Algorithms for Identification of Aerobic
Gram-Negative Bacteria*
PAUL C. SCHRECKENBERGER AND DAVID LINDQUIST
24
These algorithms are meant to assist in the identification of
organisms that are not readily identified by methods in place
in most clinical laboratories. Microbiologists planning to
identify an unknown gram-negative rod begin with colonies
on an agar plate. Our definition of “good growth on blood
agar plate (BAP)” is the presence of distinct colonies
(approximately 1 mm) on Trypticase soy agar with 5% sheep
blood after 24 h of incubation at 35°C in room atmosphere.
Poor growth indicates that more than 24 h of incubation is
necessary for the development of distinct colonies. If an
organism fails to grow on BAP after 72 h, it is considered to
show “no growth.” Morphological and phenotypic criteria
were chosen not only for their discriminatory value but also
because the methods are available in most laboratories.
Cellular morphology is determined by using a Gram stain
from a young colony on a BAP. The description of “tiny coc-
cobacilli” used for Brucella and Francisella in Table 2 implies
almost indiscernible cells resembling grains of sand. For
many organisms with pleomorphic morphologies, we chose
to represent the dominant shape.
The urea test refers to conventional Christensen’s urea
reaction after 24 h of incubation, whereas the rapid urea
result is read after 4 h. Glucose fermentation refers to an
acid reaction in the butt of a Kligler iron agar (KIA) or triple
sugar iron agar (TSI) tube. “Glucose oxidized” refers to acid
production in the upper portion of oxidative-fermentative
(OF) media. “BHIserum” refers to brain heart infusion
agar with 10% (vol/vol) serum added. The oxidase test refers
to results obtained with the N,N,N,N-tetramethyl-p-
phenylenediamine dihydrochloride reagent. Motility is best
* This chapter contains information presented in chapter 23 by Paul C.
Schreckenberger and Jane D. Wong in the eighth edition of this Manual.
371
observed by preparing a wet preparation from a young
colony on a BAP. Decarboxylase reactions are determined
by using an extremely turbid inoculum in Moeller’s media
(heavier than usual inoculum). Polymyxin B sensitivity is
indicated by any zone of inhibition surrounding a 300-U disk
on a BAP. For glucose-nonfermenting rods and other fastid-
ious organisms, the indole test is performed using the Ehrlich’s
extraction method (see chapter 50). “Esculin” refers to hydrol-
ysis of esculin in media without bile.
These algorithms are dichotomous, since many organisms
may fall into more than one group due to phenotypic vari-
ability of a given trait. The presence of two or more atypical
traits or a major variation from the ideal phenotype depicted
in an algorithm, due to antibiotic use, auxotrophy, or other
reasons, may limit the algorithm’s utility. These algorithms
are intended as a guide to presumptive identification of
an unknown isolate. The reference chapter describing the
organism should be consulted to determine the definitive
identification. To use the algorithms, start with Table 1 for
gram-negative bacteria that grow well on blood agar in 24 h
at room atmosphere and Table 2 for fastidious gram-negative
bacteria. Note that Table 1 consists of three parts, which we
have designated Table 1a, Table 1b, and Table 1c. In each
case begin in the upper left-hand column of the table; if the
test organism matches the given characteristic, then con-
tinue horizontally to the right to the next reaction. If the
reaction in the box matches your test organism, continue
moving horizontally until you reach the organisms listed in
the right-hand column. When the reaction in the box does
not match your test organism, then move down the column
vertically to find the reaction that matches. Repeat the
process until you reach the right-hand column. Be sure to
check all your reactions with the organism characteristics
given in the referenced chapter.
 372 ■
BACTERIOLOGY
TABLE 1a Identification algorithm for gram-negative bacteria that grow well on blood agar (part 1)

Yellow-pigmented colonies

Phenylalanine deaminase
Arginine decarboxylase

Growth on MacConkey
Lactose, trehalose, or
Lysine decarboxylase
Fluorescent pigment
Pigmented colonies
Glucose fermented

Sucrose fermented

Glucose oxidized

xylose fermented
Cell morphology

OF mannitol

Nitrate to gas
Polymyxin B

OF maltose
H2S in TSI

H2S in TSI
6% NaCl
Oxidase

Motility

ONPGa

Esculin
Indole

ONPG
Urea
Organism group (chapter)

Rod  Purple Chromobacterium (40)

Other   Vibrio (47)
  Aeromonas (46), Plesiomonas (45)
  Pasteurella (40), Actinobacillus (40)
  Pasteurella bettyae (40)
  EF4a (40)
 Pasteurella avium (40), Actinobacillus actinomycetemcomitans (40)
  Vibrio metschnikovii (47)
   Enterobacteriaceae (42–45)
  Providencia, Morganella (42, 45)
  Edwardsiella (42, 45)
 Pasteurella bettyae (40)
 P. bettyae (40)
 Pink Asaia, Azospirillum, Methylobacterium, Roseomonas (50)
Not pink    Shewanella spp. (50)
   Balneatrix alpica (50)
   Pseudomonas aeruginosa, P. fluorescens, P. putida (48)
  Agrobacterium Yellow Grp, O-1, O-2, Sphingomonas spp. (50)
 R   Pseudomonas-like group 2 (50)
 Burkholderia cepacia complex (49)
  B. stabilis (49)
  B. pseudomallei (49)
  Ralstonia mannolilytica (49)
 Pandoraea spp., Ralstonia spp. (49)
S   Ochrobactrum anthropi, Achromobacter groups B and E (50)
   CDC Vb-3 (48), OFBA-1, Ochrobactrum spp. (50)
 Ochrobactrum spp. (50), Acidovorax spp. (49), Pseudomonas-like
group 2 (50)
 P. mendocina (48), CDC Ic , Ochrobactrum spp. (50)
    Rhizobium radiobacter (50)
 Ochrobactrum anthropi, Achromobacter Group F, Halomonas venusta,
Inquilinus limosus (50)

24. Identification of Aerobic Gram-Negative Bacteria ■

  P. stutzeri (48), Ochrobactrum anthropi (50)
 Pseudomonas-like group 2, Herbaspirillum, CDC halophilic
nonfermenter group 1 (50)
  CDC group O-3, Inquilinus limosus, Massilia timonae (50),
Brevundimonas vesicularis (49)
  P. stutzeri (48), Achromobacter xylosoxidans (50)
 A. xylosoxidans (50), Brevundimonas diminuta (49)
aONPG, O-nitrophenyl-beta-D-galactopyranoside.

373
 374 ■
TABLE 1b Identification algorithm for gram-negative bacteria that grow well on blood agar (part 2)

NO2 reduction
decarboxylase

OF mannitol
Polymyxin B

NO3 to NO2
Curved rods
morphology

H2S in TSI

6.5% NaCl
Rapid urea

Acetamide
Pigmented

pigmented

Growth in
fermented

Arginine
oxidized
Oxidase

colonies
colonies

Glucose
Glucose

Motility

Esculin

Gelatin
Yellow-
Indole

Urea
Cell

BACTERIOLOGY
Organism group (chapter)

Rod  Not pink      R Pandoraea spp. (49)

S   Laribacter hongkongensis (50)
 P. pseudoalcaligenes, P. alcaligenes, Pseudomonas species group 1 (48)
  Bordetella bronchiseptica, Oligella ureolytica, Cupriavidus pauculus (50)
   Pseudomonas species group 1 (48), Achromobacter denitrificans (50)
 Alcaligenes faecalis (50)
   Delftia acidovorans (49)
  Achromobacter piechaudii (50)
 Bordetella avium (50, 51)
  CDC halophilic group 1 (50)
  P. pseudoalcaligenes, P. alcaligenes (48), Comamonas terrigena, C. testosteroni
(49), Achromobacter piechaudii, Cupriavidus taiwanensis (50)
 Brevundimonas diminuta, Brevundimonas vesicularis (49), Bordetella hinzii
(50, 51), P. alcaligenes (48), Cupriavidus gilardii, Cupriavidus respiraculi (50)
   Chryseobacterium indologenes/gleum, Empedobacter brevis, CDC group IIi (50)
  Bergeyella zoohelcum (50)
   Elizabethkingia meningoseptica (50)
 CDC group IIc, IIh, IIi (50)
  Weeksella virosa, Empedobacter brevis (50)
 CDC group IIe, IIg (50)
    Sphingobacterium spiritivorum, Inquilinus limosus (50)
   Sphingobacterium thalpophilum (50)
 Sphingobacterium multivorum (50)
 Sphingomonas paucimobilis, Flavobacterium mizutaii (50)
  EF-4b, Paracoccus yeei, Psychrobacter immobilis (50)
 EO-3, EO-4 (50)
   Myroides spp. (50)
 Oligella ureolytica (50)
  Alishewanella fetalis (50)
 Neisseria weaveri, N. elongata, Gilardi rod group 1 (39, 50)
TABLE 1c Identification algorithm for gram-negative bacteria that grow well on blood agar (part 3)

Glucose oxidized

Brown diffusible

Growth at 42oC
decarboxylase

decarboxylase
or OF lactose

NO2 reduced
NO3 reduced
morphology

OF maltose
Pigmented
fermented

Mannitol
Oxidase
colonies

pigment
Glucose

Motility

Gelatin
Esculin
DNase

Lysine

Lysine
Urea
Cell

Organism group (chapter)

Rod  Not pink      Burkholderia cepacia complex (48)

Both negative   Pseudomonas luteola (47)
 P. oryzihabitans (47)
 Burkholderia gladioli (48)
  Stenotrophomonas maltophilia (48)
 Sphingomonas paucimobilis (49)
  Bordetella ansorpii (50, 51)
  Kerstersia gyiorum (50)
 Bordetella trematum (50, 51)
  Acinetobacter baumannii, EO-5 (50)
  Bordetella parapertussis (50, 51)

24. Identification of Aerobic Gram-Negative Bacteria ■

  CDC NO-1 (50)
  Bordetella holmesii (50, 51)
 Acinetobacter lwoffii (50)
Diplococci  Neisseria (39), Moraxella catarrhalis (50)
 Acinetobacter (50)
Coccobacilli   Oligella ureolytica (50)
   EF-4b, Paracoccus yeei, Psychrobacter immobilis (saccharolytic) (50)
 EO-3, EO-4 (50)
  Moraxella canis, M. catarrhalis (50)
  Psychrobacter phenylpyruvica (50), Brucella spp. (52)
  Oligella urethralis (50)
  Moraxella lacunata, M. nonliquefaciens, M. osloensis, P. immobilis
(asaccharolytic) (50)
 M. atlantae, M. lincolnii, M. osloensis, P. immobilis (asaccharolytic) (50)
   Acinetobacter baumannii, EO-5 (50)
  Bordetella parapertussis (50, 51)
  CDC NO-1 (50)
  Bordetella holmesii (50, 51)

375
 Acinetobacter lwoffii (50)
376 ■ BACTERIOLOGY

TABLE 2 Identification algorithm for gram-negative bacteria with poor or no growth on blood agara

Growth H2S Cauliflower-

Growth Cellular Pigmented 6% O-shaped Require Organism group
on Urea Oxidase in like
only on: morphology colonies NaCl cells X±V (chapter)
BAP TSI colonies

Poor Tiny coccobacilli  Brucella (52)

   Francisella
philomiragia (52)
 Bordetella (50)
 Francisella (52)
Rods Pink Asaia, Azospirillum,
Methylobacterium,
Roseomonas (50)
Other  Bartonella (54)
 Haemophilus
aphrophilus (41),
Cardiobacterium,
Dysgonomonas,
Eikenella,
Kingella,
Simonsiella,
Suttonella (40)
Diplococci or  Paracoccus yeei (50)
coccobacilli
 Neisseria (39),
Moraxella (50)
None Chocolate Diplococci or Neisseria (39)
coccobacilli
Fusiform rod Capnocytophaga (40)
Rod  Bartonella, Afipia (54)
  Haemophilus (41)
 Francisella (52)
BCYE Long gram-negative Legionella (53)
rods
Regular rods  Bordetella (51)
 Francisella (52)
BHI Pleomorphic, beaded Streptobacillus (40)
serum filamentous rods
Small rods Bartonella, Afipia (54)
a Abbreviations: BCYE, buffered charcoal yeast extract agar; X, hemin; V, nicotinamide adenine dinucleotide.