MLS REVIEW NOTES DIAGNOSTIC MICROBIOLOGY

DIAGNOSTIC MICROBIOLOGY

COLONIAL MORPHOLOGY

GRAM-POSITIVE COCCI

Staphylococci:
BAP: colonies (4 mm to 8 mm) appear creamy, white or light gold, or “buttery-looking” while other
species may have gray colonies; some species may be Beta-hemolytic like S. aureus. Arranged in
clusters
Staphylococcus aureus BAP: golden yellow color and a Beta-hemolytic
Staphylococcus BAP: white, opaque, small to medium-sized pin-head, and are non-
epidermidis hemolytic
Staphylococcus BAP: white, opaque, slightly larger than pin-head, non-hemolytic although
saprophyticus some strain produce yellow pigments

Streptococci:
BAP: grayish, pinpoint, and translucent to slightly opaque white some species have muciod colonies.
Arranged in chains or pairs.
Streptococcus BAP: small, translucent, smooth, and exhibit well-defined Beta-hemolysis.
pyogenes
Sreptococcus BAP: grayish white and mucois with a small zone of Beta hemolysis.
agalactiae
Streptococcus BAP: flat with depressed centers, mucoid, and are alpha-hemolytic.
pneumoniae
Viridans streptococcus BAP: alpha hemolytic
Enterococci BAP: non-hemolytic or may be alpha- or beta-hemolytic, arranged in pairs
and long chains.
Abiotrophia and BAP: Gram-variable and pleomorphic form
Granulicatella
Aerococcus BAP: Clusters or tetrads
Gemella Gram-negative cocci in pairs clusters or short chains
Lactococcus
Leuconostoc Irregular coccoid
Pediococcus Arranged in pairs, tetrads, and clusters

GRAM-NEGATIVE COCCI

Neisseria
Gram-negative diplococci that are coffee- or kidney bead-shaped; small, grayish-white, opaque convex,
and glistening.
Neisseria gonorrhoeae CAP: small, tan-colored, translucent, and raised.
Neisseria meningitidis BAP: colonies appears bluish gray
CAP: small, tan, and mucoid
Encapsulated strains are mucoid in appearance
Moraxella catarrhalis BAP: colonies are seen as smooth, opaque, grayis-white, and have a
“hockey puck” apprearance.

GRAM NEGATIVE BACILLI

Enterobacterales
Colonies appear as large, smooth, and gray except for Klebsiella and Enterobacter with mucoid
colonies, and are non-hemolytic except for some strains of E. coli which are Beta-hemolytic.

Escherichia coli MAC: colonies appear flat and dry, and exhibit a pink color (lactose
fermenter or LF); some strains may be non-lactose or NLF.
BAP: Most strain are non-hemolytic; some are Beta-hemolytic
EMB: colonies exhibit a greenish metallic sheen.
Klebsiella pneumoniae MAC: colonies exhibit a pink color and are mucoid (LF)
Enterobacter MAC: colonies exhibit a pink color and are sometimes mucoid
Cronobacter sakazakii MAC: colonies exhibit a pink color (LF)
BHIA: colonies exhibit a tellow pigmentation
Pantoea agglomerans MAC: colonies are clear or colorless (NLF).
Serratia MAC: colonies are clear and colorless NLF; some strains may show slow or
late lactose fermenter.
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Proteus MAC: colonies are clear and colorless (NLF); exhibit a swarming
phenomenon”; and have a “burnt-chocolate” or “burnt-gunpowder” odor.
Providencia MAC: colonies are clear and colorless (NLF)
Morganella MAC: colonies are clear and colorless (NLF)
Citrobacter MAC: colonies become clear and colorless (NLF) after 24 hours; colonies
exhibits a light pinl color (LF after 48 hours.
Salmonella MAC: colonies are clear and colorless (NLF)
SSA:colonies are colorless with black centers
Shigella MAC: colonies are clear, fragile, and colorless (NLF)
SSA: colonies are colorless without black centers.
Yersinia pestis MAC: colonies are clear and colorless (NLF)
BAP: Colonies are pinpoint at 24 hours
Broth: colonies have a “stalactite-shaped” pattern
Yersinia enterocolitica MAC: colonies are clear and colorless (NLF)
CIN: colonies exhibit a “bull’s eye” appearance or dark red or burgundy
centers with transparent borders at 48 hours of incubation.
Yersinia MAC: colonies are clear and colorless (NLF)
pseudotuberculosis
Plesiomonas MAC: colonies are clear and colorless
shigelloides BAP: colonies are shiny, opaque, smooth, and non-hemolytic
Inositol-brilliant green-bile salt agar: colonies exhibit white or green
to pink color for other enterics.
HEA: colonies exhibits growth
TCBS: colonies do not exhibit growth
Edwardsiella MAC: colonies are clear and colorless (NLF)

GRAM-NEGATIVE INTESTINAL PATHOGEN (NON-ENTERIC)

Vibrio
CAP: colonies are smooth, opaque, and iridescent with a greenish blue
MAC: colonies are NLF (except for V. vulnificus which is a lactose fermenter
BAP: colonies are alpha- and beta-hemolytic
Vibrio cholerae BAP: colonies are smooth and medium-to-large-size with a greenish hue

Aeromonas
BAP: colonies are large, round, raised, white, and opaque
MAC: colonies exhibits a pink color
CIN: colonies have a “bull’s-eye” appearance

Campylobacter
Colonies are gray, flat, glistening, and irregular with a tailing effect” along the streak line or “runny
spreading” growth.

Helicobacter
CAP: colonies are gray and translucent

GRAM-NEGATIVE BACILLI (NON-FERMENTATIVE)

NON-FERMENTER
Pseudomonas BAP: colonies have a spreading growth, and are flat; have a metallic
aeruginosa sheen; have a blue-green or red-brown color; have a “grape-like” or “corn
tortilla odor: and are sometimes mucoid (observed in patient with cystic
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fibrosis);have Beta-hemolytic pattern

Acinetobacter MAC: colonies exhibit a purple color
Plump, gram-negative coccobacilli which appears in pairs; may also appear
as Gram-positive bacilli from blood culture bottles. Non-hemolytic
Stenotrophomonas BAP: colonies exbihit lavender-green to light purple pigmentation and an
maltophilia odor similar to that of ammonia
MAC: colonies exhibit a bllue color.
Burkholderia cepacia BAP: colonies exhibit a non-wrinckled yellow or yellow-green color.
Burkholderia mallei BAP: colonies are non-pigmented
Burkholderia Ashdown medium with colistin: colonies are dry, wrinkled, and have a
pseudomallei deep pink color.
Alcaligenes faecalis BAP: colonies are feather-edged, non-pigmented, alpha-hemolytic, and
have a “fruity” odor which is similar to that of apples or strawberries.
Oligella BAP: colonies are small, opaque, and white
MAC: no growth
Moraxella lacunata BAP: colonies are small and pit the agar.
MAC: colonies exhibit no growth
Chromobacterium May appear as curved bacilli. Colonies exhibits violet pigmentation
violaceum (violacein pigment)
Shewanella BAP: colonies appear mucoid and greenish (including the medium).
putrefaciens

GRAM-NEGATIVE BACILLI SMALL, PLEOMORPHIC

Haemophilus
Gram-negative, small, pleomorphic coccobacilli or rods
Haemophilus CAP: colonies are translucent, convex, tan-colored, and mucoid with a
influenzae “mousy” or “bleach-like” odor.
Haemophilus ducreyi CAP: colonies are transparent, small, non-mucoid, and tan or yellow.

AACEK GROUP

Aggregatibacter CAP: colonies are raised, convex, granula, and yellowish

aphrophilus
Aggregatibacter Colonies exhibti a “star-shaped” appearance after 48 hours of incubation.
actinomycetemcomitan
s
Cardiobacterium BAP: colonies are capnophilic and exhibit “pitting”
hominis Yeast extract: colonies exhibit a “rosette” formation and appear
filamentous
Eikenella corrodens BAP: colonies exhibit a yellow color; pit or corrode the agar; and have a
“sharp bleach” odor
Kingella spp Plump, square-ended, gram negative rods to coccobacilli that are
arranged in pairs or short chains
BAP: colonies exhibit white to yellowish-brown pigmentations
TMA: K. denitrificans resembles the growth of N. gonorrhoeae

Brucella
Small coccobacilli that are arrangede singly, pairs, or in short chains, and which have a “sandy
appearance”
BAP: colonies are small convex, translucent, yellowish and non-hemolytic; they may become brownish
in color with age.

Bordetella
Bordet-Gengou agar: colonies are smooth, glistening, and have a silver color
Bordetella pertussis Bordet-Gengou agar: colonies are small and shiny, and resemble
“mercury drops”

Francisella tularensis
Gram-negative bacilli with faint bipolar staining. Colonies are round, smooth, bluish gray to white, and
slightly mucoid.
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MLS REVIEW NOTES DIAGNOSTIC MICROBIOLOGY
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Pasteurella
BAP and CAP: colonies are gray and non-hemolytic

Legionella
BCYE: colonies appear sticky and exhibit a “rainbow color”
Legionella pneumophila BCYE: colonies are blue-green and glistenin, and have a convex
elevation.

GRAM POSITIVE BACILLI (Aerobic)

Bacillus
Microscopy: Large, boxcar-shaped, gram-positive rods with clear, unstained, central spored or “empty
spaces”

Bacillus anthracis BAP: colonies have a “Medusa head” appearance with swirling
projections; are non-hemolytic; and gain a “beaten egg white”
appearance when an inoculating loop is used.
Bacillus cerues BAP: colonies are large and feathery; have a spreading growth; have a
“frosted-glass” appearance; and a Beta-hemolytic
Bacillus subtilis BAP: colonies are large, flat, and dull with a ground glass appearance;
may be Beta-hemolytic; may exhibit pigment atoms (pink, yellow, orange,
or brown)
Bacillus pumilus BAP: colonies are large and moist; have a blister-like appearance; and
can be Beta-hemolytic

Corynebacterium
Microscopy: Slightly curved, gram-positive rods with unparallel sides and slightly wider ends that
produce a “club-like” shape
BAP: colonies have a small zone of Beta-hemolysis, although some strains are non-hemolytic.
Corynebacterium Its cells have rounded ends and “club-shaped swellings”
diphtheriae It is highly-pleomorphic cells are arranged in pairs and create X, V, Y, L
formations that closely resmebles Chinese letters
Corynebacterium Arranged in V-shaped forms and palisades
urealyticum BAP: colonies are pinpoint, white, smooth, and are non-hemolytic.
Corynebacterium Cells are arrance in parallel rows or palisades and do not exhibits any
pseudodiphtheriticum other characteristics “pleomosphism” that is similar to other
corynebacteria.
Corynebacterium Pleomorphic, club-shaped, and arranged in V-shaped forms
jeikeium BAP:Colonies appear large when added with 1% tween 80

Corynbacterium BAP: colonies have a narrow zone of Beta-hemolysis.

ulcerans CTBA: colonies have a surrounding brown halo
Loeffler’s serum agar: colonies exhibit growth
Corynebacteirum CTBA: colonies exhibit a black color and are surrounded with a brown
pseudotuberculosis halo
BAP: colonies are small are yellowish-white

Listeria Monocytogenes
Microscopy: Coccobacillary in form and are arranged singly or in short chains that resemble
streptococci
BAP: Colonies are small, smooth, translucent, grayish-blue, and are surrounded by a narrow zone of
Beta-hemolysis.

Erysipelothrix rhusiopathiae
Microscopy: Thin, phleomorphic rods with the tendency to form long filaments that are arranged in
single, short chains or in a V-shaped formation
BAP and CAP:

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MLS REVIEW NOTES DIAGNOSTIC MICROBIOLOGY
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a. Large and rough colonies - curled, slender, filamentous with a tendency to over-decolorize and
become Gram-negative bacilli
b. Small and smooth colonies - transparent, glistering, and slender rods

Arcanobacterium
BAP: colonies have a narrow zone of Beta-hemolysis; exhibitingh pitting of agar with a black opaque
dot.

Kurthia
BAP: colonies exhibits a large “medusa-head” appearance.
Nutrient agar - colonies exhibit a rhizoid growth

Aerobic Actinomyces
Microscopy: Filamentous gram-positive rods with a beaded appearance
Cells elongate to form branching filamentous forms while some organisms form hyphae on the agar
surface or into the agar.

Acid-Fast Aerobic Actinomycetes

Nocardia Microscopy: gram positive bacilli with long, thin, beaded, branching
filaments; old cultures may fragment into short, rod-shaped, coccoid
forms.
Colonies exhibit wrinkled, chalk-like, and orange-tan pigmentation.

Rhodococcus, Gordonia, and Tsukamurella

Rhodococcus equi Microscopy: coccobacilli with a “zigzag” pattern and a filamentous form
BAP: colonies exhibit a pale pink or yellow color.
Gordonia Colonies are smooth and slimy with irregular edges; but may appear as
dry or rough; and exhibit the presence of mycelia
Tsukamurella Colonies are circular with rhizoid edges; has no aerial hyphae; and exhibit
white or orange pigmentation.

Non-acid-fast, Aerobic Gram-Positive Actinomycetes

Streptomyces Colonies are dry to chalky and heaped; some colonies exhibit a grayish-
white color and a “mustry basement” odor
Actinomadura Routine agar: colonies exhibit a “molar tooth” appearance.

Mycobacteria
Microscopy: slender, sligthly curved or straight rods that have a tendency to “clump”
Egg-based agar: colonies are smooth and soft or soft or have a rough and friable growth.

Mycobacterium tuberculosis complex

Mycobacterium Colonies are slow-glowing; exhibit buff color; are raised and dry; and have
tuberculosis a “cauliflower-like” appearance. Rough colonies exhibit “cording” (curved
strands of bacilli).
Mycobacterium bovis Colonies are slow-glowing, small, granulr, rounded, and non-pigmented.

Non-tuberculous Mycobacteria (NTM) - SLOW GROWER

Mycobacterium avium Microscopy: Pheomorphi, short, coccobacilli without beading; positive
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cpmplex with periodic acid-Shoff (PAS) staining

Mycobacterium kansaii Microscopy: Long rods with distinct crossbanding
MB710- colonies are smooth to rough with dark centers and waxy edges .
Phtochromogenic colonies exbihit a dark-red crystal color.
Mycobacterium Microscopy: Long rods with cross-barring
marinum Colonies are smooth to rough, wrinkled, and yellow (photochromogenic)
Mycobacterium Microscopy: Moderately long rods without cross-banding
ulcerans Colonies are smooth, rough, and non-pigmented (6-12 weeks of
incubation).
Mycobacterium Colonies are smooth and yellowish-orange colored (scotochromogen)
gordonae
Mycobacterium xenopi Microscopy: Long and filamentous rods
MB 7H10: Colonies are small with filamentous edges
Cornmeal glycerol agar: colonies are round with branching filaments.
Mycobacterium terrae Microscopy: Short to medium coccobacilli
complex Mycobacteium triviale: colonies are rough and dry.
Mycobacterium terra: colonies are smooth
Mycobacterium nonchromogenicum: colonies are smooth to rough and
white to buff

Other NTM-Slow Growers

Mycobacterium Microscopy: Acid-fast coccoid cells
Asiatucum Dysgonic, smooth, and pogmented colonies
Mycobacterium Microscopy: Distinct acid-fast cells
genavense Dysgonic colonies; requires an extended incubation (6-8 weeks)
Mycobacterium Microscopy: Distinct acid-fast cells; short or curved, without beading,
haemophilum appears in clusters or cords
Colonies are rough to smooth and non-pigmented; the recommended
media include CAP, MHA with 5% Fildes enrichment, and LJ medium with
2% ferric ammonium citrate
Mycobacterium Microscopy: Short coccobacilli without crossbands
malmoense Non-pigmented, smooth, glistering, and opaque colonies with dense
centers
Mycobacterium Microscopy: Medium to long rods
scrofulaceum Light yellow to deep orange smooth colonies with dense centers
Mycobacterium simiae Microscopy: Short coccobacilli
Filamentous colonies; yellow and smooth colonies after an extended
incubation
Mycobacterium szulgai Microscopy: Medium to long rods with cross-barring
Yellow to orange and smooth to rough colonies

NTM-Rapid Growers
- Microscopy: Weakly-staining Gram-positive rods that resembles diphtheroids
- Colonies appear in solid media in seven days or less.
Mycobacterium Microscopy:
fortuitum - Pleomorphic : long to short, thick rods
- Old pictures: may appear partially AFB
MB7H11: colonies are branching and filamentous with aerial hyphae
MAC: colonies exhibit growth in a medium without crystal violet
Mycobacterium Microscopy: Strongly acid-fast with pleomorphism in young culture
chelonae Colonies are rough or smooth, non-pigmented, and have no filamentous
branchings
MAC: colonies exhibit growth in a medium without crystal violet.
Mycobacterium MAC: colonies exhibit growth in a medium without crystal violet
abscessus
subspabscessus
Mycobacterium Microscopy: long and tapered rods with partial acid-fastness; may be
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smegmatis beaded or avoid in form

MB7H10: Colonies are smooth or rough
MAC: colonies exhibit growth in a medium without crystal violet.

Non-cultivable NTM
Mycobacterium Microscopy: rod-shaped and exhibits “ciagr-pocket” or pocket-fence”
Leprae arrangement
Colonies exhibit growth in living tissues of the footpads of mice and
aramadillos.

GRAM POSITIVE ANAEROBIC SPORE-FORMING BACILLI

Clostridium

Clostridium perfringens Microscopy: “Boxcar-shaped” bacilli with oral, subterinal spores

BAP: colonies are dome-shaped and grayish white with double zone of
hemolysis
Litmus milk: colonies exhibit a stormy fermentation of milk.
Clostridium tetani Microscopy: Cells are with terminal spores and swollen sporangia that
have a “drumstrick” or “tennis-racket” appearance.
BAP: colonies exhibit a slow, anaerobic, heavy, smooth, and swarming
growth and have a matte surface with a narrow zone of Beta-hemolysis.
Clostridium botulinum Miscroscopy: Presence of subterminal spores
BAP: Beta-hemolytic colonies
Clostridium difficile Microscopy: chains up to 6 cells that are aligned from end to end with
oval, subterminal endospores
Cycloserine-cefoxitin-fructose agar (CCFA) - colonies exhibit a yellow color
and a “ground-glass” appearance
BAP: Colonies exhibit a “horse stable” odor; are non-hemolytic; and
produce a flurescent chartreuse under a long UV light wave.

ANAEROBIC NON-SPORE-FORMING BACILLI AND COCCI

ORGANISM MICROSCOPY CULTURE

Actinomyces Anaerobic, straight or slithly curves, Young colonies: “spider-like” or
Gram-positive rods that are banded “wooly” appearance
or beaded Old colonies: “molar tooth”
appearance
Bacteroides fragilis Plae-staining, pleomorphic, Gram- Grayish-white, circular, smooth,
negative rods with a “safety-pin” and non-hemolytic.
appearance
BACTEROIDES BILE ESCULIN:
Colonies exhibit a gray color and
growth with 20% bile, and cause
the blackening of the originally
yellow-colored agar.
Bacteroides urealyticus Pale-staining, thin, Gram-negative Colonies corrode (pit) the agar
rods
Bifidobacterium spp Gram-positive diphtheroids; that are Small, white, shiny, and convex
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MLS REVIEW NOTES DIAGNOSTIC MICROBIOLOGY
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coccoid or pointed in shape with

bifurcated (forked) ends which
resemble the shape of a “dog bone”
Clostridium septicum Gram-positive rods in young cultures Formation of rhizoid margins that
that turn resemble “ Medusa’s head”
Gram negative with age; have
subterminal spores
Eubacterium Pleomorphic, Gram-positive rods that Fluorescent chartreuse color
are seagull wing-shaped
Fusobacterium Spindle-shaped, Gram neagative The medium exhibits a green color
nucleatum rods that resembles Capnocytophaga upon air exposure; colonies have
a “breadcrumbs-like” appearance.
Lactobacillus Gram-variable rods or short Pinpoint color
coccobacilli that resemble
streptococci
Leptotrichia Large fusiform Gram-negative rods “Raspberry-like” colonies
Peptococcus niger Gram-positive cocci that are paired Small, black, and shiny colonies
singly, pairs, or in tetrads
Peptostrepcotoccus Large, Gram-positive coccobacilli in Grayish-white colonies that emits
anaerobius chains a foul odor
Porphyromonas Gram-negative coccobacilli Brown, mucoid colonies with brick
red fluorescence
Propionibacterium Diphtheroid-like, Gram-positive rods; Small, grayish-white colonies
anaerobic diphtheroids that have a palisade arrangement
Prevotella Gram-negative rods White, shiny colonies with a brick
red fluorescence
Veillonela parvula Tiny Gram-negative diplococci Red fluorescence

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