Identification flow charts

Bergey’s 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 (45°C 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

1

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 Streptomyces
acid-fast)

Branching rudimentry or absent:
Erysipelothrix Lactobacillus Eubacterium

Gram Negative
Cocci Bacilli

Neisseria Veillonella

Medium-sized cocco-bacillary:
Acinetobacter Moraxella

Tiny-coccobacillary:
Brucella Bordetella Bacteroides

Pleomorphic coccobacillary & filamentous:

Exaggerated pointed ends:

Haemophilus Bacteroides Pasteurella Francisella Actinobacillus Eikenella Cardiobacterium Flavobacterium

Fusobacterium nucleatum

Curved, comma-shaped:
Vibrio Campylobacter Spirillium

Uniformly Bacillary:

Coiled & sphero-plastic:
Streptobacillus Fusobacterium

Enterobacteriaceae Pseudomonas Aeromonas Alcaligenes Chromobacterium

2

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 + Bacillus spp. Clostridium spp Corynebacterium spp. Lactobacillus spp. Mycobacterium spp. Acid Fast +

Strict Anaerobes +
You sure you have this? Go to Bergey's.

Corynebacterium spp Lactobacillus spp. -

Clostridium spp.

See separate flowchart.

Bacillus spp.

Catalase + Corynebacterium spp. Lactobacillus spp.

Starch Hydrolysis Glucose Ferm. Activity + Corynebacterium kutsceri Corynebacterium xerosis
Acid & Gas Acid

Lactobacillus fermenti

Lactobacillus casei Lactobacillus delbrueckii

Mannitol + Lactobacillus casei Lactobacillus delbrueckii

3

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)

B. pumilus

(VP is pos., rest are neg.)

+
(add creatine as catalyst w/ reagents)

B. marinus B. sphaericus B. schlegelii

(grows at 55°C, rest no)

B. azotoformans B. larvae B. pasteurii B. popilliae

VP

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

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

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

(round spore, rest are oval)

-

Catalase

Citrate

B. badius

+

+
B. azotoformans

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

(spore is not round, rest are)

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

Cell Diamenter ! 1"m
(the width)

(containing spore)

Swollen Cell

(containing spore)

Swollen Cell

+
B. anthracis
(non-motile) (makes insecticidal protein) (colony has a rhizoidal appearance)

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

+
B. stearothermophilus B. macerans B. pantothenticus B. macquariensis B. lentus B. alcalophilus B. brevis B. circulans
(growth at 55°C-rest neg.)

B. megaterium
(citrate pos.)

+
B. sphaericus B. pasteurii B. laterosporus

B. insolitus B. marinus
(acid from glucose) (acid from glucose)

B. thuringiensis B. mycoides B. cereus

B. badius

(citrate neg.)

(catalase neg.-rest pos.)

(oval spore, rest are round)

(Motile - It is this one )

Nitrate Reduction

Citrate 6.5% NaCl Growth

+
B. pasteurii

B. sphaericus

+
B. coagulans B. licheniformis B. subtilis B. polymyxa B. alvei

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

B. pantothenticus B. circulans
(acid via arabinose neg.) (acid via arabinose pos.)

(manitol pos.) (manitol neg.)

6.5% NaCl Growth

+
B. subtilis
(no growth at 55°C) (growth at 55°C)

B. coagulans

Acid from Arabinose

+

B. macerans

(gas from glucose pos.) (gas from glucose neg., no growth < pH 7.0) (gas from glucose neg., growth < pH 7.0)

B. macquariensis
(methyl red pos.)

B. alcalophilus B. circulans

B. licheniformis

B. brevis

(methyl red neg.)

4

Identification flow charts

Gram Positive Cocci ID Flowchart

Gram Positive Cocci
Micrococcus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

Bile Esculin

Streptococcus spp. (see Bergey’s)

(growth with esculin hydrolysis)

Catalase

-

Enterococcus faecalis Enterococcus faecium Enterococcus hirae + Enterococcus mundtii

+
Micrococcus spp. Staphylococcus spp.

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

Enterococcus spp. Streptococcus spp.

Growth w/ Tellurite Gamma (!)

Mannitol Fermentation +
Staphylococcus aureus

Staphylococcus spp. Micrococcus spp.

Hemolysis

+
Enterococcus faecalis Enterococcus faecium Enterococcus hirae
(mannitol neg.)

(mannitol pos., white colony)

Enterococcus mundtii

Beta (") Yellow Pigment (Colony) +
Micrococcus spp.

(mannitol pos., yellow colony)

Alpha (#)

Staphylococcus spp.

Streptococcus pyogenes Group A Streptococcus spp. Group B

Pyrrolidonearylamidase
(PYR Test)

Glucose Fermentation +
Micrococcus varians

Novobiocin Sensitivity

Streptococcus pneumoniae Streptococcus mitis

Yes
Staphylococcus epidermidis

Streptococcus pyogenes Group A

+

No
Streptococcus spp. Group B (Streptococcus agalactiae)

- Micrococcus luteus

Staphylococcus saprophyticus

Optochin Sensitivity

Yes
Streptococcus pneumoniae

No
Streptococcus mitis

5

Identification flow charts

Gram Negative Rods ID Flowchart

Gram Negative Rods
+
Aeromonas, Pseudomonas, Vibrio

Oxidase Test

Enterobacteriaceae (See Separate Flowchartt)

Vibrio spp., Aeromonas spp.

+ Acid

Glucose Fermentation

- Acid

Pseudomonas spp.

Na+ Required for Growth

Fluorescent Diffusible Yellow Pigment Aeromonas spp. (Fluorescein)—use Psuedo F Agar

+
Vibrio spp.

+
VP Other Aeromonas spp. (See Bergey's) P. aeruginosa P. chlororaphis P. cichorii P. fluorescens P. putida P. syringae
(Oxidase Neg.)

Other Pseudomonas spp. (See Bergey's)

+
Luminescent
A. hydrophila A. salmonicida A. sobria
(subsp. masoucida) (no gas from glucose)

A. veronii

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

Other Vibrio spp. (See Bergey's)

Non-Fluorescent Diffusible Blue Pigment
(Pyocyanin)—use Psuedo P Agar

Motility Pigment P. chlororaphis P. cichorii P. fluorescens P. putida P. syringae

+
P. aeruginosa

(Yellow Colony)

+
A. hydrophila A. veronii A. salmonicida
(subsp. masoucida)

+
V. fischeri V. logei

V. orientalis V. splendidius

H2S Production Nitrate Reduction

+
A. hydrophila

A. veronii

30°C Growth

37°C Growth

+
P. chlororaphis
(Colony Pigmented)

P. putida
(Lecithinase Neg.) (Oxidase Neg.)

+
V. fischeri

+
V. logei V. orientalis

V. splendidius

P. fluorescens

P. syringae P. cichorii

(To differentiate Biovars do a lecithinase test)

(Lecithinase Pos.)

6

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 Serratia fonticola Serratia rubidaea

See Lactose Negative Flowchart for Enterobacteriaceae

(subsp. ozaenae and pneumoniae)

Indole + Citrobacter diversus Escherichia coli Erwinia chrysanthemi Klebsiella oxytoca The Rest

MR-VP

+/+
Enterobacter intermedius

-/+

Klebsiella pneumoniae
(subsp. pneumoniae)

+/Citrate
Citrobacter freundii Serratia fonticola Klebsiella pneumoniae
(subsp. ozaenae)

Enterobacter aerogenes Enterobacter cloacae Enterobacter amnigenus Erwinia carotovora Serratia rubidaea

Lysine Decarboxylase
+ Klebsiella pneumoniae subsp. pneumoniae
Non-motile, No Pigment Motile, Red pigment Motile, No pigment

+ Citrobacter diversus
(VP neg.)

Escherichia coli Serratia rubidaea

Enterobacter cloacae
Acid from sorbitol

Erwinia chrysanthemi Klebsiella oxytoca
(VP and H2S pos.) (VP pos and H2S neg.)

Enterobacter amnigenus
No acid from sorbitol

H2S Production

Enterobacter aerogenes

?? Erwinia carotovora

Lysine unknown but Gelatinase positive; none above are positive.

+ Citrobacter freundii
Motility pos.

Serratia fonticola Klebsiella pneumoniae subsp. ozaenae
Motility neg.

7

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 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

-

Edwardsiella tarda Morganella morganii Proteus vulgaris Providencia stuartii

+
??
Please See Bergey’s to ID: Shigella spp.
(boydii, dysenteriae, flexneri)

Indole Test Urease

+
Proteus mirabilis Proteus penneri
Ornithine decarb. pos. Ornithine decarb. neg., motile Ornithine decarb. neg., non-motile

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

Yersinia enterocolitica H2S

Yersinia pseudotuberculosis

+
Edwardsiella tarda Proteus vulgaris

Morganella morganii Providencia stuartii Erwinia cacticida Urease Ornithine Decarboxylase Motility

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

Shigella sonnei Yersinia pestis

+
Proteus vulgaris Edwardsiella tarda

+
Providencia stuartii Morganella morganii

Salmonella bongori Salmonella choleraesuis Serratia marcescens Serratia liquefaciens

Ornithine Decarboxylase H2S

+ +
Salmonella bongori
KCN growth pos.

Serratia marcescens
Red pigment

Shigella sonnei

Yersinia pestis

Salmonella enterica
KCN growth neg.

Serratia liquefaciens
No Red pigment

(It is this one)

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