Bergey's Manual of Determinative Bacteriology

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 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

2

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

3

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 55°C-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 55°C, 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 55°C)

-

Acid from Arabinose

-

B. coagulans

B. licheniformis

B. macquariensis

(growth at 55°C)

(methyl red pos.)

B. brevis

(methyl red neg.)

4

+

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 Bergey’s)

-

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

5

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

30°C Growth

A. veronii

+

37°C Growth

+ V. fischeri

-

P. chlororaphis

+ V. logei

V. orientalis

(Colony Pigmented)

P. fluorescens

-

(To differentiate Biovars do a lecithinase test)

V. splendidius

6

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.

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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 Bergey’s 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)

8

Shigella sonnei

Yersinia pestis