
B. pasteurii
B. marinus
B. sphaericus

B. subtilis
B. polymyxa
B. lichenifomis
B. alvei
B. coagulans

Citrate

B. badius

(containing spore)

+
B. thuringiensis

B. azotoformans
B. larvae
B. pasteurii

(grows at 55C, rest no)

(the width)

(non-motile)

(VP is pos., rest are neg.)

B. marinus
B. sphaericus
B. schlegelii

Cell Diamenter ! 1"m

B. anthracis

B. pumilus

B. polymyxa

(manitol pos.)

B. alvei

(manitol neg.)

+
B. macerans
B. macquariensis
B. alcalophilus
B. brevis
B. circulans

B. pantothenticus

(acid via arabinose neg.)

B. circulans

(acid via arabinose pos.)

6.5% NaCl Growth

+
B. subtilis

(no growth at 55C)

Acid from Arabinose

B. coagulans

B. licheniformis

B. macquariensis

(growth at 55C)

(methyl red pos.)

B. brevis

(methyl red neg.)

B. macerans

(gas from glucose pos.)

B. alcalophilus

(gas from glucose neg.,

no growth < pH 7.0)

B. circulans

(gas from glucose neg.,

growth < pH 7.0)

Identification flow charts

Gram Positive Cocci ID Flowchart

Gram Positive Cocci

Catalase

+
Micrococcus spp.
Staphylococcus spp.

Streptococcus spp.
(see Bergeys)

Micrococcus spp.
Staphylococcus spp.
Streptococcus spp.
Enterococcus spp.

Bile Esculin

(growth with esculin hydrolysis)

Streptococcus pneumoniae
Streptococcus mitis
Streptococcus pyogenes Group A
Streptococcus spp. Group B
Enterococcus spp.
Streptococcus spp.

Enterococcus faecalis
Enterococcus faecium
Enterococcus hirae
+ Enterococcus mundtii

Enterococcus spp.
Streptococcus spp.

Growth w/ Tellurite
Gamma (!)

Mannitol Fermentation
Hemolysis

+
Staphylococcus aureus

Enterococcus faecalis

Enterococcus faecium

(mannitol pos., white colony)

Staphylococcus spp.
Micrococcus spp.

Enterococcus hirae
(mannitol neg.)

Enterococcus mundtii

(mannitol pos., yellow colony)

Beta (")
Yellow Pigment (Colony)

Alpha (#)

Streptococcus pyogenes Group A

Streptococcus spp. Group B

+
Micrococcus spp.

Staphylococcus spp.

Pyrrolidonearylamidase
(PYR Test)

Streptococcus pneumoniae
Streptococcus mitis

Novobiocin Sensitivity

Glucose Fermentation
+
Micrococcus varians

Yes

Streptococcus pyogenes Group A

Staphylococcus epidermidis

- Micrococcus luteus

Streptococcus spp. Group B

(Streptococcus agalactiae)

No
Staphylococcus saprophyticus

Optochin Sensitivity

Yes
Streptococcus pneumoniae

No
Streptococcus mitis

Identification flow charts

Gram Negative Rods ID Flowchart

Gram Negative Rods

Oxidase Test

Enterobacteriaceae
(See Separate Flowchartt)

Aeromonas, Pseudomonas, Vibrio

+ Acid

Vibrio spp., Aeromonas spp.

Na+ Required for Growth

Glucose Fermentation

- Acid

Pseudomonas spp.

Fluorescent Diffusible Yellow Pigment

(Fluorescein)use Psuedo F Agar

Aeromonas spp.

VP

Vibrio spp.

+
A. hydrophila
A. salmonicida

Luminescent

Other Aeromonas spp.

(See Bergey's)

(subsp. masoucida)

P. aeruginosa
P. chlororaphis
P. cichorii
P. fluorescens
P. putida
P. syringae

Other Pseudomonas spp.

(See Bergey's)

(Oxidase Neg.)

A. sobria

(no gas from glucose)

A. veronii

+
V. fischeri
V. logei
V. orientalis
V. splendidius

Non-Fluorescent Diffusible Blue Pigment

Other Vibrio spp.

(See Bergey's)

(Pyocyanin)use Psuedo P Agar

Motility
-

Pigment

(Yellow Colony)

A. hydrophila
A. veronii

V. fischeri
V. logei

V. orientalis
V. splendidius

A. salmonicida

(subsp. masoucida)

P. chlororaphis
P. cichorii
P. fluorescens
P. putida
P. syringae

+
P. aeruginosa

H2S Production
Nitrate Reduction

+
A. hydrophila

30C Growth

A. veronii

37C Growth

+
V. fischeri

P. chlororaphis

+
V. logei

V. orientalis

(Colony Pigmented)

P. fluorescens

(To differentiate Biovars

do a lecithinase test)

V. splendidius

P. putida

(Lecithinase Neg.)

P. syringae

(Oxidase Neg.)

P. cichorii

(Lecithinase Pos.)

Identification flow charts

Family Enterobacteriaceae Lactose Positive ID Flowchart

Family Enterobacteriaceae
Lactose Fermentation

+
Citrobacter diversus
Citrobacter freundii
Enterobacter aerogenes
Enterobacter cloacae
Enterobacter amnigenus
Enterobacter intermedius
Erwinia carotovora

Erwinia chrysanthemi
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae

See Lactose Negative Flowchart

for Enterobacteriaceae

(subsp. ozaenae and pneumoniae)

Serratia fonticola
Serratia rubidaea

Indole
The Rest

+
Citrobacter diversus
Escherichia coli
Erwinia chrysanthemi
Klebsiella oxytoca

MR-VP

+/+

-/+

Klebsiella pneumoniae
(subsp. pneumoniae)

Enterobacter intermedius

+/Citrobacter freundii
Serratia fonticola
Klebsiella pneumoniae

Citrate

Enterobacter aerogenes
Enterobacter cloacae
Enterobacter amnigenus
Erwinia carotovora
Serratia rubidaea

(subsp. ozaenae)

Lysine Decarboxylase

Citrobacter diversus

Escherichia coli

Klebsiella pneumoniae subsp. pneumoniae

Enterobacter cloacae

Serratia rubidaea

Enterobacter amnigenus

Non-motile, No Pigment

(VP neg.)

Erwinia chrysanthemi

Acid from sorbitol

Motile, Red pigment

(VP and H2S pos.)

(VP pos and H2S neg.)

No acid from sorbitol

Enterobacter aerogenes

H2S Production

Klebsiella oxytoca

Motile, No pigment

??
Erwinia carotovora

Lysine unknown but Gelatinase

positive; none above are positive.

+
Citrobacter freundii

Serratia fonticola
Motility pos.

Klebsiella pneumoniae subsp. ozaenae

Motility neg.

Identification flow charts

Family Enterobacteriaceae Lactose Negative ID Flowchart

Family Enterobacteriaceae Continued

Lactose Negative Flow Chart
Edwardsiella tarda
Erwinia cacticida
Morganella morganii
Proteus mirabilis
Proteus penneri
Proteus vulgaris
Providencia stuartii
Salmonella bongori
Salmonella enterica

Edwardsiella tarda
Morganella morganii
Proteus vulgaris
Providencia stuartii

Serratia marcescens
Serratia liquefaciens
Shigella spp. (boydii, dysenteriae, flexneri)
Shigella sonnei
Yersinia enterocolitica
Yersinia pestis
Yersinia pseudotuberculosis

Erwinia cacticida
Proteus mirabilis
Proteus penneri
Salmonella bongori
Salmonella enterica
Serratia marcescens
Serratia liquefaciens
Shigella sonnei
Yersinia pestis
Yersinia pseudotuberculosis

Indole Test
Urease

??
Please See Bergeys to ID:
Shigella spp.

Proteus mirabilis

Yersinia enterocolitica

Proteus penneri

Erwinia cacticida
Salmonella bongori
Salmonella enterica
Serratia marcescens
Serratia liquefaciens
Shigella sonnei
Yersinia pestis

Ornithine decarb. pos.

(boydii, dysenteriae, flexneri)

Ornithine decarb. neg., motile

H2S

Yersinia pseudotuberculosis

Ornithine decarb. neg., non-motile

+
Edwardsiella tarda
Proteus vulgaris

Morganella morganii
Providencia stuartii

Motility

Erwinia cacticida

Shigella sonnei
Yersinia pestis

(lysine decarb. neg., the rest pos.)

Urease

Salmonella bongori
Salmonella enterica
Serratia marcescens
Serratia liquefaciens

Ornithine Decarboxylase

Proteus vulgaris

Providencia stuartii
-

Edwardsiella tarda

Ornithine Decarboxylase

Morganella morganii

H2S

+
-

+
Salmonella bongori

Serratia marcescens

Salmonella enterica

Serratia liquefaciens

KCN growth pos.

KCN growth neg.

Red pigment

No Red pigment

(It is this one)

Shigella sonnei

Yersinia pestis