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.
Bacillus spp.
You sure you have this? Go to Bergey's.
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 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)
(makes insecticidal protein)
B. mycoides
B. azotoformans B. larvae B. pasteurii
(grows at 55°C, rest no)
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. subtilis B. polymyxa B. lichenifomis B. alvei B. coagulans
(colony has a rhizoidal appearance)
B. popilliae
Catalase
(spore is not round, rest are)
B. azotoformans
Swollen Cell
Swollen Cell
(containing spore)
(containing spore)
-
B. stearothermophilus
B. megaterium
(growth at 55°C-rest neg.)
+
(citrate pos.)
B. macerans B. pantothenticus B. macquariensis B. lentus
B. badius (citrate neg.)
(acid from glucose)
B. marinus (acid from glucose)
Nitrate Reduction
6.5% NaCl Growth B. coagulans B. licheniformis B. subtilis
B. insolitus
(oval spore, rest are round)
+
-
B. pasteurii
B. sp sphaericus
Citrate
+
-
B. sphaericus B. pasteurii B. laterosporus
B. alcalophilus B. brevis B. circulans
(Motile - It is this one )
B. larvae (gelatinase pos.) B. popilliae (gelatinase neg.)
B. insolitus B. laterosporus B. pasteurii B. marinus B. sphaericus
(catalase neg.-rest pos.)
B. cereus
Citrate
+
+
B. badius
+
-
(round spore, rest are oval)
B. popilliae B. pasteurii B. lentimorbus B. azotoformans B. badius B. insolitus B. larvae B. laterosporus
(add creatine as catalyst w/ reagents)
+
B. thuringiensis
(VP is pos., rest are neg.)
B. marinus B. sphaericus B. schlegelii
+
(the width)
(non-motile)
B. pumilus
-
Cell Diamenter ! 1"m
B. anthracis
spp.
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 Streptococcus spp. (see Bergey’s)
Micrococcus s pp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.
Bile Esculin (growth with esculin hydrolysis) +
Catalase
-
+
Micrococcus spp. Staphylococcus spp.
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
-
+
Staphylococcus aureus
-
Hemolysis 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) agalactiae )
No Staphylococcus saprophyticus
Optochin Sensitivity
Yes Strept Streptoco ococcu ccuss pneumo pneumonia niaee
5
No Strept Streptoco ococcu ccuss mitis 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., Aeromonas spp.
-
Glucose Fermentation
- Acid
Pseudomonas spp.
Fluorescent Diffusible Yellow Pigment Na+ Required for Growth
(Fluorescein)—use (Fluorescein)—use Psuedo F Agar
Aeromonas Aeromonas spp.
+
+ VP
Vibrio spp.
-
+ A. hydrophila A. salmonicida
Luminescent
Other Aeromonas Aeromonas spp. (See Bergey's)
(subsp. masoucida)
-
P. P. aeruginosa P. chlororaphis P. cichorii P. fluorescens P. putida P. syringae
Other Pseudomonas 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 Non-Fluorescent Diffusible Blue Pigment
Other Vibrio 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. splen didius
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. sp lendidius
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 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
subsp. pneumoniae Klebsiella pneumoniae subsp. pneumoniae
Enterobacter cloacae
Non-motile, No Pigment
(VP neg.)
Serratia rubidaea
Erwinia chrysanthemi
Acid from sorbitol
Enterobacter amnigenus
Motile, Red pigment
(VP and H2S pos.)
No acid from sorbitol
Enterobacter aerogenes
H2S Production
Klebsiella oxytoca (VP pos and H2S neg.)
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 flexneri) Shigella spp. 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.
Erwinia cacticida Salmonella bongori Salmonella enterica Serratia marcescens Serratia liquefaciens Shigella sonnei Yersinia pestis
Proteus mirabilis Ornithine decarb. pos.
flexneri) (boydii, dysenteriae, flexneri)
Proteus penneri
Yersinia enterocolitica
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 choleraesuis Serratia marcescens Serratia liquefaciens
Ornithine Decarboxylase
+
+
Proteus vulgaris
Providencia stuartii Ornithine Decarboxylase
Edwardsiella tarda
Morganella morganii
H2S
+ Shigella sonnei
+
-
Salmonella bongori
Serratia marcescens
KCN growth pos.
Red pigment
Salmonella enterica KCN growth neg.
Serratia liquefaciens No Red pigment
(It is this one)
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Yersinia pestis
