1.1.

2 Flow Chart for the Presumptive Identification of Bacteria - 1

1.1.2 Flow Chart for the Presumptive

Identification of Selected
Bacteria from Fishes*
Dr. Emmett B. Shotts, Jr.
Department of Veterinary Medicine
University of Georgia
Athens, GA 30602
404/542-5811

his revised key for the identification of bacterial pathogens of fish was developed by Dr. Emmett
Shotts in 2001 and was designed to replace and simplify the key found in the 1994 edition of the
Blue Book. This new key provides a broad perspective of strategies useful for the biochemical
identification of bacterial fish pathogens and it will be helpful in both diagnostic work and in teaching.
Users of these new tables must keep in mind that this is a presumptive key and thus it may lead to an
incorrect identification of bacterial isolates having atypical biochemical characteristics or of species not
included in this key. It is also important to note that some bacteria require special growth media or
conditions for growth and that the initial isolations must be made using appropriate substrates and
conditions. Diagnosticians should use this key as a helpful guide, but final diagnostic decisions should
be based on more information provided in other specific bacteriology subchapters of this chapter or in
Section 1, 1.1 General Procedures for Bacteriology.

*Copyright

2001 by Emmett B. Shotts Jr.

update of Figure 6 by Ben LaFrentz
U.S. Department of Agriculture; ben.lafrentz@ars.usda.gov

2014

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 2

Figure 1: Gram Stain

Gram Stain
Purple or Blue (+)

Yellow-Pigmented Colonies

Cell Morphology
Cocci

Pink or Red (-)

Rods

No

See Figure 2

See Figure 3 Oxidase

Gram + Cocci

Gram + Rods

Reaction

Positive

Yes

See Figure 6
Yellow Pigmented
Rods

Negative

See Figure 4

See Figure 5

Aeromonas,
Pseudomonas,
and Vibrio

Gram Negative,
Oxidase Negative,
Rod-shaped bacteria

*Copyright

2001 by Emmett B. Shotts Jr.

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 3

Figure 2: Gram Positive Cocci

+
Catalase
- Pinpoint Colonies +
Obligate
Aerobe
Yes

LAP Testa

No

Hemolysis on
Blood Agar

Micrococcus Staphylococcus

Aerococcus

alpha (green)

beta (clear)

gamma (none)

P Disc +
Inhibitionb
- Bile +
Solubility

Lancefield
typeable

CAMP
test

Streptococcus
viridans
Group

Streptococcus
pneumoniae
Group

No Yes

Streptococcus
Other
Miscellaneous Nonhemolytic
iniae
Streptococcus Streptococcus Streptococcus
suspect
Lancefield Group B

a = leucine aminopeptidase test

b = ethyl hydrocuprein hydrochloride (5 g) disc

*Copyright

2001 by Emmett B. Shotts Jr.

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 4

Figure 3: Gram Positive Rods

Endospore
Formation
No

Yes

Acid Fast
Aerobe or
Facultative
Anaerobe

Anaerobe

Yes

No

Mycelia
Bacillus

Special
Media
Required

Clostridium
Yes
Nocardia

No
Mycobacterium
No

H2S
Production
Yes

Renibacterium
No
Esculin Hydrolysis

Erysipelothrix

or

Yes

No
Hemolysis

Carnobacterium
Lactobacillus

Yes
Listeria
( hemolysis
on blood agar)

(none)
Corynebacterium
*Copyright

2001 by Emmett B. Shotts Jr.

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 5

Figure 4: Aeromonas, Pseudomonas,

and Vibrio
Glucose Utilization
Oxidizer (nonfermenter)
Fermenter
Pyocyanin Pigment
No

+
O/129 Sensitivity + Novobiocin Sensitivity -

Yes

Inositol

Fluorescent Pseudomonas Vibrio

aeruginosa
Pigment
Produced
No

Yes

Miscellaneous
Nonfermenters

No

Motility

Pseudomonas
fluorescens
Group

Plesiomonas

Growth
at 35C
No

No

Yes

Yes

Yes
Pseudomonas
putida
Group
Aeromonas salmonicida
Group

Motile
Aeromonas
Group

Pigment
+
< 2 mm Colony Size > 2 mm
> 5 days Growth Rate < 5 days

Atypical

Typical
*Copyright

2001 by Emmett B. Shotts Jr.

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 6

Figure 5:Gram Negative, Oxidase Negative,

Rod-shaped bacteria
Lactose
Yes

No

+ Indole - Citrate +

Phenylamine
Deaminase
No

Escherichia

Yes

Motility

H2S
No

Enterobacter

Yes

Klebsiella
Yes

Ornithine Proteus
Decarboxylase

Citrate
No

No

+ H2S - DNAse +

Yes

Providencia Morganella
Motility at 35C

Serratia

Salmonella

Indole
No
- ONPG +
+ Maltose Malonate

Edwardsiella
ictaluri

Yes

No

Yes
Edwardsiella
tarda (H2S+)
Hafnia
Edwardsiella
hoshinae

- Lysine +
+ Arginine - Ornithine +

Yersinia
intermedia

Yersinia
ruckeri

* Copyright 2001 by Emmett B. Shotts Jr.

1.1.2 Flow Chart for the Presumptive Identification of Bacteria - 7

Figure 6: Yellow-Pigmented Rods1

+
Gliding Motility
Red Colony Color in 3% KOH

3 % NaCl
Required For Growth

Yellow

Flavobacterium
branchiophilum
(Gelatinase Positive)

Yes

No

Rhizoid Colony
Morphology

Tenacibaculum
maritimum
No

Colony Tightly
Adherent
To Agar
No

Yes

Flavobacterium
columnare

Yes

Flavobacterium psychrophilum
1

New Flavobacterium spp. and related yellow-pigmented bacteria have

been described complicating this flow chart. Definitive identification using
molecular techniques may be necessary.
*Copyright

2001 by Emmett B. Shotts Jr.

2014 by Ben LaFrentz

Updated